Experimental models of CKD

Nephrology Dialysis Transplantation, May 2013

Introduction and Aims:Kidney stone disease is associated with renal fibrosis by the unclear mechanisms. We hypothesized that calcium oxalate (CaOx), a major crystalline component of kidney stones, could induce secretion of fibrotic factors from macrophages leading to “epithelial mesenchymal transition/transdifferentiation” (EMT) of renal tubular cells. Methods:EMT markers were examined by Western blot analysis and immunofluorescence study. Level of TGF-β1 in the “secreted products of CaOx-exposed macrophages” (CaOx-M-Sup) and cellular levels of the cascade signaling molecule RhoA as well as the ubiquitinated proteins were measured. Finally, a proteasome inhibitor (MG132) was applied to examine whether the intervention of ubiquitin-proteasome pathway (UPP) could prevent EMT and changes in RhoA induced by CaOx-M-Sup. Results:Western blot analysis revealed an increased level of vimentin (mesenchymal marker) but decreased levels of E-cadherin and cytokeratin (epithelial markers) in MDCK cells treated with CaOx-M-Sup. Immunofluorescence study confirmed the increased level of vimentin and decreased level of cytokeratin, and also revealed the increased level of fibronectin (another mesenchymal marker). The data also showed decreased levels and disorganization of F-actin (cytoskeletal marker) and zonula occludens-1 (ZO-1) (tight junction marker) induced by CaOx-M-Sup. ELISA demonstrated the increased level of transforming growth factor-β1 (TGF-β1), the well-defined EMT inducer, in CaOx-M-Sup. Downstream signaling of TGF-β1 was involved as demonstrated by the decreased level of RhoA. Interestingly, pretreatment with MG132 could restore RhoA to its basal level, most likely through UPP. Moreover, MG132 successfully sustained cytoskeletal assembly and tight junction, and could prevent the cells from EMT. Conclusions:Altogether, these data demonstrate for the first time that CaOx-M-Sup could induce EMT in renal tubular cells by TGF-β1 signaling cascade via RhoA and UPP. This may be, at least in part, the underlying mechanism for renal fibrosis in kidney stone disease.

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Experimental models of CKD

Thilo Krueger 2 6 8 11 14 17 20 22 24 Peter Boor 2 6 8 11 14 17 20 22 24 Cora Schafer 1 5 7 10 13 16 19 21 23 Ralf Westenfeld 2 6 8 11 14 17 20 22 24 Vincent Brandenburg 2 6 8 11 14 17 20 22 24 Georg Schlieper 2 6 8 11 14 17 20 22 24 Willi Jahnen-Dechent 1 5 7 10 13 16 19 21 23 Markus Ketteler 0 9 12 15 Webster Jee 4 18 Xiaodong Li 3 Bill Richards 3 Jrgen Floege 2 6 8 11 14 17 20 22 24 0 Nephrology Hospital Coburg Coburg Germany 1 Biomedical Engineering RWTH University Aachen Aachen Germany 2 Nephrology and Clinical Immunology RWTH University Clinic Aachen Aachen Germany 3 Amgen Inc. Thousand Oaks CA United States 4 University of Utah Salt Lake City UT United States 5 Kawagoe Ekimae Clinic Kawagoe Japan 6 Nephrology and Hypertension Saitama Medical center, Saitama Medical University Kawagoe Japan 7 Department of Internal Medicine Daejeon Saint Mary Hospital Daejeon Republic of Korea 8 Southeast University Institute of Nephrology Nanjing China 9 Department of Internal Medicine Daejeon Sun Hospital Daejeon Republic of Korea 10 Department of Internal Medicine Dajeon Saint Mary Hospital, Catholic University Daejeon Republic of Korea 11 Division of Nephrology, Department of Medicine Showa University School of Medicine Tokyo Japan 12 Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute Boston United States 13 Nephrology Division University of Medicine and Dentistry of New Jersey New Brunswick NJ United States 14 1st Department of Critical Care Medicine and Pulmonary Diseases Research Laboratories, Evagelismos Hospital University of Athens School of Medicine Athens Greece 15 Department of Integrated Diagnostics & Therapeutics National Taiwan University Hospital Taipei Taiwan Republic of China 16 Institute of Toxicology Medical College, National Taiwan University Taiepi Taiwan Republic of China 17 Internal Medicine National Taiwan University Hospital Taiwan Taiwan Republic of China 18 Department of Dentistry Taipei Chang Gang Memorial Hospital Taiwan Taiwan Republic of China 19 Department of Internal Medicine University of Miyazaki Miyazaki Japan 20 Department of Internal Medicine University of Michigan Ann Arbor MI United States 21 Medical Research Council, Human Genetics Unit Edinburgh United Kingdom 22 Division of Nephrology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine Kaohsiung Taiwan Republic of China 23 Allergy Dongguk University Hospital, Dongguk University-Seoul 814 Siksa-Dong , Ilsandong-gu, Goyang-si Kyunggi-do Republic of Korea 24 Nephrology Dongguk University Hospital, Dongguk University-Seoul 814 Sik-sa Dong , Ilsan-Dong gu, Goyang-si Kyung-gi Province Republic of Korea EXPERIMENTAL MODELS OF CKD - SECRETED PRODUCTS OF MACROPHAGES EXPOSED TO CALCIUM OXALATE CRYSTALS INDUCE EPITHELIAL MESENCHYMAL TRANSITION OF RENAL TUBULAR CELLS VIA RhoA-DEPENDENT TGF-1 PATHWAY Introduction and Aims: Kidney stone disease is associated with renal fibrosis by the unclear mechanisms. We hypothesized that calcium oxalate (CaOx), a major crystalline component of kidney stones, could induce secretion of fibrotic factors from macrophages leading to epithelial mesenchymal transition/transdifferentiation (EMT) of renal tubular cells. Methods: EMT markers were examined by Western blot analysis and immunofluorescence study. Level of TGF-1 in the secreted products of CaOx-exposed macrophages (CaOx-M-Sup) and cellular levels of the cascade signaling molecule RhoA as well as the ubiquitinated proteins were measured. Finally, a proteasome inhibitor (MG132) was applied to examine whether the intervention of ubiquitin-proteasome pathway (UPP) could prevent EMT and changes in RhoA induced by CaOx-M-Sup. Results: Western blot analysis revealed an increased level of vimentin (mesenchymal marker) but decreased levels of E-cadherin and cytokeratin (epithelial markers) in MDCK cells treated with CaOx-M-Sup. Immunofluorescence study confirmed the increased level of vimentin and decreased level of cytokeratin, and also revealed the increased level of fibronectin (another mesenchymal marker). The data also showed decreased levels and disorganization of F-actin (cytoskeletal marker) and zonula occludens-1 (ZO-1) (tight junction marker) induced by CaOx-M-Sup. ELISA demonstrated the increased level of transforming growth factor-1 (TGF-1), the well-defined EMT inducer, in CaOx-M-Sup. Downstream signaling of TGF-1 was involved as demonstrated by the decreased level of RhoA. Interestingly, pretreatment with MG132 could restore RhoA to its basal level, most likely through UPP. Moreover, MG132 successfully sustained cytoskeletal assembly and tight junction, and could prevent the cells from EMT. Conclusions: Altogether, these data demonstrate for the first time that CaOx-M-Sup could induce EMT in renal tubular cells by TGF-1 signaling cascade via RhoA and UPP. This may be, at least in part, the underlying mechanism for renal fibrosis in kidney stone disease. 25-HYDROXYVITAMIND CAN REGULATE MINERAL METABOLISM IN A CKD MODEL OF 1HYDORXYLASE KO MICE Introduction and Aims: Supplementation with 25-hydroxyvitaminD (25D) is used in CKD patients without any knowledge of its efficacy and toxicity. Current recommendations are to supplement with 25D to achieve a certain threshold that could affect both, classical and non-classical actions of vitamin D in the body. However, their direct effect on calcium homeostasis (without conversion in calcitriol) is not fully understood. In the present work we studied the effect of 25D treatment on mineral metabolism in a model of 75% nephron mass reduction (snx) in mice lacking 1-hydroxylase (1KO). Methods: A dose response study was carried out in 10-week-old SNX 1KO mice using 25, 50 and 100 ng/g of 25(OH)2D3 to compare its efficacy activating VDR with that of a single dose of 1,25(OH)2D3(50 pg/g). Results: Sham and SNX 1KO mice receiving vehicle were both hypocalcemic. 1,25D raised blood Ca2+ levels near normal values (11,150,23mg/dl). Serum Ca2+ normalization was achieved with 25 ng/g (9,430,39mg/dl) or 50 ng/g (10,630,26mg/ dl) of 25D. The highest dose provoked marked hipercalcemia (12.460.71 mg/dl). Serum phosphate (P) levels were low in untreated sham 1KO (ko sham: 5,750,56mg/ dl) and, surprisingly were not modified by nephrectomy. Treatment with 1,25D and the two lower doses of 25D significantly raised serum P to normal levels. As expected, KO mice presented severe secondary hyperparathyroidism (PTH >3000pg/ml). PTH suppression to normal levels (100-200 pg/ml) was achieved with 1,25D (297,65 97,05 pg/ml, n=7) and the 50 ng/g dose of 25D (317,17 100,74 pg/ml, n= 7). Serum 25D increased in a dose dependent manner with 25D administration above the current recommendations to avoid vitamin D toxicity (25ng/g: 2702,21 340,02 ng/ml, 50 ng/ g: 3362,95 203,52 ng/ml and 100 ng/g: 6015,14 342,46 ng/ml). In the kidney, 1,25D effects increasing TRPV5 mRNA expression were similar to those achieved with 50 ng/ g and 100 ng/g of 25D. Calbindin-D28k mRNA and protein expression was up-regulated by 1,25D and also by any dose of 25D. In duodenum, 1,25D produced a 4-5 fold increase in TRPV6 mRNA levels and 25D increased TRPV6 levels between 1.5 and 2 folds. Calbindin-D9k mRNA and protein levels were significant up-regulated with the two highest doses of 25D with a similar potency to that of 1,25D. Conclusions: These results show that 25D administration can normalize serum Ca, P and PTH, with a potency similar to that of 1,25D without being activated by 1. However, the concentrations of 25D required in blood are extremely high, and may cause toxicity. THE LONG ACTING CALCIMIMETIC R-641 DOES NOT INDUCE ADYNAMIC BONE DISEASE IN CHRONIC KIDNEY DISEASE Introduction and Aims: Secondary hyperparathyroidism is a frequent complication of CKD and contributes to the development of renal osteodystrophy. Calcimimetics lower parathyroid hormone (PTH) secretion by activating the calcium sensing receptor in the parathyroid gland. Constant suppression of PTH secretion, however, may lead to low bone turnover adynamic bone disease. Here we investigated whether repeated administration of a long acting calcimimetic leads to adynamic bone disease in a rat CKD model. Methods: In a dose finding experiment we tested 5, 10 and 20 mg/kg of the long acting calcimimetic R-641 as a single dose in rats. CKD was induced in rats by adenine diet for 4 weeks. After 3 weeks, 10 mg/kg R-641 or vehicle treatment was started by injecting every third day for further 4 weeks. Bones were labelled with calcein (10 mg/kg) by injection at 10d and 3d prior to sacrifice. Femoral bones were subjected to histomorphometry and blood samples were collected weekly. Vascular calcification was assessed by calcium content determination from thoracic aortas. PTH-(1-84) was measured by ELISA. Results: The dose finding experiment revealed that a single dose of R-641 lowered serum calcium for over 48 hours dose dependently while suppressing PTH levels for 12 hours. Compared to healthy controls, adenine treatment increased osteoid area, osteoblast and osteoclast numbers. Adenine reduced bone area compared to controls. Treatment with R-641 for 4 weeks reduced osteoblast numbers and significantly decreased osteoid area without reaching criteria of adynamic bone disease.R-641 reduced vascular calcium content significantly (9 vs. 65 g/g). Serum PTH was significantly lower in R-641 treatment groups compared to CKD rats (939 vs. 3212 pg/ml). Conclusions: After 4 weeks treatment, a long acting calcimimetic reduced vascular calcification, improved secondary hyperparathyreoidism but did not lead to adynamic bone disease. Bone formation rate Osteoid area/bone area Bone area/trabecular area VITAMIN D DEFICIENCY IS ASSOCIATED WITH DOWNREGULATION OF KLOTHO AND DEVELOPMENT OF FIBROSIS IN A MURINE MODEL OF RENAL ISCHEMIA/ REPERFUSION INJURY Janana G. Gonalves1, Daniele Canale1, Ana Carolina de Bragana1, Maria Heloisa M. Shimizu1, Rosa Maria A. Moyses1, Lucia Andrade1, Antonio C. Seguro1 and Rildo A. Volpini1 1Nephrology School of Medicine - University of Sao Paulo So Paulo Brazil Introduction and Aims: Vitamin D deficiency (VDD) is highly prevalent in chronic kidney disease (CKD) patients. Ischemia/reperfusion-Acute Kidney Injury (IR-AKI) is considered a risk factor for CKD progression. Previous studies suggests that activation of the renin-angiotensin-aldosterone system (RAAS) and VDD could be involved in reduction of Klotho expression, an early marker of stage 1 CKD. The aim of this study was to investigate the effect of VDD on klotho expression and fibrosis development in a model of IR-AKI. Methods: Male Wistar rats (180-200g) were randomly divided into four groups (n=8 each): control (C) and ischemic (IR) fed a standard diet; VDD and VDD+IR, fed a free-vitamin D diet. On day 28, IR and VDD+IR rats were submitted to 45-minute clamping of both renal arteries. On day 90, we measured inulin clearance (GFR); Mean arterial blood pressure (MAP), renal blood flow (RBF) and calculated renal vascular resistance (RVR). We also measured serum levels of 25-hydroxyvitamin D (25(OH)D, ng/mL) and proteinuria. In renal tissue, we immunoblotted for klotho and performed imunohistochemical assay for collagen IV and fibronectin. We estimated fibrosis by fractional interstitial area (FIA) and fibronectin/collagen IV expression by score ranging from 0 to 4 according to the extension of staining. Data are expressed as mean SEM. Table 1. Hemodynamic and biochemical data and fractional interstitial area, Western Blot and Immunohistochemical studies. Results: Vitamin D levels were similar in C (15.41) and IR (150.6) groups and <1.5 ng/ mL in VDD groups. GFR, RBF and RVR were not different among the studied groups. MAP was markedly elevated in VDD, IR and VDD+IR groups, reflecting a possible interaction on RAAS. Also, VDD, IR and VDD+IR groups showed larger areas of fibrosis and higher scores for fibronectin and collagen IV and decreased levels of klotho. Conclusions: The increased proteinuria/fibrosis and fibronectin/collagen IV expression in VDD+IR rats suggests progressive renal injury. Downregulation of klotho expression is a possible marker of chronification. Our study shows a very plausible role of VDD in this pathway as a potential stimulus for fibrosis. (FAPESP/CNPq). ap<0.05 vs C; bp<0.05 vs VDD; cp<0.05 vs IR. BLOCKADE OF THE HOMEOSTATIC CHEMOKINE STROMAL DERIVED FACTOR-1 / CXCL-12 PREVENTS GLOMERULOSCLEROSIS DURING KIDNEY DISEASE Simone Romoli1, Adriana Migliorini1 and Hans-Joachim Anders1 1Nephrologisches Zentrum Klinikum der Universitaet Muenchen Medizinische Klinik und Poliklinik IV Mnchen Bavaria Germany Introduction and Aims: Stromal Derived Factor (SDF)-1 (also known as CXCl-12) is a homeostatic chemokine, which promotes the homing of hematopoietic stem cells (HBSC) to the bone marrow and of innate immune cells to sites of tissue injury. Similarly, SDF-1 regulates the survival and migration of CD133/CD24+ human renal progenitor cells (RPCs) to the site of injury during glomerular repair.Therefore, here we studied the role of SDF-1 in acute and chronic glomerular disorders such as anti-GBM nephritis and Alport nephropathy. Methods: C57BL/6 male mice were procured from Jackson Laboratories (Bar Habour, MA). Collagen 4A3-deficient mice with Alport nephropathy (Alport mice) were bred in house. All experimental procedures were approved by the local government authorities. Anti-GBM