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• W2070960669 abstract "Calcimimetics increase the sensitivity of the parathyroid calcium-sensing receptor to extracellular calcium for efficient control of hyperparathyroidism. Recent studies suggest that there are beneficial effects of calcimimetics beyond the control of bone and mineral homeostasis. Here, we tested whether the calcium-sensing receptor is also expressed and functionally relevant in podocytes. Analysis of microarray data using Gene Set Enrichment Analysis found that the calcimimetic R-568 influenced various pathways related to oxidative stress, cytoskeletal regulation, cell proliferation, and survival in cultured podocytes. R-568 induced a dose- and time-dependent phosphorylation of the ERK1/2–P90RSK–CREB signaling cascade, and induced pro-survival phosphorylation of BAD and Bcl-xl, thus reducing puromycin aminonucleoside (PAN)-induced podocyte apoptosis by half. Moreover, R-568 preserved the actin cytoskeleton in podocytes exposed to PAN and improved recovery from exposure to cytochalasin D, a reversible inhibitor of actin polymerization. In rats, co-administration of R-568 prevented the proteinuria caused by a single dose of PAN and attenuated the glomerulosclerosis and loss of GFR caused by repetitive puromycin treatment. Hence, calcimimetics limit podocyte damage by antiapoptotic and cytoskeleton-stabilizing effects and may constitute a new approach in the prevention and treatment of glomerular disease. Calcimimetics increase the sensitivity of the parathyroid calcium-sensing receptor to extracellular calcium for efficient control of hyperparathyroidism. Recent studies suggest that there are beneficial effects of calcimimetics beyond the control of bone and mineral homeostasis. Here, we tested whether the calcium-sensing receptor is also expressed and functionally relevant in podocytes. Analysis of microarray data using Gene Set Enrichment Analysis found that the calcimimetic R-568 influenced various pathways related to oxidative stress, cytoskeletal regulation, cell proliferation, and survival in cultured podocytes. R-568 induced a dose- and time-dependent phosphorylation of the ERK1/2–P90RSK–CREB signaling cascade, and induced pro-survival phosphorylation of BAD and Bcl-xl, thus reducing puromycin aminonucleoside (PAN)-induced podocyte apoptosis by half. Moreover, R-568 preserved the actin cytoskeleton in podocytes exposed to PAN and improved recovery from exposure to cytochalasin D, a reversible inhibitor of actin polymerization. In rats, co-administration of R-568 prevented the proteinuria caused by a single dose of PAN and attenuated the glomerulosclerosis and loss of GFR caused by repetitive puromycin treatment. Hence, calcimimetics limit podocyte damage by antiapoptotic and cytoskeleton-stabilizing effects and may constitute a new approach in the prevention and treatment of glomerular disease. The G protein-coupled calcium-sensing receptor (CaSR) is expressed by parathyroid hormone (PTH)-producing cells and by cells lining the kidney tubule. It has an essential role in maintaining calcium homeostasis by sensing alterations of circulating calcium concentrations and coupling the information to pathways that modify PTH secretion or renal cation handling. Mutations in the CaSR gene may cause hypocalciuric hypercalcemia, hypoparathyroidism, or hyperparathyroidism. The CaSR is also involved in the regulation of various cellular functions such as cellular proliferation,1.Molostvov G. Fletcher S. Bland R. et al.Extracellular calcium-sensing receptor mediated signalling is involved in human vascular smooth muscle cell proliferation and apoptosis.Cell Physiol Biochem. 2008; 22: 413-422Crossref PubMed Scopus (46) Google Scholar differentiation,2.Whitfield J.F. The calcium-sensing receptor–a driver of colon cell differentiation.Curr Pharm Biotechnol. 2009; 10: 311-316Crossref PubMed Scopus (19) Google Scholar membrane voltage,3.Huang C. Sindic A. Hill C.E. et al.Interaction of the Ca2+-sensing receptor with the inwardly rectifying potassium channels Kir4.1 and Kir4.2 results in inhibition of channel function.Am J Physiol Renal Physiol. 