Experimental models of CKD

Nephrology Dialysis Transplantation 28 (Supplement 1): i185–i197, 2013 doi:10.1093/ndt/gft114 EXPERIMENTAL MODELS OF CKD SP338 SECRETED PRODUCTS OF MACROPHAGES EXPOSED TO CALCIUM OXALATE CRYSTALS INDUCE EPITHELIAL MESENCHYMAL TRANSITION OF RENAL TUBULAR CELLS VIA RhoA-DEPENDENT TGF-β1 PATHWAY Rattiyaporn Kanlaya1, Kitisak Sintiprungrat1 and Visith Thongboonkerd1 1 Medical Proteomics Unit, R&D Department Siriraj Hospital, Mahidol University Bangkok Thailand 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. SP339 25-HYDROXYVITAMIND CAN REGULATE MINERAL METABOLISM IN A CKD MODEL OF 1αHYDORXYLASE KO MICE Noelia Torremadé1, René Bindels2, Joost Hoenderop2, Elvira Fernandez1, Adriana Dusso1 and Jose M. Valdivielso1 1 Nephrology Research Department IRBLleida, University Hospital Arnau de Vilanova Lleida Spain, 2Department of Physiology Nijmegen Centre for Molecular Life Sciences Nijmegen The Netherlands 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 1α-hydroxylase (1αKO). Methods: A dose response study was carried out in 10-week-old SNX 1αKO 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 1αKO mice receiving vehicle were both hypocalcemic. 1,25D raised blood Ca2+ levels near normal values (11,15±0,23mg/dl). Serum Ca2+ normalization was achieved with 25 ng/g (9,43±0,39mg/dl) or 50 ng/g (10,63±0,26mg/ dl) of 25D. The highest dose provoked marked hipercalcemia (12.46±0.71 mg/dl). Serum phosphate (P) levels were low in untreated sham 1αKO (ko sham: 5,75±0,56mg/ 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 1α. However, the concentrations of 25D required in blood are extremely high, and may cause toxicity. SP340 THE LONG ACTING CALCIMIMETIC R-641 DOES NOT INDUCE ADYNAMIC BONE DISEASE IN CHRONIC KIDNEY DISEASE Thilo Krueger1, Peter Boor1, Cora Schafer2, Ralf Westenfeld1, Vincent Brandenburg1, Georg Schlieper1, Willi Jahnen-Dechent2, Markus Ketteler3, Webster Jee4, Xiaodong Li5, Bill Richards5 and Jürgen Floege1 1 Nephrology and Clinical Immunology RWTH University Clinic Aachen Aachen Germany, 2Biomedical Engineering RWTH University Aachen Aachen Germany, 3 Nephrology Hospital Coburg Coburg Germany, 4University of Utah Salt Lake City UT United States, 5Amgen Inc. Thousand Oaks CA United States 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 (...truncated)