nephritis was induced by single intravenous injection of anti-GBM serum in C57BL/6 mice at 6-7 week of age. Control mice received inactive NOX-A 12 and treatment mice received active NOX-A 12, a SDF-1 inhibitor (20mg/kg s.c.), (Noxxon, Germany), thrice a week. Alport mice received the treatment starting from week 4 of their age. Murine immortalized renal progenitor cells (mRPCs) and human RPCs (RPCs) were used for in-vitro cell culture studies. Histology, immunohistochemistry, ELISA, qRTPCR was used for further analysis. Results: Anti-GBM sera induced proteinuria in a dose dependent manner in C57BL/6 mice. Treatment with active NOX-A 12 significantly reduced the proteinuria levels at early phase (day 2) after injection of 35l and 50l anti-GBM sera in C57BL/6 mice compared to inactive NOX-A 12. However, the extent of proteinuria remains unaffected at later phase of the disease, day 28 in C57BL/6. Similarly, treatment of NOX-A 12 had no effect on survival of Alport mice compared to inactive NOX-A 12 group. Histology analysis of the glomerular lesions in the kidney at the end of the anti-GBM sera study revealed no significant changes in control and treatment groups. Similarly, no significant difference in the number of podocytes was observed. In-vitro, recombinant SDF-1 markedly decreased the proliferation capability of mRPCs as well as RPCs in MTT assay. Moreover, recombinant SDF-1 inhibited the differentiation of mRPCs and RPCs into podocytes in the presence of podocyte differentiation medium, as observed by lower mRNA expression of nephrin, CD2AP and WT-1 in the RNA extracted from the differentiated cells with or without recombinant SDF-1. Conclusions: Taken together, SDF-1 suppresses the differentiation of RPCs into podocytes and its blockade in kidney disease protects the kidney from damage in early phase but has no effects on the later phases of Ig anti-GBM. Counter, block it in Alport's disease isnt effective on survival ratio.Thus, SDF-1 blockade is beneficial in early phase but not in the later phase of the glomerular diseases. The reasons for these differential roles of SDF-1 and its mechanism are still unclear and warrants further studies. URINARY EXCRETION OF PODOCYTE'S DAMAGE AND SELF-DEFENSE FACTORS IN PATIENTS WITH CHRONIC GLOMERULONEPHRITIS (CGN) Olga Eskova1, Natalia Neprintseva1, Natalia Tchebotareva1, Irina Bobkova1 and Lidiya Kozlovskaya1 1I.M. Sechenov First Moscow State Medical University Moscow Russian Federation Introduction and Aims: The imbalance between local injury factors and defense mechanisms determines the tissue damage in CGN. The most interest of recent studies was focused on the effector link of renal inflammation, but self-defense mechanisms of kidney cells, including podocytes (Pdc), are poorly studied. The aim of the study was to evaluate in patients ( pts) with different CGN courses the podocyturia (PdcU) level (as a marker of Pdc's injury), urinary excretion of proinflammatory interleukin-6 (IL-6) and heat shock protein-27 (HSP-27) (as a protective intracellular protein, which helps to keep structure of Pdc's actin cytoskeleton). Methods: 68 CGN pts were studied: 20 inactive CGN (I group), 23 active CGN with proteinuria (PU) 1-3 g/d (II group), 25 CGN with nephrotic syndrome (NS) (III group), including 11 pts with moderate NS (PU-3-5 g/d ), 7 pts with severe NS (anasarca, PU 6-12 g/d, hypoalbuminemia < 20 g/L), and 7 pts - with NS in combination with nephritic syndromeand impaired renal function. 8 healthy subjects were studied as control. PdcU was estimated by urinary flow cytometry, urinary IL-6 and HSP-27 levels were measured by ELISA. Results: The PdcU, urinary IL-6 and HSP-27 levels in active CGN were higher than in control. These indices in III pts group were significantly higher than in II and I groups (PdcU (cells/ml): I-6,2 [3,7;6,8], II-6,8 [3,2;17,4], III-16 [8,0;38,4] p<0,05. IL-6 (ng/ml): I-5,35 [0,41; 13,1], II-5,44 [1,3; 12,8], III- 9,97 [1,95;25,6] p<0,05. HSP-27 (ng/ml): I-0,72 [0,65;0,98], II-0,76 [0,68;1,14], III-1,1[0,73; 1,83] p<0,05). In active CGN urinary HSP-27 excretion correlated directly with PU (Rs=0,27, p<0,05) and negatively with serum albumin level (Rs=-0,22, p<0,05). In CGN with severe NS PdcU and urinary HSP-27 levels were higher than in CGN with moderate NS (168 [19; 782] cells/ml vs 7,87 [4,2; 30] cells/ml and 2,28 [0,93; 4,02] ng/ml vs 1,07 [0,71; 1,72] ng/ml respectively, p<0,01). Urinary IL-6 excretion in progressive CGN course was significantly raised compared to pts with NS and normal renal function (22 [10,2; 92,5] ng/mL vs 8,27 [1,48; 16,35] ng/ml, p<0,05). Exactly in these pts urinary IL-6 correlated positively with serum creatinine level (Rs=0,53, p < 0,01) and a negatively - with GFR (Rs=-0,43; p < 0,05). We consider that revealed direct correlation between PdcU and urinary IL-6 (Rs=0,39, p<0,05) reflects Pdc's injury during a local immune inflammation. At the same time, correlation between the PdcU and urinary HSP-27 (Rs=0,35, p<0,05) in NS indicates the reciprocal activation of mechanisms, which limit Pdc's structural damage and glomerular permeability. Conclusions: Active Pdc damage in proteinuric forms of CGN and activation of intracellular protection in response to inflammation and Pdc injury can be proposed according to our data. Insufficiency of some kidney self-defense mechanisms can be considered as a risk factor for CGN progression. NEPHROPROTECTIVE EFFECT OF RAS-ANTAGONISTS IN MICE CARRYING R140Q PODOCIN MUTATION Ivana Simic1, Mansoureh Tabatabaeifar1, Tanja Wlodkowski1, Helga Denc1, Geraldine Mollet2, Corinne Antignac2 and Franz Schaefer1 1Pediatric Nephrology Center for Pediatrics and Adolescent Medicine, University of Heidelberg Heidelberg Germany, 2Inserm U983 and Dept. of Genetics Hopital Necker Paris France Introduction and Aims: Mutations in the NPHS2 gene, encoding the podocyte protein podocin, are the most common cause of hereditary nephrotic syndrome. Recently, we generated an inducible knock-in mouse carrying the R140Q podocin mutation, the murine analogue of the most common human mutation R138Q. R140Q-podocin hemizygous mice develop nephrotic-range proteinuria, i | Abstracts focal-segmental glomerulosclerosis und progressive renal failure. The aim of this study was to test the antiproteinuric und nephroprotective efficacy of RAS antagonists in this mouse model. Methods: C57BL/6 mice with Nphs2Flox/R140Q, Cre+genotype were injected with Tamoxifen or vehicle for 5 days to induce hemizygosity for R140Q-mutant podocin. The animals (8 per group) were treated prophylactically with the ACE inhibitor ramipril (R), the AT1 receptor blocker candesartan (C), the combination of ramipril and candesartan (R+C), the non-RAS antihypertensive amlodipine (A) or remained untreated with either tamoxifen induction (sick controls) or vehicle injections (healthy controls). Weight, blood pressure and proteinuria were monitored once weekly. Biochemical and histopathological changes were examined after 4 weeks. Results: Blood pressure was elevated in sick animals and more markedly reduced by RAS antagonists than by amlodipine (mean arterial pressure: healthy controls, 85; sick controls, 96; R, 69; C: 66; R+C, 50; A, 81 mm Hg). Proteinuria was markedly attenuated in animals treated with RAS antagonists (by 76% in C, 77% in R and 78% in R+C relative to sick controls) but not in those receiving amlodipine. After 4 weeks, mice receiving R+C were normo-albuminemic (R, 27; C, 30; R+C, 31; A, 24 g/l; sick controls, 19; healthy controls, 32 g/l) and serum creatinine was increased less than in untreated animals (R, 0.12; C, 0.12; R+C, 0.14; A, 0.13; sick control, 0.16; healthy control, 0.11 mg/dl). Histopathologically, animals treated with RAS antagonists scored lower glomerular sclerosis indices (R+C, 0.91; sick controls, 1.47; healthy controls, 0.25). The average number of podocytes per glomerulus was reduced by 50% in sick animals but was preserved in R+C animals (R+C, 73; A, 62; sick controls, 39; healthy controls, 75). Whereas podocin mRNA expression was preserved or even increased in all diseased animals, Western blot analysis showed a subtotal loss of podocin protein in all induced animals irrespective of pharmacological treatment. Conclusions: In mice carrying the most common human podocin mutation, administration of RAS antagonists markedly attenuates proteinuria and podocyte loss and delays glomerulosclerosis despite persistently increased intracellular degradation of mutant podocin protein. Our findings suggest that RAS blockade provides effective pharmacological nephroprotection in this hereditary podocytopathy. PODOCYTE DYSFUNCTION IN CYSTINOSIS IS ASSOCIATED WITH INCREASED PODOCYTE MOTILITY AND PODOCYTURIA Ivanova A. Ekaterina1, Laura Giardino2, Maria Pia Rastaldi2, Lambertus Van den Heuvel1 and Elena Levtchenko1 1Pediatrics KU Leuven Leuven Belgium, 2Renal Research Laboratory Fondazione IRCCS Policlinico & Fondazione D'Amico Milano Italy Introduction and Aims: Cystinosis is an autosomal recessive disorder caused by mutations in the CTNS gene that encodes a lysosomal cystine transporter and results in high cystine levels in the patients' lysosomes. A major consequence of cystinosis is the development of severe renal disease, characterized by general proximal tubular dysfunction (renal Fanconi syndrome) progressing towards end-stage renal failure. Despite the general belief that renal proximal tubular cells are the primary targets in cystinosis, several lines of evidence, such as the presence of glomerular proteinuria and morphologic podocyte changes, point to early podocyte dysfunction in this disorder. Methods: We analyzed urine samples from healthy donors (n=9) and cystinosis patients (n=14). Conditionally immortalized podocyte cell lines were generated from freshly voided urine of healthy volunteers and cystinosis patients and characterized for expression of specific markers (CD2AP, synaptopodin, nephrin, podocin) by qPCR. Results: We demonstrated a significant level of podocyturia in cystinosis using qPCR test to quantify the amount of specific podocyte mRNA sequences in urine (mean expression values calculated as Ct normalized by creatinine for each sample, p<0.05 for CD2AP and synaptopodin). Also, urine samples from cystinosis patients provided more viable podocytes suitable for further culturing in comparison with healthy donors. Cystinosis podocyte cell lines accumulated cystine (mean cystine content was 15.6 nm/mg protein vs 0.47 in controls). Using viable podocytes extracted from urine of both healthy donors and cystinosis patients, we established several conditionally immortalized podocyte cell lines and characterized them by the expression of typical podocyte markers (CD2AP, synaptopodin, nephrin and others). Cystinosis podocytes demonstrated increased motility in wound-healing assay as compared to the control. Also, the actin cytoskeleton, as shown by phalloidin and alpha-actinin staining was altered in cystinosis cells. Conclusions: Together our data demonstrate important changes in podocyte function in cystinosis that can underlie glomerular proteinuria associated with this disease. Future studies will enter mechanisms of podocyte motility, cytoskeletal assembly, and endocytosis to find molecular therapeutic targets and improve cystinosis phenotype. RABBIT POLYCLONAL ANTIBODY INDUCED BY GENETIC IMMUNIZATION WITH MOUSE NEPHRIN cDNA CAUSED FOCAL SEGMENTAL GLOMERULOSCLEROSIS (FSGS) WITH NEPHROTIC SYNDROME IN MICE Chikako Okina1, Tomoko Okamoto1, Mariko Kamata1, Junya Murano1, Kei Kobayashi1, Kazuhiro Takeuchi1, Fumi Kamata1, Takeshi Sakai1, Shokichi Naito1, Togo Aoyama1, Takashi Sano1, Yasuo Takeuchi1 and Kouju Kamata1 1Nephrology in Internal Medicine Kitasato University School of Medicine Sagamihara Kanagawa Japan Introduction and Aims: Glomerular lesion induced by in vivo administration of specific antibody against mouse nephrin protein are investigated. Methods: Thirty microgram of expression vector containing nephrin cDNA of full length, immunoglobulin (Ig) motifs 1-8 or fibronectin motif are administered 4 times every 2 weeks into 9 New Zealand White rabbits, respectively. Control vector is also administered into 3 rabbits in the same manner. Purified rabbit IgG obtained at 8 wks are administered once into C57BL/6N mice, intravenously. Urinary protein excretion and pathological findings of mouse kidney are evaluated. Results: All 20 mice with 1-4mg IgG produced by full-length nephrin cDNA showed massive proteinuria from day 1 with a dose-dependent manner. All 6 mice with 4mg IgG and 6 out of 7 mice with 2mg IgG produced by full-length cDNA showed a glomerular lesion of FSGS. Remaining 8 mice with 1-2 mg IgG produced by full-length cDNA had a minor glomerular abnormality. None of 12 mice with 4mg IgG produced by Ig motifs 1-8 and fibronectin motif cDNA showed a significant proteinuria. Conclusions: Polyclonal specific antibody against mouse nephrin protein induces glomerular FSGS lesion and nephritic syndrome in mice. LACK OF MURINE DOUBLE MINUTE (MDM)-2 IN PODOCYTES CAUSES AUTOPHAGIC CELL DEATH AND FOCAL SEGMENTAL GLOMERULOSCLEROSIS Introduction and Aims: Podocytes are terminal differentiated epithelial cells of the glomerular filtration barrier that can hardly be replaced upon loss. Like neurons, they seem to survive many years or even decades and it remains a miracle, how they manage to stand the many hemodynamic, toxic, and immunologic insults that occur during lifetime. The E3-ubiquitin ligase murine double minute (MDM)-2 is a non-redundant element of NF-kB signaling and the master negative regulator of tumor suppressor gene p53-mediated cell cycle arrest. We have recently shown that MDM2 blockade can prevent adriamycin-induced podocyte loss, proteinuria, and glomerulosclerosis. In these studies, MDM2 blockade restored p53 and prevented podocyte mitotic catastrophe, because podocyte mitosis leads to podocyte detachment and death. The role of the intense MDM2 expression in non-stressed podocytes, however, is unknown today. We hypothesized, that MDM2 would be required to prevent p53 overactivation, a state that may cause premature senescence or even podocyte loss. Methods: We generated and phenotypically characterized podocyte-specific MDM2-knockout mice (Podocin-Cre/MDM2flox). Results: Comparison of podo-MDM2 -/- mice with their heterozygous +/- littermates revealed lack of MDM2 in podocytes upon immunostaining of renal sections and by glomerular isolate qPCR. Nephrin/WT-1+ podocyte counts were identical in 3 weeks old mice of both groups. The podocyte/glomerulus ratio increased with aging in the heterozygous control mice while it declined in podo-MDM2 KO mice by week 14 of age. Relative podocytopenia in podo-MDM2 KO mice resulted in proteinuria and progressive focal segmental glomerulosclerosis. Electron microscopy of podo-MDM2-/- renal tissue displayed the typical abnormalities of glomerulosclerosis but also specific ultrastructural changes in the podocytes, such as massive vacuolization and ER abnormalities suggesting involvement of excessive autophagy and autophagic podocyte death. Conclusions: In contrast to the pathogenic (mitogenic) role of MDM2 in podocyte injury, podocytes need MDM2 during homeostasis to avoid excessive autophagy. MDM2 seems to be an essential regulator of podocyte injury and homeostasis. UP-REGULATION OF TRPC6 IN PODOCYTES ASSOCIATED WITH THE DEVELOPMENT OF GLOMERULAR HYPERFILTRATION IN RATS WITH 5/6 NEPHRECTOMY Introduction and Aims: Recent works reveal that TRPC6 functions as one of key factors for the regulation of podocyte signaling, especially for