2007; 292: F1073-F1081Crossref PubMed Scopus (66) Google Scholar and apoptosis.4.Lin K.I. Chattopadhyay N. Bai M. et al.Elevated extracellular calcium can prevent apoptosis via the calcium-sensing receptor.Biochem Biophys Res Commun. 1998; 249: 325-331Crossref PubMed Scopus (87) Google Scholar, 5.Hurtel-Lemaire A.S. Mentaverri R. Caudrillier A. et al.The calcium-sensing receptor is involved in strontium ranelate-induced osteoclast apoptosis. New insights into the associated signaling pathways.J Biol Chem. 2009; 284: 575-584Crossref PubMed Scopus (181) Google Scholar Extracellular signal-regulated kinase, mitogen-activated protein kinases,6.Molostvov G. James S. Fletcher S. et al.Extracellular calcium-sensing receptor is functionally expressed in human artery.Am J Physiol Renal Physiol. 2007; 293: F946-F955Crossref PubMed Scopus (109) Google Scholar, 7.Ogata S. Kubota Y. Satoh S. et al.Ca2+ stimulates COX-2 expression through calcium-sensing receptor in fibroblasts.Biochem Biophys Res Commun. 2006; 351: 808-814Crossref PubMed Scopus (33) Google Scholar intracellular calcium,8.Young S.H. Rozengurt E. Amino acids and Ca2+ stimulate different patterns of Ca2+ oscillations through the Ca2+-sensing receptor.Am J Physiol Cell Physiol. 2002; 282: C1414-C1422Crossref PubMed Scopus (66) Google Scholar phospholipase,5.Hurtel-Lemaire A.S. Mentaverri R. Caudrillier A. et al.The calcium-sensing receptor is involved in strontium ranelate-induced osteoclast apoptosis. New insights into the associated signaling pathways.J Biol Chem. 2009; 284: 575-584Crossref PubMed Scopus (181) Google Scholar, 7.Ogata S. Kubota Y. Satoh S. et al.Ca2+ stimulates COX-2 expression through calcium-sensing receptor in fibroblasts.Biochem Biophys Res Commun. 2006; 351: 808-814Crossref PubMed Scopus (33) Google Scholar and potassium channels9.Vassilev P.M. Kanazirska M.P. Ye C. et al.A flickery block of a K+ channel mediated by extracellular Ca2+ and other agonists of the Ca2+-sensing receptors in dispersed bovine parathyroid cells.Biochem Biophys Res Commun. 1997; 230: 616-623Crossref PubMed Scopus (7) Google Scholar have been reported to mediate the CaSR effect. Furthermore, the CaSR binds to filamin A through its carboxy-terminal domain. Filamin A, an actin cross-linking protein, provides mechanical strength to the actin cytoskeleton.10.Hjalm G. MacLeod R.J. Kifor O. et al.Filamin-A binds to the carboxyl-terminal tail of the calcium-sensing receptor, an interaction that participates in CaR-mediated activation of mitogen-activated protein kinase.J Biol Chem. 2001; 276: 34880-34887Crossref PubMed Scopus (162) Google Scholar, 11.Huang C. Wu Z. Hujer K.M. et al.Silencing of filamin A gene expression inhibits Ca2+ -sensing receptor signaling.FEBS Lett. 2006; 580: 1795-1800Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar In glomerular visceral epithelial cells (podocytes), the actin cytoskeleton is a highly dynamic contractile apparatus critical for the integrity of the cells’ characteristic foot processes. Alterations of the actin cytoskeleton lead to reversible foot-process effacement and proteinuria, followed by podocyte apoptosis and eventual irreversible glomerular sclerosis.12.Faul C. Asanuma K. Yanagida-Asanuma E. et al.Actin upregulation of podocyte structure and function by components of the actin cytoskeleton.Trends Cell Biol. 2007; 17: 428-437Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar Pharmacological activation of the CaSR can be achieved by phenylalkylamine compounds such as R-568 and cinacalcet. These agents allosterically bind to the parathyroid and tubular CaSR, increase its sensitivity to extracellular calcium, and reduce plasma PTH, calcium, and phosphate levels.13.Strippoli G.F. Tong A. Palmer S.C. et al.Calcimimetics for secondary hyperparathyroidism in chronic kidney disease patients.Cochrane Database Syst Rev. 2006; 18: CD006254Google Scholar Cinacalcet is the first calcimimetic approved for treatment of uremic hyperparathyroidism. Recent animal work moreover suggests beneficial effects of calcimimetics beyond control of bone and mineral homeostasis. In subtotally nephrectomized rats, the calcimimetic R-568 markedly reduced proteinuria, blood pressure, glomerulosclerosis, and the progression of renal failure.14.Ogata H. Ritz E. Odoni G. et al.Beneficial effects of calcimimetics on progression of renal failure and cardiovascular risk factors.J Am Soc Nephrol. 