Bone & mineral metabolism - II, Urolithiasis (T26-T56)

Poster Presentations T25 ERYTHROPOIETIN GENE EXPRESSION IS UPREGULATED IN ONLY A SUBSET OF HIF ALPHA POSITIVE CLEAR CELL RENAL CARCINOMA M.S. Wiesener, M. Gläser, C. Warnecke, U.A. Frei, H.-J. Gröne, K.-U. Eckardt. Nephrology and Medical Intensive Care, Charité, Humboldt University, Berlin; Cellular and Molecular Pathology, German Cancer Research Centre, Heidelberg Polycythemia is a classical feature of tumor patients with renal cell carcinoma (RCC), yet being rare with less than 5% of reported cases. The reason for polycythemia is most likely due to overproduction of erythropoietin (EPO) in tumor cells, which has been reported in single cases in the past. Transcriptional regulation of EPO occurs through hypoxia inducible factors (HIF), of which the alpha-subunits determine the activity, being rapidly destroyed in the presence of oxygen. The majority of RCC are of the clear cell type, of which most associate with an inactivation of the tumor suppressor gene von Hippel Lindau (VHL), the suspected cause of tumorigenesis. The VHL protein is also responsible for destruction of HIF alpha subunits in normoxia, mediating ubiquitination and proteasomal lysis. We have recently demonstrated, that the majority of RCC overexpress HIF-1alpha and that its abundance correlates with the expression of two target genes, glucose transporter1 and VEGF. To determine the frequency of EPO overexpression, we collected 60 RCC and performed mRNA protection assays, as well as immunoblotting for HIF1 and –2alpha. Expression of HIF and EPO in RCC was observed in 74% and 25% of cases, resp., EPO expression thus being considerably more frequent than clinically observed polycythemia. All of the EPO producing tumors were of the clear cell type and showed HIFalpha overexpression. Thus activity of HIF seems necessary but, in striking contrast to other target genes, not sufficient for EPO induction in renal cancer. Identification of the additional regulatory mechanisms may provide important insights into the regulation of EPO expression. 197 Bone & mineral metabolism – II, Urolithiasis T26 ROLE OF CALCIUM-SENSING RECEPTOR GENE IN CALCIUM AND PHOSPHATE METABOLISM IN END-STAGE RENAL DISEASE Dariusz Moczulski, Marta Pasierb, Ewa Zukowska-Szczechowska, Sylwia Gorczynska-Kosiorz, Wladyslaw Grzeszczak. Department of Internal Medicine, Diabetology and Nephrology, Silesian School of Medicine, Zabrze, Poland The calcium-sensing receptor (CASR) is involved in the calcium and phosphate metabolism. It regulates secretion of parathyroid hormone, calcium reabsorption in kidney and influences the function of osteoblasts and osteoclasts. It is well known that some patients with end stage renal disease (ESRD) are predisposed to the extensive progression of secondary hyperparathyroidism. It has been postulated that the genetic factors might explain this susceptibility. Some reports from Japanese population indicated that the progression of secondary hyperparathyroidism might be predicted by the genetic variants in the CASR gene. Therefore, we decided to examine the association between the polymorphism in the CASR gene and the calcium and phosphate metabolism in ESRD. We collected 87 patients with ESRD treated with heamodialysis. In each patient two frequent polymorphisms in the CASR gene were genotyped: A to G base change in the codon 990 in intracellular domain and T to C base change of intron 4. The genotyping was performed using the PCR-based protocol and digestion of the PCR product with the specific restriction enzyme. The patients were divided into subgroups based on the CASR genotype. When we compared the serum parathyroid hormone, plasma calcium and inorganic phosphate among the subgroups no differences were observed. We also did not find any correlation between the genetic variants and serum parathyroid hormone, plasma calcium and inorganic phosphate, when we adjusted for age, gender, body mass index and duration of renal replacement therapy. In conclusion, our study indicates that the calcium and phosphate metabolism is not associated with the genetic variants in the calcium sensing receptor gene in end stage renal disease. Our findings do not confirm the positive correlation observed in Japanese population. T27 VARIABLE DIALYSATE CALCIUM (DCa++): INFLUENCE ON BONE METABOLISM OF HEMODIALYZED PATIENTS Dimitrios Arvanitis 1 , Aristidis Mantakas 2 , Macroui Sonikian 1 , Aliki Hadjilouka 2 , Dimosthenis Vlassopoulos 1 . Controversy still exists over the adequacy of DCa++ concentrations for the control of sec. hyperparathyroidism. We studied the effect of variable DCa++ concentration in 19 HD pts (iPTH<200pg/ml): group A, 5 parathyroidectomised (PTX 7/8) pts (4M/1F) vs. group B, 14 pts (12M /2F) not PTX. A/B time on HD was 142 (70-174)/49.5 (17-142) months, P:0.001). A and B were dialyzed with high/low Ca++(3.5/2.5/3.5 mEq/l) for 3 subsequent semesters. Before and after each period, we measured: Ca++, phosphates, alc phosphatase (AP)-bone sub fraction (APBF), iPTH, vit. D, osteocalcine (OC). Group A: on low DCa++, iPTH rose (28.9±26.8/78.7±118.8 pg/ml, P:0.04), on high DCa++ it dropped (78.7±118.8/50.2± 52.7 pg/ml, P:0.04). OC changes were similar (68.7±17.2/117± 34.4/57.2±29.2 ng/ml, P:0.04). Group B: on low DCa++ IPTH changes were similar to A (104.9±94.4/173.2±128.1 pg/ml, P: 0.001), but were not inversed on high DCa++ (177.6±157.7 pg/ ml). OC followed iPTH changes (99.8±66.9/163.9±88.2/88.3± 64.9 ng/ml, P<0.03). Vit. D levels dropped high and rose with low DCa++ (25.5±11.0/17.4±7.3/22.1±8.3 ng/ml, P:0.01). APBF rose with low DCa++ (18.6±18.1/ 26.8±20.4 µg/l, P:0.003). Low DCa++ stimulates bone turnover. This is reversible by high DCa++ only in parathyreoidectomised pts. DCa++ controls OC levels in all pts and vitamin D in those with intact parathyroids. PPARγ is a nuclear transcription factor that modulates the growth of epithelial (gastric and colon) cancer cells. As a preliminary step to this study, we investigated by immunohistochemistry whether PPARγ was expressed in renal tumor tissue (5 clear cell adenocarcinomas, 3 papillary carcinomas, 1 sarcomatoid carcinoma and 1 oncocytoma), and we found that all the tested tumors expressed PPARγ in the nucleus and in the cytoplasm. Then, we investigated whether PPARγ inhibits renal tumor cell growth by affecting the expression of MET and the HGFinduced cell proliferation. The expression of MET mRNA was evaluated through quantitative Real Time PCR in a renal carcinoma cell line (Caki 1) in culture, before and after conditioning (24h) with the PPARγ agonist 15d-PGJ2 (1 and 10 µM). The proliferation rate of Caki 1 cells (1x105) stimulated with recombinant HGF (50 ng/ml), with and without 15d-PGJ2 (10µM), was analyzed after 24, 48, 72, 96 hours of culture by the bromodeoxyuridin test. Treatment with 15d-PGJ2 significantly reduced MET mRNA expression in Caki 1 cells (-70% 10µM; -30% 1µM vs MET mRNA in basal conditions; p<0.005). HGF stimulated Caki 1 cell growth (...truncated)