the receptor of pressure load. The aim of this work was to assess the changes in the expression of TRPC6 in podocyte in accordance to the development of glomerular hyperfiltration (GHF) in 5/6 nephrectomized rats. Methods: Each one-third of upper and lower pole of right kidney was surgically removed, and left kidney was totally removed one week after. Ras were sacrificed 6 weeks after removal of left kidney to sample the remaining kidney for histological, immunohistochemical and molecular biological analysis. Isolated glomeruli were used for gene expression analysis by RT-PCR. Results: Estimated glomerular volume, urine albumin-to-Cr ratio (ACR) and expression score of desmin were all significantly increased in 5/6 nephrectomized (N) rats comparing to sham operated (S) rats, indicating glomerular hypertrophy and development of podocyte injury associated with GHF. Electron microscopic observation showed deformity of podocytes including effacement of foot process in N. Development of glomerular sclerosis in N was shown by FGS score (0.0 in S, 26.0 in N). Similarly, gene expression of TRPC6 was significantly increased in N (100.55.8 in S, 156.215.7% in N). Double immunostaining with TRPC6 and WT-1 antibodies confirmed co-localization of both antigens, and indicated an increase in the number of TRPC6 positive podocyte in N. Additionally, double immunostaining with TRPC6 and synaptopodin also showed an increase in TRPC6 expression above the capillary wall in N associated with blunted immunoreactivity and disruption of synaptopodin staining along the capillary. Changes in gene expression of TRPC6 in podocytes were also studied by use of in situ hybridization. Conclusions: In the present study, involvement of TRPC6 in the development of glomerular sclerosis resulted from GHF was confirmed by in vivo disease model. Effects of TRPC6 signaling-blockade is now under studying. MODEL FOR IDIOPATHIC FSGS: PROTEINURIA AFTER INJECTION OR OVEREXPRESSION OF CARDIOTROPHIN-LIKE CYTOKINE 1 IN MICE Virginia J. Savin1, Mukut Sharma1, Changli Wei3, Jochen Reiser3, Ellen T. McCarthy2, Ram Sharma1 and Jean-Francois Gauchat4 1Nephrology Kansas City VA Medical Center Kansas City MO United States, 2Kidney Institute University of Kansas Medical Center Kansas City KS United States, 3Medicine Rush University Medical Center Chicago IL United States, 4Pharmacology University of Montreal Montreal QC Canada Introduction and Aims: Idiopathic focal segmental glomerulosclerosis (FSGS) is associated with recurrence after transplantation due to a circulating permeability factor or factors (N Engl J Med, 334:878-883, 1996). We have shown the effects of FSGS plasma and its fractions on glomerular permeability in vitro and in vivo and have used state-of-the-art proteomics to identify cardiotrophin-like cytokine-1 (CLC-1 or CLCF1), a member of the IL-6 family, as a candidate for the active substance. CLC-1, like FSGS patient plasma, increases albumin permeability (Palb) of isolated glomeruli and appears to act via its known receptor complex and activation of the JAK/STAT signaling pathway. Monoclonal antibody to CLC-1 markedly diminished the effect of active FSGS plasma on Palb. We hypothesize that a new model of FSGS can be based on the effects of CLC-1 in mice. We used acute injection, prolonged infusion or expression by electroporation to determine the potential utility in creating a murine model of human recurrent FSGS. Methods: All studies were done in C57BL6 mice. rCLC-1 (R&D Systems) was injected intraperitoneally (IP), one dose, 10 g/kg, or infused by minipump for 28 days, 40 g /kg/day. A construct containing CLC-1 was administered by electroporation in the hind limb of mice. Measurements included urinary albumin/creatinine, pJAK2 and pSTAT3 in peripheral blood cells (PBC) and in kidney homogenate. Glomerular histology was assessed. Results: Albuminuria was induced promptly by rCLC-1 after either injection or electroporation. Peak albuminuria occurred by 7 days of expression and was 3-5 fold increased vs. baseline. IP administration of rCLC-1increased pJAK2 and pSTAT3 of PBC within 15 min. and renal pJAK1 and pSTAT3 remained upregulated for at least 72 hours after injection. Albuminuria and kidney pJAK2 and pSTAT3 were markedly increased after 28 days of infusion. At that time, mesangial matrix was increased in a focal pattern. Conclusions: We conclude that CLC-1 mimics many of the renal effects of the active fraction of plasma from patients with idiopathic FSGS and may play an important role in its etiology. These effects include induction of albuminuria and expansion of mesangial matrix. The relationship between effects of CLC-1 and those of other candidates such as circulating urokinase receptor (suPAR) (Nat Med 17:952-60, 2011) is not known. A murine model based on administration or overexpression of CLC-1 may permit us to define mechanisms of injury and to test potential therapeutic agents prior to use in clinical trials of FSGS. URINARY LOSS OF PACAP AS A CAUSE OF INCREASED RISK OF THROMBO-EMBOLIC DISEASE IN CONGENITAL NEPHROTIC SYNDROME Introduction and Aims: Thrombotic complications occurring in up to 15% of patients represent a severe burden in nephrotic syndrome (NS). The underlying mechanisms are mainly unraveled in regard of venous thrombosis, while elevated blood platelet count and hyperaggregability increase the risk of arterial thrombosis. A role of the neuropeptide PACAP ( pituitary adenylate cyclase-activating polypeptide) as an inhibitor of megakaryocyte maturation and platelet function has recently been established. PACAP interferes with the regulation of apoptosis in megakaryocytes, via stimulation of NFB signaling. We assumed that urinary losses of PACAP bound to ceruloplasmin in NS might lead to PACAP deficiency, leading to thrombocytosis and increased platelet reactivity. The aim of this study was to investigate plasma PACAP levels in relation to blood platelet counts and aggregability in patients with congenital NS (CNS). Methods: Four patients with CNS of the Finnish type, aged 0.5-19 months were tested. Plasma and urinary levels of PACAP were measured semi-quantitatively by western blot. In two patients, platelet aggregation tests were performed by a platelet aggregometer before and after bilateral nephrectomy. Hematopoietic stemcells were isolated from the same two patients and differentiated into colony forming unit (CFU) megakaryocytes. Results: All patients had plasma PACAP deficiency (14-40% of control, p<0.001) and excessive urinary PACAP excretion. In three patients both kidneys were removed as a routine treatment of CNS. PACAP plasma levels progressively increased during the first days after nephrectomy and blood platelet counts normalized. In one patient, platelet aggregation tests were performed. In analogy to PACAP deficient mice, an increased platelet aggregation response to collagen was found in nephrotic state, while platelets isolated after bilateral nephrectomy showed normal reactivity towards collagen. In two patients hematopoietic stem cells were isolated before and after bilateral nephrectomy and differentiated in vitro into CFU megakaryocytes. During nephrotic state there was, in both patients, a significant decrease of CFU megakaryocytes after addition of recombinant PACAP in comparison with the condition without recombinant PACAP. After bilateral nephrectomy, there was a significant decrease of CFU megakaryocytes in comparison with the condition before bilateral nephrectomy and there was no difference between conditions with and without addition of recombinant PACAP. Conclusions: Urinary PACAP losses leading to blood PACAP deficiency explain increased platelet count and aggregability via stimulating megakaryopoesis. This mechanism is likely to underlie arterial thrombosis in CNS. MITOCHONDRIAL DYSFUNCTION IN PODOCYTE REDUCES ALPHA ACTININ-4 AND SYNAPTOPODIN IN PODOCYTE, WHICH INDUCES PROTEINURIA Introduction and Aims: Our previous report showed that Crif1deletion induce severe mitochondrial destruction in mice podocyte. And it resulted in massive albuminuria and effacement of foot process in mice. Actin cytoskeleton architecture and dynamics in podocyte are important constituents of the glomerular filtration barrier. There have been few studies about the relation of mitochondria and actin cytoskeleton in podocytes of glomerulus. We evaluated the changes of actin cytoskeletal proteins and architecture in mitochondrial injured podocyte. Methods: We used immortalized mouse podocyte cell line. Crif1 silencing(si)RNA treatment was used for inducing mitochondrial injury. We divided podocytes into 3 groups; control podocytes, scrumble(sc) RNA treated podocytes, Crif1 siRNA treated podocytes. We checked the expression of mitochondrial respiratory complex IV, WT-1, and Crif1 for mitochondrial dysfunction in podocytes. We evaluated the expression of alpha actinin 4, synaptopodin, nephrin, ZO-1, and colfillin using western blot. Using confocal microscopy, we examined actin cytoskeleton architecture and mitochondria of podocyte. For evaluation of cell migration, we performed scratch assay. Results: Crif1 siRNA treatment reduced the expressions of mitochondrial respiratory complexIV and O2 consumption in cultured podocytes. Alpha actinin-4 and synaptopodin were significantly decreased in Crif1 siRNA treated podocyte compared to control and Crif1 scRNA treated podocytes. There were no differences in nephrin and ZO-1. Crif1 siRNA treated podocyte showed an enhanced formation of dot-like alpha actinin-4 and an increase of fragment mitochondria in confocal microscopy compared to scRNA treated podocytes. Podocyte migration was increased in Crif1 siRNA treated podocytes. Conclusions: With the above results, it is speculated that mitochondrial dysfunction induced by crif1 inhibition reduces alpha actinin-4 and synaptopodin in podocyte. THE SPHINGOSINE-1-PHOSPHATE RECEPTOR AGONIST FTY720 IMPROVED EXPRESSION OF ENOS IN KIDNEYS FROM SUBTOTALLY NEPHRECTOMIZED RATS Introduction and Aims: It has become evident that decreased bioavailability of endothelial nitric oxide (NO) produced from endothelial NO synthase (eNOS), referred to as endothelial dysfunction, plays a crucial role in further progression of kidney damage .Recently, sphingosine 1-phosphate (S1P) has been highlighted as an endothelial barrier-stabilizing mediator by the eNOS/NO pathway. FTY720 is a S1P analog originally developed as a novel immunosuppressant. The phosphorylated form of FTY720 binds to S1P receptors to exert S1P-like biological effects, suggesting i | Abstracts endothelial barrier promotion by FTY720. In the study, we investigated the effects of FTY720 on the levels of nitric oxide (NO) and the expression of eNOS in the renal tissue from subtotally nephrectomized rats. Methods: Seven days after surgery, Sprague-Dawley rats were allocated to the following groups: Sham operate surgery, subtotally nephrectomy (SNX) +vehicle, and SNX +FTY720 (0.3mg/kg body wt).Rats were killed on week 12 after surgery and blood, urine and kidneys were collected for analyses. Blood pressure was detected before killed. The renal histological changes were investigated by light and transmission electron microscopy. The expressions of eNOS were detected by immunohistochemical staining, RT-PCR and Western blot. Results: FTY720 significantly attenuated the rise in blood pressure, proteinuria, serum creatinine and urea nitrogen in SNX(P<0.01). FTY720 treatment prevented the downregulation of the level of nitric oxide(NO) in the kidney tissue from SNX. SNX Rats had greater glomerular sclerosis index and vascular lesions index than sham rats (P<0.01), FTY720 treatment ameliorated these effects (P<0.01). There was mitochondrial vacuolation in endothelial cells in glomerulus and widening of subendothelial spaces in SNX by Electron microscopy , these abnormalities were attenuated by FTY720.Immunohistochemistry showed the tissue localization of eNOS protein in the sham rat kidneys was present in the cytoplasm and and peripheral membrane of endothelial cells in glomeruli and vasculature. Semiquantitative analyses of staining intensity of eNOS in the glomeruli and vascular showed expression of eNOS was lower in SNX rats compared with the sham (P<0.01) , whereas FTY720 abrogated this change (P<0.01). FTY720 treatment improved eNOS mRNA expression measured by RT-PCR (P<0.01) and protein expression (P<0.01) by Western blot compared with the untreated SNX. Conclusions: FTY720 ameliorates endothelial injury in glomeruli and vasculature in kidneys from subtotally nephrectomized rats, which suggested a potential value of FTY720 in preventing progression of chronic kidney disease. OLTIPRAZ ATTENUATES RENAL FIBROSIS INDUCED BY UNILATERAL URETERAL OBSTRUCTION IN MICE Introduction and Aims: Renal fibrosis plays an important role in progression of chronic kidney disease. Recent study showed that nuclear factor E2-related factor 2 (Nrf2) activation increase glomerular filtration rate in diabetic patients. It is known thatoltipraz, Nrf2activator, reduces oxidative stress, inflammation and fibrosis in various animal models including liver cirrhosis. We investigated whether oltipraz attenuate renal fibrosis induced by unilateral ureteral obstruction (UUO)in mice. Methods: Male C57BL/6 mice(8 weeks old, 24-26g) were divided into 4 groups; sham operated mice, oltipraz-treated sham mice(10mg/kg, intraperitoneal injectiondaily), untreated UUO mice, and oltipraz-treated UUO mice.We measured renal expressions of HO-1andNrf2 1 week after UUO surgery. Light microscopic examination of obstructed kidneys and immunohistochemistry for TGF- and SMA wereperformed for the evaluation of renal fibrosis 1 week after UUO. Results: Renal gene and protein expressions ofNrf2 and HO-1 in oltipraz-treated UUO mice were significantly higher than those of sham operated and UUO mice ( p < 0.01, p<0.01). The levels of TGF- and-SMAmRNA expression of oltipraztreated UUO mice were significantly lower than those of untreated UUO mice( p< 0.05). Oltiprazalso decreased significantly renal protein expressions ofTGF- and-SMA in immunohistochemistry compared to untreated UUO mice. RenalF4/80 positive cells in oltipraz treated UUO mice were significantly lower than that of untreated UUO mice ( p< 0.05). Conclusions: In conclusion, the results of the present study suggest that oltipraz attenuates renal fibrosis induced by unilateral ureteral obstruction in mice. TRANSPLANTATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS AMELIORATES CRESCENTIC GLOMERULONEPHRITIS IN WISTAR-KYOTO RATS Introduction and Aims: Multipotent mesenchymal stem cells (MSC) have become a promising therapeutic approach in many clinical conditions. The hypothesis that MSCs can provide a potential therapy for human anti-glomerular basement membrane (GBM) glomerulonephritis (GN) was tested. Methods: Nephrotoxic serum nephritis was induced in Wistar-Kyoto (WKY) rats on day 0. Groups of animals were given either human MSCs (hMSCs) (3 x 106) or vehicle (HBSS) by intravenous injection on day 4; all rats were sacrificed at either day 7 or day 13. Some rats with nephritis were administered hMSCs that were fluorescently labeled using conjugated red fluorochrome CellTracker CM-DiI for identification in histopathological sections. Renal morphological investigations were performed at sacrifice. Results: At 6 h after labeled hMSC administration, hMSCs were localized in glomeruli and the tubulointerstitium. Body weight was comparable between the two treatment groups throughout the study. Serum creatinine level was significantly lower in the MSC-treated