2003; 14: 959-967Crossref PubMed Scopus (131) Google Scholar As similar findings were obtained by parathyroidectomy, it was assumed that the renoprotective effect observed with calcimimetics is related to suppression of PTH secretion and a sustained reduction in blood pressure.15.Odenwald T. Nakagawa K. Hadtstein C. et al.Acute blood pressure effects and chronic hypotensive action of calcimimetics in uremic rats.J Am Soc Nephrol. 2006; 17: 655-662Crossref PubMed Scopus (58) Google Scholar Here, we propose a direct nephroprotective action of R-568 at the glomerular podocyte level. We demonstrate that the CaSR is expressed by differentiated murine podocytes and its pharmacological activation induces transcriptional and post-translational pathways promoting cell survival and functional integrity. Consistent with these podocyte protective actions, R-568 is demonstrated to prevent proteinuria, reduce glomerulosclerosis, and conserve global renal function in a model of subacute podocyte damage. The CaSR was expressed both on the RNA and on the protein level in differentiated cultured murine podocytes, but not in cells in the undifferentiated state. Immunocytochemical and immunogold staining of cultured podocytes showed the CaSR to be localized at the endoplasmic reticulum (ER), the periphery of the cytoplasm, and the cell membrane. In addition, there was evidence for vesicular trafficking from the ER to the cell membrane (Figure 1a). Electron microscopy studies with immunogold labeling in mice kidney sections demonstrated localization of the CaSR close to the slit diaphragm of the podocytes (Figure 1b and c). Co-staining of the CaSR and podocin in human renal tissue identified CaSR in human podocytes (Figure 1d). CaSR abundance was similar in murine-cultured podocytes, isolated glomeruli, and whole kidney lysates (112±23%, 127±11%, and 121±11% of β-actin control; Figure 2a). Sucrose gradient ultracentrifugation showed enrichment of CaSR proteins in caveolin-poor membrane fractions (Figure 2b).Figure 2Renal calcium-sensing receptor (CaSR) abundance and localization. CaSR abundance is similar in whole kidney tissue lysate, isolated glomeruli and in murine cultured podocytes (a). Sucrose density gradient centrifugation of cultured mice podocyte, followed by western blotting, demonstrated that the CaSR is not found in the caveolin-1 and 2 enriched membrane fractions (b).View Large Image Figure ViewerDownload (PPT) Microarray experiments and a functional analysis of microarray data using Gene Set Enrichment Analysis (GSEA) were performed to reveal the impact of R-568 on podocytes. R-568 influenced various pathways related to oxidative stress, cytoskeletal regulation, cell proliferation, and survival (Table 1). The gene set ‘response to oxidative stress’ was enriched by R-568. The upregulation of glutamate cysteine ligase catalytic subunit (Gclc) and glutamate cysteine ligase modifier subunit (Gclm), two rate-limiting enzymes for the synthesis of glutathione, implies a potential anti-oxidative effect of R-568. Also, the gene set ‘regulation of cytoskeleton organization and biogenesis’ was enriched in the treated group. A cytoskeleton-stabilizing effect of R-568 is indicated by the upregulation of genes responsible for microtubule stabilization (Mid1ip1, Arhgef2, Apc, Mapt and Clasp1), for actin filament formation (Cdc42ep2 and Arf6), and for stress fiber formation (Arhgef10l, Tsc1 and Nf2). The upregulation of gene set ‘cell cycle arrest GO0007050’ and downregulation of gene set ‘apoptosis_GO’ imply that R-568 enhances cell cycle control and suppresses apoptosis in podocytes.Table 1Cellular pathways regulated by R-568 in podocytesGene setNESNPFDRResponse to oxidative stress1.6570.0020.045Regulation of cytoskeleton organization and biogenesis1.6350.00980.049Apoptosis_GO−1.512<0.00010.099Cell cycle arrest GO00070501.4390.05410.122Activation of JNK pathway1.7990.0040.023Regulation