rats than in the HBSS-treated rats on day 13 ( p < 0.05). When compared to vehicle treatment, MSC-treated rats had reduced proteinuria on day 7 ( p < 0.05) and 13 ( p < 0.01). MSC treatment also decreased kidney weight (Day 7: p < 0.05; Day 13: p < 0.01) and glomerular tuft area (Day 7: p < 0.05; Day 13: p < 0.05). ED1-positive macrophages ( p < 0.05), CD8-positive cells ( p < 0.05), and apoptotic cells ( p < 0.001), assessed by TUNEL staining, in glomeruli were significantly reduced by MSC treatment on day 7. In the MSC-treated rats, there was an inhibition of IL-1b expression in glomeruli that was co-localized with glomerular macrophages. Renal cortical mRNA for TNF-a ( p < 0.0001), IL-1b ( p < 0.001), and IL-17 ( p < 0.01) was decreased, whereas IL-4 ( p < 0.05) and Foxp3 ( p < 0.01) was increased in the MSC-treated group on day 7. hMSC treatment was associated with a significant decrease in the serum IL-17 level ( p < 0.05) and a prominent increase in the serum IL-10 level ( p < 0.01) in rats with nephritis. Collagen type I ( p < 0.01), type III ( p < 0.01), and TGF-b ( p < 0.05) mRNA were significantly decreased by MSC treatment on day 13. The urinary total collagen level was significantly lower in the MSC-treated group ( p < 0.05). Conclusions: The present results demonstratedthat anti-inflammatory and immunomodulatory effects were involved in the mechanism of attenuating established experimental anti-GBM GN by hMSCs, which resulted in blunting subsequent development of chronic kidney disease.These results suggest that hMSCs are a potential new therapeutic approach in patients with anti-GBM GN. ALK1 HETEROZYGOUS DISRUPTION IMPAIRS TGF-/SMAD SIGNALING AND INCREASES RENAL FIBROSIS FOLLOWING URETERAL OBSTRUCTION Jose M. Muoz-Felix1,2,4, Jose M. Lopez-Novoa1,2,4 and Carlos Martinez-Salgado1,2,3,4 1Department of Physiology and Pharmacology University of Salamanca Salamanca Spain, 2Cardiovascular Research Biomedical Research Institute of Salamanca (IBSAL) Salamanca Spain, 3Hospital Universitario de Salamanca Instituto de Estudios de Ciencias de la Salud de Castilla y Len (IECSCYL) Salamanca Spain, 4Institute Queen Sophie for Renal Research (IRSIN) Fundacion Renal Iigo Alvarez de Toledo Madrid Spain Introduction and Aims: An excessive accumulation of extracellular matrix (ECM) in the renal interstitium, myofibroblast activation, cell infiltration, tubular apoptosis and proliferation are characteristic features of tubulointerstitial fibrosis, which is one of the common end points of chronic renal insufficiency. ALK1 (activin receptor-like kinase I) is a type I receptor for the profibrotic cytokine transforming growth factor- 1 (TGF1), with a pivotal role in endothelial proliferation and migration. ALK1 promotes Smad1/5 signaling, antagonizing ALK5/Smad2/3 signaling in endothelial cells. ALK1 potentiates ECM protein synthesis in scleroderma fibroblasts. Nevertheless, the role of ALK1 in obstructive nephropathy is unknown. The purpose of this study is to assess the role of ALK1 activation on obstructive nephropathy. Methods: We performed unilateral ureteral obstruction (UUO), an obstructive nephropathy experimental model, in haploinsufficient (ALK1+/-) and control (ALK1+/+) mice in order to analyze the role of ALK1 haploinsufficiency 15 days following UUO. Kidney ultrastructure was analyzed by hematoxylin-eosin staining. We analyzed tubulointerstitial fibrosis by Masson's trichrome and Red Sirius staining. Phospho-Smad1, phospho-Smad2 and phospho-Smad3 expression were evaluated by western blot and immunostaining. Results: Phospho-Smad1, phospho-Smad2 and phospho-Smad3 expressions were increased following UUO in both ALK1+/+ and ALK1+/- obstructed kidneys. Phospho-Smad1 expression was lower, while phospho-Smad3 was higher in obstructed kidneys from ALK1+/- mice than in wild type mice. No differences in Smad2 phosphorylation were found between obstructed kidneys from ALK1+/+ and ALK1+/mice. Tubulointerstitial fibrosis was higher in obstructed kidneys from ALK1+/- mice than in wild type mice. Conclusions: ALK1 heterozygous disruption modifies TGF-/Smad signaling leading to increased renal fibrosis after 15 days of ureteral obstruction. Thus, activation of the ALK1 signalling pathway seems to be protective against renal fibrosis. ANATOMICAL AND FUNCTIONAL IMAGING OF RENAL VASCULATURE IN EXPERIMENTAL RENAL FIBROSIS Josef Ehling1,2, Janka Bb ckov1,3,4, Felix Gremse2, Fabian Kiessling2, Jrgen Floege3, Twan Lammers2 and Peter Boor1,3 1Institute of Pathology RWTH University Aachen Germany, 2Department of Experimental Molecular Imaging, Helmholtz Institute for Biomedical Engineering RWTH University Aachen Germany, 3Division of Nephrology RWTH University Aachen Germany, 4Institute of Molecular Biomedicine Comenius University Bratislava Slovakia Introduction and Aims: Renal fibrosis is associated with rarefaction of the renal vasculature and the latter might contribute to fibrosis progression. To date, mostly histological methods have been used to analyze the state of renal vasculature in fibrosis. The aim of this study was to evaluate functional in vivo tests of the renal microvasculature, such as non-invasive, longitudinal blood volume determination, in murine models of renal fibrosis. Methods: We sequentially analyzed two models of renal fibrosis: murine unilateral ureteral obstruction (UUO, days 1, 3, 5, 7 and 10) and Alport mice (6 and 8 weeks old). We assessed renal blood volume (rBV) using in vivo contrast-enhanced micro computed tomography (CT); visualized vasculature using ex vivo high-resolution CT (spatial resolution 4 m) after in vivo perfusion of the renal vasculature with a cast agent; and analyzed vascular permeability using extravasation of Evans blue. Results: Kidney volume assessed by CT showed expansion of obstructed UUO kidneys due to hydronephrosis (starting at day 1 and reaching maximum on day 3) and compensatory hypertrophy of the contralateral kidneys (obvious at day 7). The rBV slightly increased in contralateral kidneys on day 5 (+12%, p=0.14, n=4 per group) and remained stable thereafter, whereas rBV in obstructed kidneys was significantly decreased on day 3 (-33%, p<0.01) and further decreased until day 10 (-66%, p<0.01) in comparison to contralateral kidneys. In Alport mice, a slightdecrease in kidney volume could be observed from week 6 to week 8. Compared to wild type littermates, Alport mice demonstrated a significantly reduced rBV at the age of 6 and 8 weeks (-41% and -53%, respectively, both p<0.01, n=5 per group). Results were validated using immunohistochemistry (CD31, VEGFR2, Meca-32). 2D slices and 3D volume renderings of high-resolution ex vivo CT scans demonstrated a significant reduction of mainly small renal blood vessels in both models of renal fibrosis (UUO at day 10 and Alport at week 8) in comparison to healthy kidneys. Compared to contralateral kidneys and wild type littermates, the extravasation of Evans blue was higher in obstructed UUO kidneys (+112%, p<0.01) and with high variability also in Alport mice (+143%, p=0.17). Conclusions: We established methods for the in vivo assessment of the renal vasculature in mice. Renal fibrosis in both models was characterized by a progressive reduction of the overall number and functionality of renal blood vessels. These data not only lay the basis for better understanding of renal fibrosis progression but also for intervention studies targeted at maintaining the number and function of renal microvessels. IRAK-M IN RENAL FIBROSIS UPON UNILATERAL URETERAL OBSTRUCTION Introduction and Aims: Innate immune activation via IL-1R and Toll-like receptors contributes to acute kidney injury but data on their role in chronic kidney disease (CKD) are conflicting. Activation of various innate immunity receptors results in IRAK-1/IRAK-4-mediated signaling and secretion of pro-inflammatory cytokines such as IL-12, IL-6 or TNF-, all of which are implicated in renal injury and elevated in CKD. Interleukin-1 receptor-associated kinase-M (IRAK-M) is a member of the IRAK family that lacks kinase activity. Therefore, IRAK-M a negative regulator of IL-1R/TLR signalling and, hence, blocks macrophage polarization towards the proinflammatory M1 phenotype and rather fosters regulatory macrophages differentiation. As renal fibrosis in CKD either involves predominantely M1 or M2 macrophages depending on the predominating tissue microenvironment, we speculated that IRAK-M would have a regulatory role on tubulointerstitial fibrosis and CKD progression, e.g. upon unilateral ureteral obstruction (UUO). Methods: UUO was performed in 6-8 week old, sex- and age-matched wildtype and IRAK-M-deficient mice. Contralateral kidneys served as intraindividual control. Results: The expression of IRAK-M increased within two days after UUO in obstructed versus unobstructed kidneys. Compared to WT mice, IRAK-M -/- mice displayed reduced postobstructive tubulointerstitial disease as determined by light microscopy, immunohistochemistry and intrarenal mRNA expression of proinflammatory and profibrotic mediators. Furthermore, immunostaining revealed significantly reduced numbers of F4/80+ macrophages in IRAK-M-deficient versus wildtype mice. We did not observe a clear phenotype switch of macrophage upon unilateral ureteric obstruction as assessed by flow cytometry. Conclusions: Taken together, these results strongly support a pathogenic role for IRAK-M in postobstructive renal fibrosis and identify this molecule as a potential therapeutic target. TWEAK INDUCED RENAL INFLAMMATION BY EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION Sandra Rayego-Mateos1, Jose Morgado1, Ana Belen Sanz1, Satoru Eguchi2, Janos Pato3, Gyorgy Keri3, Jesu s Egido1, Alberto Ortiz1 and Marta Ruiz-Ortega1 1Universidad Autnoma de Madrid. IIS-Fundacin Jimnez Daz Madrid Spain, 2Temple University Philadelphia United States, 3Semmelweis University, Vichem Chemie Budapest Hungary Introduction and Aims: The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK), binds to its receptor Fn14 to elicit multiple biological activities, including stimulation of cell growth/apoptosis, angiogenesis and induction of inflammatory cytokines. EGFR transactivation involves the release of EGFR ligands, including heparin binding EGF-like growth factor (HB-EGF) and transforming growth factor- (TGF-) , by metalloproteinases of the ADAM family, of which ADAM-17 is the best characterised member. Our aim was to investigate whether TWEAK could transactivate EGFR in the kidney and the involvement of this molecular pathway in TWEAK mediated-renal responses. Methods: In vitro studies were done in tubular epithelial cells. The in vivo effect of TWEAK was evaluated by intraperitoneal injection of recombinant human TWEAK (0.5 g/per mouse) into C57BL6 mice. Results: In tubular epithelial cells TWEAK induced EGFR phosphorylation, via Fn14 binding, as shown by gene silencing. The blockade of ADAM17, by pharmacological inhibition with TAPI-2, or by ADAM17 gene silencing, inhibited TWEAK-mediated EGFR transactivation and downstream signalling, including ERK activation and upregulation of proinflammatory factors. Pre-treatment of cells with CRM197, that neutralize HB-EGF binding to EGFR, or TGF- blockade by a specific neutralizing antibody,diminished TWEAK-induced EGFR phosphorylation. Incubation with recombinant HB-EGF or TGF-; increased proinflammatory gene expression. Tweak-injected mice presented increased EGFR phosphorylation levels in the kidney, mainly located in tubular epithelial cells. The treatment of with the EGFR kinase inhibitor erlotinib markedly diminished renal EGFR activation and inhibited TWEAK-induced renal changes observed at 24 hours, including ERK activation (a downstream EGFR signalling), up-regulation of proinflammatory factors and interstitial inflammatory cell infiltration. Conclusions: EGFR transactivation required binding of TWEAK to the Fn14 receptor, ADAM17 activation, and release of the EGFR ligands HB-EGF and TGF-. In the kidney,TWEAK transactivates EGFR signalling pathway leading to renal ERK activation and induction of inflammatory cell infiltration. PBI-4050, A NOVEL FIRST-IN-CLASS ANTI-FIBROTIC COMPOUND, INHIBITS CTGF AND COLLAGEN I EXPRESSION IN NORMAL RAT KIDNEY FIBROBLASTS AND HUMAN KIDNEY PROXIMAL TUBULE EPITHELIAL CELLS, AND REDUCES KIDNEY FIBROSIS IN 5/6-NEPHRECTOMIZED RATS Martin Leduc1, Lilianne Geerts1, Brigitte Grouix1, Franois Sarra-Bournet1, Alexandra Felton1, Liette Gervais1, Shaun Abbott1, Jean-Simon Duceppe1, Boulos Zacharie1, Christopher Penney1, Pierre Laurin1 and Lyne Gagnon1 1ProMetic BioSciences Inc. Laval QC Canada Introduction and Aims: Interstitial fibroblasts are the principal effector cells of organ fibrosis. Transforming growth factor (TGF)--activated myofibroblasts express -smooth muscle actin (-SMA), secrete connective tissue growth factor (CTGF) that functions as a downstream mediator of TGF- action on fibroblastic cell types, and are important in the synthesis of collagen I. Also, evidence suggests that renal tubular epithelial cells can undergo epithelial to mesenchymal transition (EMT) to become matrix-producing fibroblasts under pathologic conditions. The aim of this study was to investigate the effect of PBI-4050, a first-in-class anti-fibrotic compound, on the expression of fibrosis markers in TGF-1-stimulated normal rat kidney fibroblasts (NRK-49F) and human kidney proximal tubule epithelial cells (HK-2) by qPCR. Methods: The effect of PBI-4050 on the mRNA expression of fibrotic markers was analyzed by qPCR in TGF-1-stimulated NRK-49F and HK-2 cells. Results: Activation of NRK-49F cells with TGF-1 resulted in a strong increase in the mRNA expression of -SMA (4 times) and CTGF (2 times) but not of collagen I. Treatment with PBI-4050 significantly reduced the TGF-1-induced overexpression of -SMA (80%) and CTGF (40%), and reduced basal collagen I (30%) mRNA expression. In HK-2 epithelial cells, PBI-4050 significantly reduced TGF-1-induced overexpression of CTGF (30%) and collagen I (50%) mRNA expression. These results correlate with in vivo results observed in a CKD/ESRD model. In 5/6-nephrectomized rats, oral administration of PBI-4050 (200 mg/kg) significantly decreased the mRNA overexpression of CTGF (50%), -SMA (30%) and collagen I (50%) in the remnant kidney. Conclusions: These results indicate a direct effect of PBI-4050 on fibroblasts and epithelial cells as observed by an inhibition of CTGF, -SMA and collagen I mRNA expression, and this is translated by a reduction of fibrosis in the kidney. MECHANISM OF GLOMERULAR HO-1 REGULATION Introduction and Aims: A number of independent observations on the role of the heme:HO-1 system in the kidney have emphasized the renoprotective effect of HO-1 induction. However, intrinsic glomerular cells apparently have a limited ability to i | Abstracts upregulate HO-1 when compared to tubular cells as shown by experimental models of glomerular injury in which potent HO-1 inducers such as cytokines and pro-oxidant radicals including superoxide (O2), hydrogen peroxide (H2O2) and peroxynitrite (ONOO), are overproduced in the glomerular milieu; a robust HO-1 induction was consistently found not in glomerular cells but at tubular sites downstream of the glomerular capillary. This raises the question of whether HO-1 induction in glomeruli is subject to tight regulation. Here, we address this question in normal rat glomeruli using the natural HO-1 substrate/inducer, heme (hemin). Methods: Glomeruli were isolated from wild type (WT), male, Sprague-Dawley (SD) rats, hmox1+/- SD, and SD with glomerular epithelial cell (GEC)-targeted HO-1 overexpression (GEC+/-). hmox1+/- rats were obtained using Zinc Finger Nuclease (ZFN) technology designed to target a specific HO-1 sequence within Exon 3. GEC+/- rats were obtained using Sleeping Beauty Transposon mediated transgenesis. Glomeruli from WT, hmox1+/- and GEC+/- rats were treated with defined concentrations of Heme (Hemin) for 18 h. Glomerular protein lysates were assessed for HO-1 levels by western blotting. HO-1 mRNA levels were assessed by quantitative Real-time PCR amplification. Results: Constitutive HO-1 protein expression was observed in wild type, hmox1+/- and GEC+/-glomeruli. In hmox1+/- rat glomeruli, a 60-70% HO-1 depletion was achieved compared to WT. In WT glomeruli, low Hemin (6-200 M) concentrations increased HO-1 synthesis (mRNA and protein) in a dose dependent manner, up to 2-fold. At higher hemin concentrations (400 M), HO-1 synthesis (mRNA and protein) was reduced to constitutive levels while at 800 M there was an even higher reduction (5-fold below constitutive levels). In hmox1+/- glomeruli, Hemin (400 M) failed to reduce HO-1 while at 800 M HO-1 synthesis reduction was attenuated compared to WT. In GEC+/glomeruli, the inhibitory effect of hemin on HO-1 synthesis was markedly increased. Specifically, HO-1 was barely detectable at 400 or 800 M Hemin compared to WT while 200 M Hemin had an inhibitory rather than a stimulatory effect on HO-1 (shift of the inhibitory effect to lower concentrations). Conclusions: Heme-mediated HO-1 synthesis in glomeruli is regulated by HO-1 levels achieved, pointing towards a negative feedback regulatory mechanism. This mechanism is clearly operational in GEC in which, under injury conditions, may serve to prevent HO-1 protein from reaching levels that may cause cytotoxicity due to the release of catalytically active Fe++. AUTOPHAGY INDUCTION PROMOTES ARISTOLOCHIC ACID-I-INDUCED RENAL INJURY Introduction and Aims: Ingestion of aristolochic acid (AA) causes AA nephropathy first by inducing tubular apoptosis in the acute phase. Crosstalk between autophagy and apoptosis might orchestrate the fate of tubular cells in acute AA nephropathy. We tested this hypothesis by acute administration of AA in vivo and in vitro. Methods: Autophagy was induced as enhanced Atg5 and LC3-II expressions in the kidneys of AA-I-treated rats. Punctuate LC3-GFP dots and autophagosomes were elucidated in the acute AA-I nephropathy. Next, the normal rat renal proximal tubular epithelial cells (NRK52E) were used to investigate the mechanisms of autophagy involved in acute AA-I nephropathy. The median lethal dose (LD50) of AA-I (100 mM) was applied in in vitro study. Results: Cleavage of poly (ADP-ribose) polymerase (PARP), nuclear condensation, and fragmentation were significantly observed in the AA-I-treated NRK52E cells. In addition, AA-I induced Atg5 and LC3-II expressions and punctuated LC3-GFP dots. Autophagy flux by using lysosome inhibitor E64 induced the accumulation of LC3-II, which further promoted apoptosis as shown by enhancing PARP cleavage. Inhibition of autophagy by 3-methyl adenine led to attenuating AA-I-induced apoptosis as indicated by decreasing PARP cleavage, nuclei condensation, and decreasing number of cells negative for acridine orange/ethidium bromide staining. Furthermore, knockdown of Atg5 by short hairpin RNA attenuated LC3-II expression and PARP cleavage in NRK52E cells. Conclusions: We suggest that an Atg5-dependent autophagy, which promotes renal tubular cell apoptosis, is induced in the acute phase of AA-I-induced nephropathy. ASSESSMENT OF ERYTHROPOIETIN EFFECTS IN THE PROGRESSION OF EXPERIMENTAL CHRONIC KIDNEY DISEASE Fernando F. Carvalho1, Vicente P. Teixeira1, Waldemar S. Almeida1 and Nestor Schor1 1Nephrology Division Unifesp So Paulo Brazil Introduction and Aims: The Erythropoietin (EPO) is an endogenous glycoprotein produced primarily in kidney and its main function is stimulate the production of blood cells to transport oxygen to the tissues. The EPO has been used primarily to treat anemia caused by chronic kidney disease. Recent studies have shown a renoprotective EPO effect on ischemic and chronic kidney disease. The mechanisms of renoprotection include inhibition of apoptosis, inflammation and induction of angiogenesis. Thus, the aim of this study is to evaluated the influence of EPO on progression of kidney disease in experimental chronic kidney disease. Methods: Male Wistar rats weighing 280-300g underwent 5/6 nephrectomy and were divided into two groups: (NX) only nephrectomized(n=6) and (NX-EPO) nephrectomized(n=6) and treated with a weekly dose of erythropoietin (250UI/kg/ip).All animals were sacrificed 8 weeks after surgery. Hematocrit, serum creatinine, proteinuria, indirect blood pressure measurement, glomerular score and tubular lesion and immunohistochemical analysis were assessed. For immunohistochemical of desmin expression, a semiquantitative 04 system, with 0 being negative stained and 4 the most positive stained, was used. The immunohistochemical expression of PCNA was determined by counting positive tubular cells in 10 randomly selected fields (400x). Results: The NX-EPO group showed significant improvement in serum creatinine (NX 1.6 0.4 versus NX-EPO 0.8 0.1, P 0.001) and protein/urine creatinine ratio (NX 11.2 6.0 versus NX-EPO 4.1 2.2, P = 0.021). Histopathological results demonstrate a lower rate of glomerular sclerosis (NX=33% versus NX-EPO=17%) and tubulointerstitial fibrosis (grade III NX versus grade I NX-EPO) according to Banff classification. Immunohistochemistry results reveal an increased expression of desmin in NX group (NX grade 3.4 vesus NX-EPO grade 2.0) and a lower PCNA expression in tubular cells (NX= 8.0 versus NX-EPO= 3.0).There were no significant differences in hematocrit and blood pressure between the two groups. Conclusions: Our study suggests a beneficial effect of EPO in the model of progression of experimental chronic renal disease reflected by improvement of serum creatinine, proteinuria and attenuation of glomerular lesion score. We observed a lower expression of desmin in podocytes and lower expression of PCNA in tubular cells regardless of its effect on hematocrit and blood pressure. MITOCHONDRIAL HOMEOSTASIS IS IMPEDED BY DEGRADATION AND AUTOPHAGY IN OXIDATIVE STRESS-INDUCED RENAL CELL INJURY David M. Small1,2, Nigel C. Bennett1,2, Jeff Coombes3, David W. Johnson4 and Glenda C. Gobe1,2 1School of Medicine Univ of Queensland Brisbane QLD Australia, 2CKDR Translational Research Institure Brisbane QLD Australia, 3Sch Human Movement Studies Univ of Queensland Brisbane QLD Australia, 4Nephrology Princess Alexandra Hosp Brisbane QLD Australia Introduction and Aims: Oxidative stress deregulates mitochondrial genes and has a key role in the development of many kidney diseases. Balancing degradation of defective mitochondria with renewal of healthy mitochondria is vital for renal cell health. p62 (indicates faulty mitochondria) and the nuclear transcription factorperoxisome proliferator-activated receptors (PPAR) and (oxidative stress-responsive, mitochondrial biogenesis) need investigation. The aim of this project was to investigate the molecular and structural changes of mitochondrial homeostasis and biogenesis in oxidative stress-induced renal proximal tubular (PT) epithelial injury. Methods: HK-2 PT cells were treated with hydrogen peroxide (H2O2; 0.2, 0.4, 0.6, 0.8, 1.0mM) for 2h and 18h. p62, phospho-PPAR/PPAR, PPAR and LC3-II (autophagy) (Western blot; densitometry), and cellular ATP (luciferase-based assay) were analyzed. Mitochondrial biogenesis was quantified (MitoTracker Red; analysis software). PPAR agonists and antagonists were used to determine protective or cytotoxic effects. Results: Following 2h H2O2 exposure, p-PPAR/PPAR, p62 and PPAR decreased ( p<0.05), along with ATP and MitoTracker Red ( p<0.001, p<0.05, respectively). These results indicate early mitochondrial dysfunction and degradation. Following 18h H2O2 exposure, p62 and LC3-II expression increased ( p<0.05), p-PPAR/PPAR was normal, and ATP and MitoTracker Red remained low ( p<0.001, p<0.05, respectively). Apoptosis increased progressively with H2O2 in a concentration and temporal manner. Results suggest mitochondrial biogenesis is impeded by degradation and autophagy resulting in progressive loss of renal cells after oxidative stress. However, modulation of the PPARs had no effect on results. Conclusions: Oxidative stress promotes mitochondrial destabilisation in HK-2 cells by increasing p62, and perhaps by early loss of PPAR activation. Failure to remove damaged mitochondria via autophagy, or defective p62, may lead to a spiralling cycle of oxidative stress, with progressive deterioration of tubular function in kidney disease. C5b-9 MEMBRANE ATTACK COMPLEX PLASMATIC LEVELS IN DIFFERENT FORMS OF ACUTE KIDNEY INJURY Nuria Montero1, Alejandra Prada1, Marta Riera1, Mara Orfila1, Julio Pascual1, Eva Rodrguez1 and Clara Barrios1 1Nephrology Hospital del Mar-IMIM Barcelona Spain Introduction and Aims: Complement pathway is involved in the pathophysiology of several kidney diseases. The final component C5b-9 membrane attack complex (MAC) has been unequivocally demonstrated to be involved in the development of experimental membranous nephropathy and in animal models of ischemia-reperfusion. After injury, complement is activated leading to the production of proinflammatory cytokines such as interleukin-6 and generated neutrophils recruitment producing NGAL (Neutrophil Gelatinase-Associated Lipocalin) release. Methods: 77 plasma samples were collected in a tertiary hospital; 61% of them (47 patients) had AKI and 38% (30 patients) had a normal renal function. We obtained samples for 26 septic patients (47% AKI), 23 trasplant kidney patients (ischemia-reperfusion model; 70% AKI), 15 patients under colistin treatment (nephrotoxic model; 46% AKI) and 13 patients with several possible AKI risk factors (50% of them developed finally multifactorial AKI) .Samples were tested for IL-6 and MAC using ELISA kit and NGAL were tested by means to inmunofluorescence assay. Results: Plasmatic MAC level was statistically different in patients with AKI as compared to normal renal function controls, regardless of the etiology of AKI (501 247 mAU/mL vs 388150 mAU/mL; p 0.015). Plasmatic IL-6 levels were significant higher in a AKI patients compared to normal kidney function (10,472,8 pg/mL vs 7,373,0 pg/mL p=0,02) and NGAL levels were also significantly higher in AKI patients (570,5305 ng/mL vs 292,5233 ng/mL p< 0,001). No relevant differences in the three biomarkers were detected in the different etiological subgroups. Conclusions: Our data show that in AKI, regardless etiology, the complement system is activated, leading the pro-inflammatory cytokine stimulation (IL-6) and could produce releasing of NGAL from neutrophils. TIMP-1 INHIBITION AMELIORATES RENAL FIBROSIS IN TGF- TRANSGENIC MICE Gabor Kokeny1,2, Krisztina Fazekas1, Laszlo Rosivall1,2 and Miklos M. Mozes1,2 1Department of Pathophysiology Semmelweis University Budapest Hungary, 2International Nephrology Research and Training Center Semmelweis University Budapest Hungary Introduction and Aims: We have previously shown that CBAxB6-TGF transgenic mice are characterized by hepatic expression of Alb/TGF transgene results in 10 fold elevation of circulating TGF- levels. These mice develop massive proteinuria, severe glomerulosclerosis, moderate interstital fibrosis and premature uremic death by the age of 21 days. Analysis of renal matrix related molecules (MMPs, TIMPs, Smads, matrix components) revealed striking increase solely in TIMP-1 mRNA expression (90 fold as compared to wild type) (Kokeny et al, Clin Kidney J 2011, 4 (S2): 421-429). To confirm the role of elevated TIMP-1 expression in the development of renal fibrosis we examined the effect of TIMP-1 inhibition in this model. Methods: Eight days old male CBAxB6-TGF transgenic mice (n=5) were treated daily with TIMP-1 neutralizing antibody (2 ug/day, R&D Cat: AF980) intraperitoneally for 5 days. IgG treated male CBAxB6-TGF transgenic mice served as control (n=4). At the age of 14 days, urinary protein/creatinine ratio (P/C), serum creatinine, urea, and plasma TGF- levels were determined . Kidneys were collected for renal histology (glomerulosclerosis index (GSI) on PAS stained slides). Data were statistically analyzed using Mann-Whitney test. Results: Plasma TGF- levels and body weights of treated and control mice were comparable (9.541.36 vs 9.511.03 grams and 5012 vs 5714 ng/ml, respectively). Renal hypertrophy, as characterized by relative kidney weight was decreased in the treated animals as compared to controls (7.40.7 vs 9.21.5 mg kidney/g body weight, p<0.05). Urinary protein/creatinine ratio was significantly lower in treated mice (treated: 6.71.6 vs control: 14.25.9, p<0.05). TIMP-1 inhibition also lowered serum creatinine by 30% and serum urea by 50% in treated animals (1.40.1 vs 2.10.4 mg/dl and 5125 vs 11039 mg/dl, respectively, p<0.05) and ameliorated glomerular hypertrophy, mesangial proliferation and expansion (glomerulosclerosis index in treated: 1.500.25 vs control: 2.540.16, p<0.05). Conclusions: Our preliminary data demonstrate that inhibition of TIMP-1 significantly ameliorates the progression of TGF- induced renal fibrosis in this model. UP-REGULATION OF ALK1 IN MOUSE KIDNEY FOLLOWING URETERAL OBSTRUCTION Jose M. Muoz-Felix1,2,3, Jose M. Lopez-Novoa1,2,3 and Carlos Martinez-Salgado1,2,3,4 1Department of Physiology and Pharmacology University of Salamanca Salamanca Spain, 2Cardiovascular Biomedical Research Institute of Salamanca (IBSAL) Salamanca Spain, 3Institute Queen Sophie for Renal Research (IRSIN) Fundacin Renal Iigo Alvarez de Toledo Madrid Spain, 4Hospital Universitario de Salamanca Instituto de Estudios de Ciencias de la Salud de Castilla y Len (IESCYL) Salamanca Spain Introduction and Aims: Tubulointerstitial fibrosis, one of the common end points of chronic renal insufficiency, is characterized by an excessive accumulation of extracellular matrix (ECM) in the renal interstitium, myofibroblast activation, cell infiltration, tubular apoptosis and proliferation. Transforming growth factor-beta 1 (TGF-1) is considered a fundamental profibrotic cytokine. ALK1 (activin receptor-like kinase I) is a type I receptor for TGF-1 with a pivotal role in endothelial proliferation and migration. Others receptors such as ALK5 and endoglin are overexpressed in experimental models of renal fibrosis. Nevertheless the expression levels and the role of ALK1 in renal fibrosis are unknown. Methods: We performed unilateral ureteral obstruction (UUO) in mice, an obstructive nephropathy experimental model, in order to analyze the expression of ALK1 following 15 days UUO by western-blot and immunofluorescence, and the co-expression with other proteins such as -SMA (myofibroblast marker) and CD68 (macrophage marker) in order to elucidate which cells expressed ALK1 following UUO. We also cultured renal fibroblasts from haploinsufficient (ALK1+/-) and control (ALK1+/+) mice in order to analyze the role of ALK1 in ECM protein (collagen I, fibronectin) expression. Results: ALK1 expression is increased following UUO. In non-obstructed kidneys the expression of ALK1 is restricted to glomerular cells, some interstitial fibroblasts and smooth muscle cells of small blood vessels. In obstructed kidneys, ALK1 expression is mainly located in the tubulointerstitial area. Double immunostaining with ALK1/ -SMA and ALK1/CD68 showed that ALK1 is expressed in both myofibroblasts and infiltrated macrophages. ALK1 is expressed in cultured renal fibroblasts, and ALK1 heterozygous renal fibroblasts showed higher expression of collagen I and fibronectin, suggesting that ALK1 downregulates ECM protein expression. Conclusions: Summarizing, ALK1 upregulation following UUO may be considered as a protective mechanism against renal fibrosis due to its ability to downregulate ECM protein expression. REGULATION OF TUBULAR CELL PHENOTYPE BY THE LIM PROTEIN HIC-5 Nick Hornigold2, Jeremy Hughes3 and Andrew Mooney1 1Renal Unit St James's University Hospital Leeds United Kingdom, 2CRUK Clinical Centre St James's University Hospital Leeds United Kingdom, 3Centre for Inflammation Research University of Edinburgh Edinburgh United Kingdom Introduction and Aims: We have previously reported the role of the LIM protein, Hic-5 in the regulation of mesangial cell phenotype during progressive chronic kidney disease, and shown that it has an important role in controlling pathophysiological phenotype change in experimental glomerulosclerosis. During those studies, we also identified a subset of tubular cells that constitutively express this protein (Kidney International 2010). Therefore we undertook a series of in vivo and in vitro experiments to investigate its role in tubular phenotype regulation in health and disease. Methods: NRK52E rat tubular cells and MDCK canine tubular cells were transfected with a Hic-5 overexpression construct. Wild type cells and Hic-5 transfected cells were then compared in assays for proliferation, apoptosis, adhesion and cell motility. In separate experiments, rats were subject to unilateral uereteric obstruction (UUO) and sacrificed at 7, 14 and 21 days. Immunostaining for Hic-5 was undertaken. Results: NRK52E and MDCK cells do not express Hic-5 in culture, and have a dedifferentiated (neither proximal nor distal tubular) phenotype. Forced over-expression of Hic-5 results in cells with increased proliferation, decreased susceptibility to apoptosis, increased adhesion and reduced motility. In health, tubular cells of distal phenotype constitutively express Hic-5, but following UUO, this expression is down-regulated and lost by 14 days, associated with tubular cell apoptosis and progressive disease. Conclusions: Hic-5 regulates important phenotypical characteristics in a subset of tubular cells including proliferation, attachment and susceptibility to cell death. These findings, and the loss of expression during experimental UUO supports its role as a mediator of cell loss during tubular injury. CHRONIC EFFICACY OF LISINOPRIL IN IMPROVING KIDNEY BIOMARKERS, FUNCTION AND STRUCTURE IN UNx MALE ZSF1 RATS Agnes Benardeau1, William Riboulet1, Anthony Vandjour1, Bjoern Jacobsen2, Christian Apfel1 and Karin Conde-Knape1 1pRED, CVM F Hoffmann_La Roche AG Basel Switzerland, 2pRED, NCS F Hoffmann-La Roche AG Basel Switzerland Introduction and Aims: Diabetic nephropathy (DN) is a complex pathology leading to ESRD. Development of treatments that prevent ESRD requires translatable models and evaluation of biomarkers. UNx reduces GFR, increases proteinuria in animal models and human. Therefore we aim to explore UNx as a mean of exacerbating disease in this model. i | Abstracts Methods: We used hypertensive and metabolic syndrome male ZSF1 rats that we submitted to unilateral nephrectomy (UNx, at 12w of age) to exacerbate DN and associated diseases. We characterized metabolic, renal and cardiac changes in ZSF1 rats within 12w post UNx and compared them to age-matched Sham ZSF1 rats. Urine and plasma parameters were measured for determining relevant biomarkers (BMs). Echography method was used for describing the evolution of kidney and heart size, and cardiac function. Impact of aging and UNx on kidney, liver, eyes and heart structure was determined by histology and IHC analysis. Sensitivity of the UNx_ZSF1 rat model to antihypertensive drugs (ACEi, Lisinopril@30mg/kg as Food Admix) was evaluated during chronic treatment of ZSF1 rats ( for 3 months) on kidney BMs, function and structure. Results: Our data showed that UNx did neither exacerbate SBP of ZSF1 rats nor change LV mass, cardiac function or heart rate up to 16W after surgery compared to age-matched Sham ZSF1 rats despite development of cardiac hypertrophy with aging. Glucose intolerance and insulin resistance was unchanged by UNx. UNx induced further hyper TG, hypercholesterolemia and elevated NEFAs in plasma of ZSF1 rats and induced hepatocellular vacuolation. UNx increased right kidney size, kidney weight and KW/BW ratio compared to age-matched Sham ZSF1 rats. UNx reduced urine volume, Creatinine Clearance and deteriorated several plasma (BUN, KIM-1, Cyst-C, FGF-21, FGF-23 and b2M) and urine BMs (Albuminuria, Cyst-C, and TGFb1). UNx increased severity of overall kidney lesions compared to age-matched controls including mainly mesangial expansion, glomerulosclerosis, glomerular vacuolation, tubulointerstitial inflammation, proteinaceous casts and thickening of basement membrane. Lisinopril significantly reduced hypertension (SBP: -22% vs. Vehicle) of UNx_ZSF1rats already 3W after the start of the treatment, reduced LV mass and right kidney size. Lisinopril normalized albuminuria, reduced KIM-1, b2M, TGFb1 and Cyst-C excretion. Lisinopril reduced kidney fibrosis and inflammation. Conclusions: Our data confirms that the ZSF1 rat model develops DN with similarities to human morphology and recapitulates features of metabolic syndrome observed in human. UNx worsened DN progression in male ZSF1 rats as compared to Sham. Lisinopril significantly reduced diabetic renal injury (structure, functions and improvement of BMs), reduced BP, hypertrophy and dyslipidemia. Sensitivity of the ZSF1 UNx to kidney for exploring DN is confirmed. PBI-4419, A NOVEL FIRST-IN-CLASS ANTI-FIBROTIC COMPOUND, INHIBITS TGF--INDUCED EPITHELIAL-MESENCHYMAL TRANSITION OF HUMAN RENAL TUBULAR HK-2 CELLS Brigitte Grouix1, Alexandra Felton1, Franois Sarra-Bournet1, Martin Leduc1, Lilianne Geerts1, Liette Gervais1, Shaun Abbott1, Jean-Franois Bienvenu1, Jean-Simon Duceppe1, Boulos Zacharie1, Christopher Penney1, Pierre Laurin1 and Lyne Gagnon1 1ProMetic BioSciences Inc. Laval QC Canada Introduction and Aims: Emerging evidence suggests that renal tubular epithelial cells can undergo epithelial to mesenchymal transition (EMT) to become matrix-producing fibroblasts (myofibroblasts) under pathologic conditions. The aim of this study was to investigate the effect of our novel, first-in-class anti-fibrotic compound, PBI-4419, on renal EMT. Methods: The effect of PBI-4419 on transforming growth factor-beta (TGF-1)-induced EMT was analyzed on human proximal tubule epithelial cells (HK-2). To assess the progression of EMT, the expression level of the pro-epithelial marker E-cadherin and the mesenchymal/pro-fibrotic markers, connective tissue growth factor (CTGF) and collagen I, was determined by quantitative real-time PCR. EMT was induced by TGF-1 as indicated by the downregulation of E-cadherin along with the upregulation of CTGF and collagen I transcript expression. Results: TGF-1-induced EMT was significantly inhibited by PBI-4419 (5 M) as demonstrated by an increase of E-cadherin and the decrease of CTGF and collagen I expression. Interestingly, it was also observed that in the absence of TGF-1, PBI-4419 alone supported an epithelial phenotype by upregulating E-cadherin and by downregulating basal expression of CTGF and collagen I. We further demonstrate the activity of PBI-4419 on EMT with renal tissues from 5/6-nephrectomized rats; markers for fibrosis (CTGF and collagen I) and a marker for myofibroblasts (-smooth muscle actin: -SMA) were assessed. CTGF, collagen I and -SMA mRNA expression was significantly upregulated in the remnant kidney. Treatment with PBI-4419 resulted in a significant reduction in the expression of CTGF (60%, p<0.001, down to the control level of the sham animals), collagen I (60%, p<0.0001) and -SMA (30%, p<0.05) in the remnant kidney. Conclusions: In conclusion, these results support that PBI-4419 has the potential as a novel therapeutic agent in the prevention or reduction of fibrosis in kidney diseases by inhibiting EMT. INDOXYL SULFATE SUPPRESSES TRANSACTIVATION OF HYPOXIA-INDUCIBLE FACTOR 1 AND DYSREGULATES HYPOXIA RESPONSE IN RATS WITH CHRONIC KIDNEY DISEASE AND EXPERIMENTAL HEART FAILURE Tetsuhiro Tanaka1, Junna Yamaguchi1 and Masaomi Nangaku1 1Division of Nephrology and Endocrinology University of Tokyo School of Medicine Tokyo Japan Introduction and Aims: Chronic hypoxia in the tubulointerstitium serves as a final common pathway in progressive renal disease. Circumstantial evidence suggests that HIF-1, while obviously expressed in the ischemic tubules, may be functionally suppressed in a CKD milieu. In this study, we hypothesized that indoxyl sulfate (IS), a uremic toxin, impairs cellular hypoxia response. Methods: In vitro, the expression of HIF-1, Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2) and HIF-1 target genes was measured by immunoblotting and real-time PCR. Binding of HIF-1 to the target gene promoters was evaluated by chromatin immunoprecipitation (ChIP) assays. Association of the cofactor p300 with HIF-1 was assessed by mammalian two-hybrid assays. mRNA stability of CITED2 was measured by actinomycin D treatment. The role of MAP kinase pathways was evaluated by using specific inhibitors. In vivo, the effect of IS on the expression of HIF-1 target genes was investigated in rats with adriamycin nephropathy as well as the remnant kidney, in the presence or absence of an oral adsorbent, AST-120. Additionally, the expression HIF-1 target genes was evaluated in the isoproterenol-induced heart failure (HF) model in rats treated with or without indole. Results: In human proximal tubular cells (HK-2), IS reduced the hypoxic induction of HIF-1-target gene mRNA and protein. This effect was not accompanied by quantitative changes of the HIF-1 protein, but was associated with the functional impairment of the HIF-1 C-terminal transactivator domain (CTAD). Among factors which impede the recruitment of transcriptional co-activators to the HIF-1CTAD, CITED2 was markedly upregulated by IS, via mechanisms of posttranscriptional mRNA stabilization involving the extracellular signal-regulated kinase (ERK)1/2 pathway. In vivo, the incommensurate expression of HIF-target proteins was demonstrated in the ischemic tubules of several CKD models, such as adriamycin nephropathy and the remnant kidney, which was offset by an oral adsorbent of indole, AST-120, signifying the involvement of IS. Further, the induction of angiogenic, hypoxia-inducible genes was blunted in rats with experimental heart failure, when they were given indole in parallel. Conclusions: Results of these studies uncover a novel role of IS in modulating the transcriptional response by HIF-1, and provide insight into molecular mechanisms underling progressive nephropathies as well as the cardio-renal-anemia syndrome. INDOXYL SULFATE DOWNREGULATES RENAL EXPRESSION OF Nrf2 THROUGH ACTIVATION OF NF-B Toshimitsu Niwa1, Dilinaer Bolati1, Hidehisa Shimizu1, Maimaiti Yisireyili1 and Fuyuhiko Nishijima2 1Nagoya University Graduate School of Medicine Nagoya Japan, 2Biomedical Research Laboratories, Kureha Co. Tokyo Japan Introduction and Aims: Indoxyl sulfate is accumulated in the serum of uremic patients, accelerating the progression of kidney failure. In uremic rat kidney, the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its related genes is downregulated. The present study aimed to determine whether indoxyl sulfate affects Nrf2 expression in the kidney. Methods: Effects of indoxyl sulfate on expression of Nrf2 were determined using human proximal tubular cells (HK-2 cells) and following animals: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). AST-120, an oral sorbent which reduces serum level of indoxyl sulfate, was administered to uremic rats to determine its effect on the expression of Nrf2. Results: Indoxyl sulfate downregulated Nrf2 expression in HK-2 cells. The indoxyl sulfate-induced downregulation of Nrf2 expression was alleviated by an inhibitor of nuclear factor-B (NF-B) ( pyrrolidine dithiocarbamate), and small interfering RNA specific to NF-B p65. DN+IS, DH, and DH+IS rats showed decreased expression of Nrf2 and heme oxygenase-1 (HO-1), an antioxidant gene and a target of Nrf2, and increased expression of 8-hydroxydeoxyguanosine (8-OHdG), a marker of reactive oxygen species in the kidneys compared with DN. Thus, indoxyl sulfate as well as hypertension suppressed expression of Nrf2 in rat kidneys. AST-120 increased the expression of Nrf2 and HO-1, and suppressed expression of 8-OHdG in the kidneys compared with control uremic rats. Conclusions: Indoxyl sulfate downregulates renal expression of Nrf2 through activation of NF-B. CYTOTOXIC EFFECTS OF p-CRESOL IN RENAL EPITHELIAL TUBULAR CELL A. Brocca1,2, G. Virz1,2, M. de Cal1,2 and C. Ronco1,2 1Nephology Dept. S. Bortolo Hospital Vicenza Italy, 2International Renal Research Institute Vicenza-IRRIV Vicenza Italy Introduction and Aims: Uremic syndrome is characterized by a deterioration of kidney function due to the accumulation of uremic toxins. These are characterized by low molecular weight and different hydrophobicity; they can exist in free water-soluble form or bind reversibly to serum protein. Uremic toxins are particularly difficult to remove by conventional dialysis treatments and are the major causes of mortality in CKD patients. One of the uremic toxin is p-cresol, a fenol protein-bound lipofile, by-product of protein catabolism. It is produces by intestinal bacteria. Uremia causes a modification of intestinal flora, increasing the number of p-cresol bacteria productors. Furthermore, increase of plasmatic p-cresol concentration leads to development of CKD. p-cresol cytotoxic effect in monocytes is well known, particularly in macrophages, but it's still poorly understand what determines in epithelial renal cell. Our aim is to evaluate in vitro effect of p-cresol on renal tubular epithelial cell line (LOC), in terms of apoptosis and necrosis, to better understand the pathophysiological impact of this toxin. Methods: We perform the detection of apoptosis and necrosis in LOC, incubated for 24 hours in medium with increasing concentration of p-cresol, by Annexin V/ Propidium Iodide Cytofluorimetric assay. We used scalar concentration of p-cresol, from 40 mg/L (up level in uremic patient) to 2.5 mg/l. In addition, we evaluated Caspase-3 levels by ELISA kit and the detect DNA Ladder, that showing the typical apoptotic DNA fragmentation. All experiments were performed 5 times. Results: p-cresol concentrations 20 mg/L cause the necrosis of >60% of cells; instead at concentration 5 mg/L the percentage is comparable to control.We observed a positive trend, but no significant relationship between Caspase-3 levels and p-cresol concentration.DNA Ladder is present in cells treated with low concentration of p-cresol and decreases at increasing concentration of toxin, when the necrosis is almost the only type of cell death. We can better understand the pathophysiology of this phenomenon increasing the number of experiments. Conclusions: In conclusion, our data show that p-cresol has a cytotoxic effect on LOC. In particular, p-cresol causes cellular death in renal tubular cells, determining necrosis in almost total cultured cells at the maximal concentration. At lower concentration, p-cresol determines cell death through apoptosis. This data is supported also by the detection of Caspase-3 activation. It will be interesting to extend the analysis considering muscular cells to estimate the damage of uremic toxin in muscle tissue (uremic atrophy) or studying a possible development of uremic syndrome in Cardiorenal Syndrome. mesangial cells, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, -3 PUFAs, significantly counteracted the up-regulation of pro-fibrotic genes induced by stimulatory agents. Our aim was to investigate in renal tubular cells in vitro, the pro-fibrotic effect of CyA and a possible protective action of the fish oil component DHA. Methods: HK-2 cells (ATCC, # CRL-2190) of human proximal tubular origin were grown in appropriate medium, subcultured in 6-well plates and grown to sub-confluence. Subsequently, the cells were exposed to CyA at 5 g/ml alone or simultaneously with DHA 50 M for 24 hrs. In order to test the possible toxic effect of such treatment, morphology and viability were assessed at light microscopy. Gene expression analysis was performed by RT-PCR to evaluate renal fibrosis markers such as TGF, CTGF, FN, COLIV and MCP-1. Results: CyA, induced a significant up-regulation of all pro-fibrotic genes considered: TGF +149%, CTGF +130% FN +129%, COLIV +158, MCP-1 +127% ( p<0.05). On the contrary, the effect of DHA treatment resulted in a significant down-regulation: TGF -10%, CTGF 36%, FN 26%, COLIV 15%, MCP-1 27% with respect to controls (untreated cells). When DHA was used in simultaneous treatment with CyA, it exerted a significant inhibitory effect on CyA-induced upregulation of all genes: TGF 50%, CTGF 50%, FN 55%, COLIV 60%, MCP-1 52% ( p<0.01 vs CyA-treated cells). Conclusions: Our in vitro results of fibrogenic cytokines upregulation substantiate clinical reports of CyA-induced kidney toxicity. Moreover, we demonstrated a beneficial effect of the fish oil active component DHA on the pro-inflammatory cytokine imbalance induced by CyA at renal tubular level. The favourable action of omega-3 PUFA on CyA-altered pro-fibrotic cytokines profile may suggest their use in kidney transplanted patients for the non-pharmacological management of adverse outcomes such as tubular atrophy, tubular dysfunction, and interstitial fibrosis characteristic of chronic CyA nephropathy. DIFFUSION-WEIGHTED MRI DOES NOT DETECT KIDNEY FIBROSIS IN A RAT MODEL OF FIBROSIS Peter Boor1,2, Michael Perkuhn3,4, Martin Weibrecht3,4, Stephanie Zok2, Ina V. Martin2, Felix Schoth3, Tammo Ostendorf2, Christiane Kuhl3 and Jrgen Floege2 1Institute of Pathology RWTH University Aachen Germany, 2Division of Nephrology RWTH University Aachen Germany, 3Department of Radiology RWTH University Aachen Germany, 4Research Laboratories Philips Technologie GmbH Innovative Technologies Aachen Germany Introduction and Aims: Diffusion-weighted (DW) magnetic resonance imaging (MRI) measures MRI signal loss induced by random movement of water molecules, i.e. tissue image based on water diffusion. DW-MRI signal loss can be numerically expressed as apparent diffusion coefficient (ADC). Random water movement is restricted in cells, i.e. the higher the cellular content or density of a tissue, the lower the ADC value. Experimental and clinical studies suggested that ADC was reduced in and correlated with experimental renal fibrosis or the stage of chronic kidney disease (CKD). We tested the feasibility of DW-MRI as an imaging tool to measure renal fibrosis. Methods: We used rats with unilateral ureteral obstruction (UUO), a model of tubulointerstitial fibrosis. We performed DW-MRI of kidneys (using clinical 3T TX MR) before, on days 3 and 5 after UUO as well as after release of obstruction and sacrifice and calculated renal cortical ADC values. Results: Obstructed kidneys had reduced ADC compared to the non-obstructed kidneys on both day 3 and more so on day 5. Surgical release of the obstruction had no effect on this difference. No obvious differences in ADC were observed in animals measured immediately versus 5 hours after obstruction-release, suggesting that OMEGA-3 FATTY ACIDS FOR THE PREVENTION OF CYCLOSPORIN A-INDUCED PROXIMAL TUBULAR TOXICITY IN VITRO Giovanna Priante1, Estella Musacchio2, Chiara Valvason1, Leonardo Sartori2, Antonio Piccoli1 and Bruno Baggio1 1Department of Medicine - DIMED, University of Padova Nephrology Unit Padova Italy, 2Department of Medicine - DIMED, University of Padova Clinica Medica I Padova Italy Introduction and Aims: Cyclosporin A (CyA) is a potent immunosuppressant widely employed in the prevention of organ transplant rejection as well as in the treatment of autoimmune diseases. However, chronic CyA nephrotoxicity, characterized by early disturbances of proximal tubule structure and function progressing to tubular atrophy and interstitial fibrosis, is a universally recognized side effect of CyA therapy. Epidemiological and clinical studies clearly demonstrate that fatty acids (PUFAs) and their metabolites play an important role as autocrine and paracrine mediators in various patho-physiological conditions at different tissue and organ levels including kidney disease. In this context, we recently demonstrated that in cultured human i | Abstracts parenchymal edema did not contribute to ADC values. After sacrifice, ADC in both kidneys dropped to less than 50% compared to those measured in living animals. More importantly, the ADC of obstructed kidneys were significantly higher compared to non-obstructed kidneys. The ADC after sacrifice correlated closely with tubular dilation, interstitial expansion and fibrosis. Conclusions: Our data indicate that low ADC values measured by DW-MRI do not reflect fibrosis in vivo but rather non-random water movement in kidneys, i.e. perfusion, tubular flow and water reabsorption. Ex vivo, i.e. after cessation of all non-random water movement, fibrotic kidney cortex is characterized by increased DW-MRI signal (i.e. higher ADC values) compared to non-fibrotic kidneys. This is most likely due to expansion of space which allows random water movement in fibrotic tissue, i.e. tubular dilation, cell atrophy and widening of interstitial space. DW-MRI seems not to be a useful method for specific assessment of renal fibrosis. EFFECT OF SPIRONOLACTONE ON THE COURSE OF RENAL FAILURE AND CARDIOVASCULAR SYSTEM IN RATS WITH 5/6 NEPHRECTOMY Aigul Karabaeva1, Ashot Essaian2, Olga Beresneva2, Marina Parastaeva2, Ivan Kayukov2 and Alexey Smirnov2 1Institute of Cardiology and Therapy Alma-Aty Kazakhstan, 2St.-Petersburg State Pavlov Medical University St.-Petersburg Russian Federation Introduction and Aims: To evaluate the effects of the spironolactone on the course of experimental chronic renal failure, blood pressure (BP) and myocardial hypertrophy in Wistar rats with 5/6 nephrectomy (NE). Methods: Animals were divided into 3 Groups: Group 1 sham-operated (control, C; n=12); Group 2 NE rats (n=10); Group 3 NE rats receiving spironolactone (0.2 mg/ day; n=9). The animals were observed during 2 months after the NE. Serum urea (Ur, mmol/l),creatinine (Cr, mmol/l), potassium (K+, mmol/l) and plasma aldosterone concentration (PAC, pg/ml) levels were investigated. Mean BP (mm Hg) was measured in the awaked rats by the tail cuff method. The degree of left ventricular hypertrophy was estimated as a ratio: left ventricular mass/body mass (LVH, mg/g). Results: NE in rats from Groups 2 and 3 was associated with significant rise of Ur (18.6 2.5 in Group 2; 18.7+0.9 in Group 3 vs 6.10.2 in C; p<0.05 in both cases), Cr (0.07 0.003 in Group 2; 0.070.003 in Group 3 vs 0.040.002 in C; p<0.05 in both cases) and K+ (7.910.3 in Group 2; 7.910.3 in Group 2; 7.730.2 in Group 3 vs 4.510.2 in C; p<0.05 in both cases). There were no statistically significant differences in the levels of Ur, Cr and K+ between Groups 2 and 3. BP in Groups 2 (151.32.7) and 3 (151.3 2.3) were significantly higher than in C (121.21.8; p<0.001 in both cases). There were no statistically significant differences in the levels of BP between Groups 2 and 3. In Group 3 LVH (2.520.06) did not differ significantly from those in the C (2.35 0.09; p=NS). However in Group 2 (2.800.11) it was significantly higher than in C ( p<0.05). There were no statistically significant differences in the LVH between Groups 2 and 3. PAC in Group 3 (281.739.0 was significantly higher, than in C (145.417.4; p<0.05). There were no differences in the levels of PAC between Groups 2 (207.632.9) and 3 or between Group 2 and C ( p=NS in both cases). Conclusions: In rats with NE spironolactone has cardioprotective effect independent of the influence on BP, degree of renal dysfunction or PAC level. P2-RECEPTORS ACTIVATION INCREASES GLOMERULAR PERMEABILITY FOR ALBUMIN WITHOUT CHANGES IN ALBUMIN EXCRETION IN URINE Introduction and Aims: An association between albuminuria and progression of renal disease is postulated. Albumin is filtered through glomerular barrier and reabsorbed in proximal tubule, thus, dysfunction of glomerular and tubular processes may result in increased excretion of albumin. Microalbuminuria is a popular laboratory marker of early damage of glomeruli. The glomerular barrier consists of endothelial cells, basement membrane and podocytes. Activity of these cells is modified by extracellular nucleotides via P2-receptors in para-/autocrine manner. P2-receptors are expressed on cells of glomerular barrier and their expression is modified in pathophysiological conditions e.g.diabetes, hypertension associated with increased albumin excretion in urine. The aim of the study was to investigate the effect of P2-receptors activation on glomerular capillary permeability for albumin. Methods: The osmotic pumps with 2-methylthioATP (2-MeSATP), specific and non-selective agonist of P2-receptors or buffer were implanted into rats for 7 days and animals were kept in metabolic cages to collect urine. Isolated glomeruli were incubated with buffer or 2-MeSATP and continuously observed with use of video-microscopy (Olympus IX51) before and after the medium was replaced by oncopressive medium.Glomerular volume was derived from the area of glomerular image calculated using CellSens Dimension Software (Olympus). Reflection coefficient of albumin (salb) was calculated as the ratio of the V for the experimental glomeruli to the V of control glomeruli in response to identical oncotic gradients: salb=Vexp./ Vcontrol. Glomerular permeability for albumin (Palb) was calculated as Palb=1-salb. Albumin concentration in urine was measured by ELISA. Results: Volume changes of isolated glomeruli occurred within 2 minutes and were maintained for at least 5 minutes in response to changes of oncopressive medium containing 5% bovine serum albumin (BSA) to 1% BSA and next to 5% BSA: V5%=1.08 0.13 nl, V1%=1.220.14 nl and V5%=1.080.13 nl. Palb was 0.100.04. 2-MeSATP (10 min) induced concentration-dependent increase in Palb to 0.480.03 (10-10M), 0.54 0.04 (10-8M) and to 0.800.06 (10-6M) in control glomeruli. Palb in glomeruli isolated from rats exposed to 2-MeSATP were significantly increased 0.350.04 vs. 0.13 0.02. Interestingly, there was no difference in urine albumin excretion before and after exposure to 2-MeSATP 87.524.2 mg/24h vs. 68.19.9 mg/24h. Conclusions: P2-receptors activation increases glomerular permeability to albumin without changes in albumin excretion in urine. Thus, dysfunction of glomeruli precedes changes in albumin excretion in urine and this suggests that microalbuminuria is not a marker of early damage of glomeruli. QUANTITATIVE REAL-TIME PCR ALLOWS miRNA AND mRNA EXPRESSION ANALYSIS OF PARIETAL EPITHELIAL CELLS AND GLOMERULAR CRESCENTS Clemens L. Bockmeyer1, Karen Kokowicz1, Putri A. Agustian2, Stephanie Zell1, Juliane Wittig1 and Jan U. Becker1 1Institute for Pathology Hannover Medical School Hannover Lower Saxony Germany, 2Clinic for Nephrology and Hypertension Hannover Medical School Hannover Lower Saxony Germany Introduction and Aims: Glomerular crescents develop from parietal epithelial cells (ParEpCs). The presence and the extent of crescents correlate with a dismal prognosis. So far, studies on ParepCs were limited because ParEpCs could not be analyzed by quantitative real time-PCR (qRT-PCR) for mRNAs and miRNAs. This proof-of-principle study shows that our improved laser microdissection and RT-PCR techniques can overcome this limitation and enable mRNA and miRNA expression analysis of ParEpCs and the crescents derived thereof. Methods: In a first pilot study we isolated three compartments (i) glomerulus, (ii) ParEpCs and (iii) periglomerular tissue from four paraffin embedded renal biopsies with IgA glomerulonephritis (n=2) and mild tubulointerstitial nephritis with normal glomeruli (n=2). After preamplification we quantified the compartment markers WT1 ( podocytes), PAX-2 (ParEpCs) and CD45 (leukocyte infiltrates) as well as small nuclear RNA reference transcripts (RNU48 and snRNU6) by qRT-PCR. Relative mRNA expression was calculated to the mean expression of three different reference genes (GAPDH, PGK1, POLR2A). Results are given as the mean of the relative expression levels. Results: Mean relative expression levels were as follows: WT1 in glomeruli: 8.5, in ParEpCs: 2.8, WT1 in periglomerular tissue: 0.0. PAX-2 in glomeruli: 0.1, in ParEpCs: 2.3, in periglomerular tissue: 2.7. CD45 in glomeruli: 0.0, in ParEpCs: 0.0, in periglomerular tissue: 0.0. RNU48 and snRNU6 were expressed at a quantifiable level in all three compartments with cTvalues between 16 and 26. Conclusions: Our results show that it is possible to selectively isolate ParEpcs from other kidney compartments for mRNA and miRNA expression studies. The improved laser microdissection and RT PCR techniques allow to study whether crescents and ParEpCs display different mRNA and miRNA profiles. This enables advances in the research on ParEpC pathology and crescent formation not only in patients with Schoenlein Henoch purpura. MODERATE CALORIE RESTRICTION IS EFFECTIVE IN PREVENTING PROGRESSION IN THE AA-4EBP1 RAT MODEL SYSTEM Introduction and Aims: FSGS prevalence is increasing world-wide in association with the world-wide obesity epidemic. Podocyte depletion drives progression in glomerular diseases. Progressive podocyte loss leading to progression and ESKD can also be triggered by growth-dependent glomerular enlargement, as demonstrated using the podocin promoter-driven AA4EBP1 transgenic rat model. In this model system proteinuria, glomerulosclerosis and progression to ESKD is triggered by body growth alone and can be completely prevented by 40% calorie restriction to prevent weight gain and glomerular enlargement (Fukuda et al, JASN, 2012). Methods: We evaluated whether a moderate 20% calorie restricted diet would be as effective as a 40% CR diet in reducing progression. To test this hypothesis we used the 300g male uni-nephrectomized podocin-AA4EBP1 transgenic rats maintained on either an ad-lib regular chow diet (AL), a 20% calorie restriction diet (20%cr), or a 40% calorie restriction diet (40%cr). Morphometry was used to measure glomerular tuft volume, podocyte number per tuft, podocyte density and glomerulosclerosis. Results: On the AL diet rats gained 7330g over 16 weeks of observation. Rats on the 20%cr diet gained 314g and on the 40%cr diet lost 6730g of weight(al vs 20%cr; p<0.01, al vs 40%cr; p<0.01, 20%cr vs 40%cr; p<0.01). 24hr urine protein was +64 24mg in the al, -3.52.6mg in the 20% cr and -2.44.2mg in the 40% CR (al vs 20%cr; p<0.05, al vs 40%cr; p<0.05). The % glomerulosclerosis was 22.910.7 in the AL, 2.2 3.4 in the 20% cr and 1.41.6 in the 40% CR (al vs 20%cr; p<0.05, al vs 40%cr; p<0.05). The glomerular tuft volume (m3) was for AL 1,323,979508,892, 20%cr 1,249,867156,960 and for 40%cr 677,42937,091 (al vs 40%cr p<0.05, 20%cr vs 40%cr p<0.05). The total podocyte volume (m3)(GLEPP1 staining positive volume) was for AL 701,368267,563, for 20%cr 643,09248,627 and for 40% CR 407,33728,553 (ALvs 40%cr; p<0.05, 20%cr vs 40%cr; p<0.05). The podocyte density (m3) (glomerular tuft volume/number of podocytes) was for AL 10,7984,808, for 20% CR 8,7421,151 and for 40% cr 6,0831,259 (AL vs 40%cr; p<0.05, 20%cr vs 40%cr; p<0.05). The GLEEP1 negative (non-podocyte) tuft volume (m3) was for AL 622,611 275,885, for 20% cr 606,775110,610 and for 40% CR 270,09239,722 (20%cr vs 40% cr; p<0.05). Conclusions: Moderate (20%) calorie restriction was not as effective as 40% CR but provided significant protection compared to AL fed rats. These data show that, in this model system, dietary intervention at a moderate level to maintain a stable weight can reduce development of glomerulosclerosis compared with continued ad lib feeding. NGAL AND ANESTHETICS: PROTECTIVE ROLE OF SEVOFLURANE Maria Rosaria Fazio1, Valentina Donato1, Silvia Lucisano1, Valeria Cernaro1, Rosaria Lupica1, Domenico Trimboli1, Gaetano Montalto1, Carmela Aloisi1, Anna Teresa Mazzeo2 and Michele Buemi1 1Department of Internal Medicine, Division of Nephrology University of Messina Messina Italy, 2Department of Anesthesiology University of Messina Messina Italy Introduction and Aims: NGAL (neutrophil gelatinase-associated lipocalin), a protein produced by neutrophils and the proximal renal tubule, is overexpressed in conditions of acute renal suffering, constituting a more sensitive and specific diagnostic marker of creatinine in the diagnosis of AKY. The aim of this study was to analyze serum and urinary NGAL in patients undergoing neurosurgery and sedated with two different i | Abstracts anesthetic procedures in order to assess any property nephrotoxic and/or renal protection by anesthetics. Methods: The study was conducted on 20 patients divided by the anesthetic procedure used: total intravenous anesthesia (type A) and balanced anesthesia (intravenous +inhalation) (type B). The anesthetic drugs used in type A are propofol and remifentanil, in addition to type B was used Sevoflurane. All patients were performed preoperatively (T0), 1 hour (T1), 2 hours (T2) and 24 hours (T3) after the end of surgery, blood samples and urine for the dosage of NGAL and serum creatinine. Results: At time T2 we have found, in both groups, increases in serum and urinary NGAL ( p = 0.002) compared to baseline. In addition, individuals undergoing anesthesia type B have blood levels and urinary NGAL significantly lower than those treated with anesthesia type A ( p <0.0001).The serum creatinine remained unchanged, however, at all times of observation ( p> 0.05). Conclusions: The increased levels of NGAL indicates that exposure to anesthetics results in a renal insult. Moreover, this biomarker may have a prognostic role as the lowest levels were found in group B, or the group treated with sevoflurane, known volatile anesthetic with nefroprotettive property. IMPACT OF THE NEW DERIVATIVES OF POLYISOPRENOID ALCOHOLS - NEW COMPONENTS OF DRUG CARRIERS - ON RENAL MORPHOLOGY AND MORPHOMETRIC ANALYSIS OF MALE SPRAGUE-DAWLEY RATS Olga Gawrys1, Krzysztof H. Olszynski1, Marta Kuczeriszka1, Katarzyna Gawarecka2, Ewa Swiezewska2, Marek Chmielewski3, Marek Masnyk3, Janina Rafaowska4 and Elzbieta Kompanowska-Jezierska1 1Mossakowski Medical Research Centre Polish Academy of Sciences Warsaw Poland, 2Institute of Biochemistry and Biophysics Polish Academy of Sciences Warsaw Poland, 3Institute of Organic Chemistry Polish Academy of Sciences Warsaw Poland, 4Mossakowski Medical Research Centre Polish Academy of Sciences Warsaw Poland Introduction and Aims: In treatment of many diseases serious side effects of drugs are a major problem, which physicians and patients are forced to deal with. Beside searching for new active substances, seeking for substances improving already existing drugs seems to be good strategy. Reducing harmful effects of drugs could be accomplished by providing faster drug passing through the biological membranes, so the active substance would reach faster its destination. New derivatives of polyisoprenoid alcohols, called amino-prenols, were proven to possess such lipofecting properties. In this study we investigate if these compounds do not per se cause untoward effects on the living organism so they can be used as components of liposomal drug carriers. Methods: Male SpragueDawley rats received for four weeks daily, subcutaneous injections (0,5 ml) of liposomes built of dioleoyl phosphatidylethanolamine (DOPE) (L, n=13), liposomes built of DOPE and amino-prenols (ratio 10:1) (L+P, n=13) or water solvent (W, n=12). After four weeks heart and kidneys were harvested for histological and morphometric analysis. Results: During four weeks all animals were characterized by good health and activity. The 4-week increments in body weight were for L:1094, L+P:974 and W:1024 % (inter-group differences not significant). Left ventricular to body weight ratio was the highest in the L group (2.370.08 mg/g) and significantly different from W (2.140.04 mg/g) but not from the L+P group (2.220.03 mg/g). The values for L and L+P groups were not significantly different. Kidney weights expressed as a percent of body weight were nearly identical in all groups (L: 0.430.01; L+P:0.430.01; W: 0.440.01 %). All measured values of urinary albumin excretion (UAE) were in the normal range for Sprague-Dawley rats; only in W group significant increase in UAE after 4 weeks was observed (from 0.230.07 to 0.550.14 mg/day). Renal tissue slices stained with hematoxylin-eosin did not indicate explicit signs of kidney damage in all groups; inter-group differences in the microscopic images of the renal cortex and medulla were not observed. After subcutaneous administration amino-prenols did not affect the function of renal excretory system of normal Sprague-Dawley rats. Conclusions: Morphology and morphometric analysis showed no negative changes in renal structures caused by tested compounds, therefore we find them suitable as a component of liposomal drug carriers. INDOXYL SULFATE IMPAIRS MITOCHONDRIAL BIOGENESIS AND FUNCTION IN HUMAN ENDOTHELIAL CELLS Introduction and Aims: Endothelial dysfunction is the hallmark of the chronic kidney disease (CKD). The uremic toxin, indoxyl sulfate (IS), has recently been reported to induce endothelial dysfunction in patients with CKD. Although some evidence shows that IS causes oxidative stress to endothelium, the mechanisms by which IS causes endothelial dysfunction are yet to be examined. Our study aimed to investigate the impacts of IS on the mitochondrial function and biogenesis in human endothelium. Methods: Human umbilical vein endothelial cells (HUVEC) were cultured with different treatments and were grouped as control, IS (125g/ml), IS+VitC (200M), IS +N-acetylcysteine (NAC) (10mM) and IS+Resveratrol (10M). In these groups, reactive oxygen species (ROS) production was calculated by flow-cytometry, mitochondrial membrane potential was estimated by the mean fluorescence intensity of rhodamine 123 and the mitochondrial DNA content was measured by quantitative real time PCR. Western blots were performed to assay the expression of the key mitochondrial biogenesis regulators, PGC1, Tfam and NRF1. Results: IS caused enhanced ROS production, reduced mitochondrial membrane potential and decreased mitochondrial DNA content in HUVEC cells. These effects were partially rescued by the antioxidants, VitC and NAC and by the known biogenesis stimulator, resveratrol. In response to IS, the expression of PGC1 in HUVEC cells was upregulated whereas those of Tfam and NRF1 remained unchanged. Conclusions: Our results demonstrated that IS causes mitochondrial dysfunction in HUVEC cells by both oxidative stress and impaired mitochondrial biogenesis. We also offered novel insights that resveratrol protects endothelial mitochondria from IS-mediated injuries. THE SIGNIFICANT ROLE OF CURCUMIN IN EPITHELIAL-MESENCHYMAL TRANSITION OF RENAL TUBULAR EPITHELIAL CELLS Introduction and Aims: Epithelial mesenchymal transition (EMT) is an essential process during embryogenesis and organ development, and is characterized by loss of epithelial cell morphology, markers and cellular adhesion, and appearance of mesenchymal properties such as migration and invasion. EMT is also involved in several adult pathologies, especially in cancer and fibrosis. EMT of tubular epithelial cells is known to play a key role in the process of renal fibrosis. In vitro, TGF-b1 can stimulate tubular epithelial cells to undergo myofibroblastic transition, and finally to become fibrogenic cells. Curcumin has been known to have an anti-inflammatory and antifibrotic effect in various organs such as heart, liver and lung, but the exact mechanisms are not clear yet. Moreover, there's insufficient knowledge about the role of curcumin on EMT process. The aim of this study is to reveal the roles of curcumin on renal tubular EMT induced by TGF-b1. Methods: Renal tubular epithelial cell line, HK-2 cells, were examined to assess the effect of curcumin on TGF-b1-induced EMT. Morphological and phenotypic changes were analyzed via confocal microscopy. Additionally, cell lysates were used to estimate epithelial (E-cadherin) or mesenchymal (a-SMA) markers, metalloproteinases, and various interleukines(ILs). Transcription factors, such as snail-1, were also measured by quantitative RT-PCR. Results: Renal tubular epithelial cells showed characteristic features of EMT after TGF-b1 stimulation; loss of cadherin and disruption of cell-cell contact with a concomitant induction of mesenchymal marker, a-SMA. Moreover, both a migratory property and proliferative activity were observed. These changes, however, were attenuated significantly by an administration of curcumin. The expression of transcription factor, snail-1, was also suppressed markedly in a curcumin treated cells. Additionally, curcumin treatment caused a significant decrease of VEGF, IL-6, IL-10, IL-12 and MMP-3 level, but there was no significant change in IL-5, IL-8, IL-13 and MMP-9. Conclusions: This study suggests that curcumin attenuates TGF-b1-induced EMT in renal tubular cells through the downregulation of transcription factor, snail-1 with affecting various ILs and MMPs. Considering that curcumin has been taken safely in a form of food, further study will be focused to evaluate the clinical effects of curcumin in patients with AKI or CKD.

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Rattiyaporn Kanlaya, Kitisak Sintiprungrat, Visith Thongboonkerd, Noelia Torremadé, René Bindels, Joost Hoenderop, Elvira Fernandez, Adriana Dusso, Jose M. Valdivielso, Thilo Krueger, Peter Boor, Cora Schafer, Ralf Westenfeld, Vincent Brandenburg, Georg Schlieper, Willi Jahnen-Dechent, Markus Ketteler, Webster Jee, Xiaodong Li, Bill Richards, Jürgen Floege, Janaína G. Gonçalves, Daniele Canale, Ana Carolina de Bragança, Maria Heloisa M. Shimizu, Rosa Maria A. Moyses, Lucia Andrade, Antonio C. Seguro, Rildo A. Volpini, Simone Romoli, Adriana Migliorini, Hans-Joachim Anders, Olga Eskova, Natalia Neprintseva, Natalia Tchebotareva, Irina Bobkova, Lidiya Kozlovskaya, Ivana Simic, Mansoureh Tabatabaeifar, Tanja Wlodkowski, Helga Denc, Geraldine Mollet, Corinne Antignac, Franz Schaefer, Ivanova A. Ekaterina, Laura Giardino, Maria Pia Rastaldi, Lambertus Van den Heuvel, Elena Levtchenko, Chikako Okina, Tomoko Okamoto, Mariko Kamata, Junya Murano, Kei Kobayashi, Kazuhiro Takeuchi, Fumi Kamata, Takeshi Sakai, Shokichi Naito, Togo Aoyama, Takashi Sano, Yasuo Takeuchi, Kouju Kamata, Dana Thomasova, Hauke A. Bruns, Helen Liapis, Hans-Joachim Anders, Takatsugu Iwashita, Hajime Hasegawa, Kaori Takayanagi, Taisuke Shimizu, Juko Asakura, Shinpei Okazaki, Yuta Kogure, Minoru Hatano, Hiroaki Hara, Megumi Inamura, Mizuki Iwanaga, Tomoyuki Mitani, Tetsuya Mitarai, Virginia J. Savin, Mukut Sharma, Changli Wei, Jochen Reiser, Ellen T. McCarthy, Ram Sharma, Jean-Francois Gauchat, Benedicte Eneman, Kathleen Freson, Lambertus Van den Heuvel, Chris Van Geet, Elena Levtchenko, Dae Eun Choi, Jin Young Jeong, Yoon Kyung Chang, Ki-Ryang Na, Kang Wook Lee, Young Tai Shin, Hai-Feng Ni, Jun-Feng Chen, Ming-Hui Zhang, Ming-Ming Pan, Bi-Cheng Liu, Kang Wook Lee, Jin Young Jeong, Dae Eun Choi, Yoon Kyung Chang, Seong Suk Kim, Ki-Ryang Na, Young Tai Shin, Taihei Suzuki, Masayuki Iyoda, Kei Matsumoto, Yuki Shindo-Hirai, Yoshihiro Kuno, Yukihiro Wada, Yasutaka Yamamoto, Takanori Shibata, Tadao Akizawa, Jose M. Muñoz-Felix, Jose M. Lopez-Novoa, Carlos Martinez-Salgado, Josef Ehling, Janka Bábíčková, Felix Gremse, Fabian Kiessling, Jürgen Floege, Twan Lammers, Peter Boor, Maciej Lech, Roman Günthner, Georg Lorenz, Mi Ryu, Regina Gröbmayr, Heni Susanti, Koichi S. Kobayashi, Richard A. Flavell, Hans-Joachim Anders, Sandra Rayego-Mateos, Jose Morgado, Ana Belen Sanz, Satoru Eguchi, Janos Pato, Gyorgy Keri, Jesũs Egido, Alberto Ortiz, Marta Ruiz-Ortega, Martin Leduc, Lilianne Geerts, Brigitte Grouix, François Sarra-Bournet, Alexandra Felton, Liette Gervais, Shaun Abbott, Jean-Simon Duceppe, Boulos Zacharie, Christopher Penney, Pierre Laurin, Lyne Gagnon, Maria G. Detsika, Pu Duann, Elias A. Lianos, Ka Ian Leong, Chih-Kang Chiang, Ching-Chin Yang, Cheng-Tien Wu, Li-Ping Chen, Kuan-Yu Hung, Shing-Hwa Liu, Fernando F. Carvalho, Vicente P. Teixeira, Waldemar S. Almeida, Nestor Schor, David M. Small, Nigel C. Bennett, Jeff Coombes, David W. Johnson, Glenda C. Gobe, Nuria Montero, Alejandra Prada, Marta Riera, María Orfila, Julio Pascual, Eva Rodríguez, Clara Barrios, Gabor Kokeny, Krisztina Fazekas, Laszlo Rosivall, Miklos M. Mozes, Jose M. Muñoz-Felix, Jose M. Lopez-Novoa, Carlos Martinez-Salgado, Nick Hornigold, Jeremy Hughes, Andrew Mooney, Agnes Benardeau, William Riboulet, Anthony Vandjour, Bjoern Jacobsen, Christian Apfel, Karin Conde-Knape, Brigitte Grouix, Alexandra Felton, François Sarra-Bournet, Martin Leduc, Lilianne Geerts, Liette Gervais, Shaun Abbott, Jean-François Bienvenu, Jean-Simon Duceppe, Boulos Zacharie, Christopher Penney, Pierre Laurin, Lyne Gagnon, Tetsuhiro Tanaka, Junna Yamaguchi, Masaomi Nangaku, Toshimitsu Niwa, Dilinaer Bolati, Hidehisa Shimizu, Maimaiti Yisireyili, Fuyuhiko Nishijima, A. Brocca, G. Virzì, M. de Cal, C. Ronco, Giovanna Priante, Estella Musacchio, Chiara Valvason, Leonardo Sartori, Antonio Piccoli, Bruno Baggio, Peter Boor, Michael Perkuhn, Martin Weibrecht, Stephanie Zok, Ina V. Martin, Felix Schoth, Tammo Ostendorf, Christiane Kuhl, Jürgen Floege, Aigul Karabaeva, Ashot Essaian, Olga Beresneva, Marina Parastaeva, Ivan Kayukov, Alexey Smirnov, Irena Audzeyenka, Malgorzata Kasztan, Agnieszka Piwkowska, Dorota Rogacka, Stefan Angielski, Maciej Jankowski, Clemens L. Bockmeyer, Karen Kokowicz, Putri A. Agustian, Stephanie Zell, Juliane Wittig, Jan U. Becker, Ryuzoh Nishizono, Madhusudan P. Venkatareddy, Marboob A. Chowdhury, Su Q. Wang, Akihiro Fukuda, Larysa T. Wickman, Yan Yang, Roger C. Wiggins, Maria Rosaria Fazio, Valentina Donato, Silvia Lucisano, Valeria Cernaro, Rosaria Lupica, Domenico Trimboli, Gaetano Montalto, Carmela Aloisi, Anna Teresa Mazzeo, Michele Buemi, Olga Gawrys, Krzysztof H. Olszynski, Marta Kuczeriszka, Katarzyna Gawarecka, Ewa Swiezewska, Marek Chmielewski, Marek Masnyk, Janina Rafałowska, Elzbieta Kompanowska-Jezierska, Wen-Chin Lee, You-Ying Chau, Lung-Chih Lee, Chien-Hua Chiu, Chien-Te Lee, Jin-Bor Chen, Woo-Kyung Kim, Sung Joon Shin. Experimental models of CKD, Nephrology Dialysis Transplantation, 2013, i185-i197, DOI: 10.1093/ndt/gft114