of MAPKKK cascade1.7430.00810.033Abbreviations: FDR, false discovery rate; MAPKKK, kinase of the kinase of the mitogen-activated protein kinase; NES, normalized enrichment score, a positive NES value indicates enrichment in the R-568-treated group; NP, nominal P value. Open table in a new tab Abbreviations: FDR, false discovery rate; MAPKKK, kinase of the kinase of the mitogen-activated protein kinase; NES, normalized enrichment score, a positive NES value indicates enrichment in the R-568-treated group; NP, nominal P value. GSEA suggests that the MAPK pathway mediates the effect of R-568 on podocytes (Table 1). We therefore examined the response of different MAPK members to R-568 and their impact on prosurvival genes. ERK1/2 was phosphorylated in response to R-568 exposure in a dose- (4–50 nmol/l) and time-dependent manner (Figure 3a and b). Increasing the concentration above 10 nmol/l did not lead to a further activation of ERK1/2. Maximal phosphorylation was obtained after 10 min of R-568 and returned to baseline within 60 min. The p38MAPK showed a biphasic response pattern with phosphorylation after 5 min and again after 30 min of R-568 exposure, while the third member of the MAPK family, JNK, showed no response at any time point (data not shown). The downstream kinase 90RSK was phosphorylated in response to R-568 (10 nmol/l) in a similar time pattern as ERK1/2 and led to phosphorylation of the transcriptional factor cAMP response element binding protein (CREB; Figure 3c), a well-established activator of prosurvival genes. R-568 (10 nmol/l) increased the amount of pBAD in a time-dependent manner; maximal phosphorylation occurred within 6–12 h. Bcl-xl was phosphorylated in a similar, time-dependent manner by R-568 (Figure 4). The specific MEK1/2 inhibitor (UO126, 10 μmol/l), a kinase upstream of ERK1/2, prevented the R-568-induced phosphorylation of ERK1/2 and CREB (Figure 5). Intracellular cAMP was dose- and time-dependently reduced by R-568 (10 nmol/l, 5 min: 80±16%; P=NS, 10 min 35±13%, P<0.05; 100 nmol/l, 5 min: 35±4%, P<0.05; 10 min: 34±3%, P<0,05 as compared with untreated control). The activity of the Rho kinases RhoA and Rac1 was increased by R-568 (+44±1% and +127±10%, compared with untreated control, both P<0.05).Figure 5R-568-dependent phosphorylation of ERK1/2 and cAMP response element binding protein (CREB) is MEK-dependent. Murine podocytes were treated with R-568 (10 nmol/l; R) for the indicated time intervals in the presence and absence of the MEK 1/2 inhibitor U0126 (10 mmol/l; I), indices indicate the treatment time in minutes. C, control.View Large Image Figure ViewerDownload (PPT) Fluorescence-activated cell sorting analyses were performed to prove that R-568 protects podocytes from stress-induced apoptosis. PAN (30 μg/ml) increased apoptosis in cultured podocytes from a baseline rate of 20% to 46±5 and 74±9% after 48 and 60 h of incubation, respectively (Figure 6). Addition of R-568 (10 nmol/l) reduced the PAN-induced increase in apoptosis rate by 57±3 and 50±5% after 48 and 60 h, respectively (both P<0.05). Incubation of podocytes with PAN (30 μg/ml) for 72 h resulted in cytoplasmatic shrinking, increased apoptosis, and a partial disruption of actin filaments. Actin was localized peripherally; focal contacts were diminished; and the cell size and the actin content per cell were reduced (Figure 7, Table 2). Administration of R-568 (10 nmol/l) increased cell size, actin density, and actin content per cell. Co-incubation with PAN and R-568 completely prevented cell shrinking, actin depletion, and partially preserved cytoskeletal structure. In podocytes treated with ERK1/2, p38, and JNK inhibitors, the actin cytoskeleton appeared unaltered. The protective action of R-568 against PAN was preserved. In contrast, in podocytes treated with the rho-kinase inhibitor H1152, the actin cytoskeleton was largely degraded and R-568 co-administration lost its protective effect against PAN. Exposure of podocytes to the protein kinase A inhibitor H89 partially inhibited actin cytoskeleton formation and the protective action of R-568 (Figure 7).Table 2Cell size and actin content in podocytesControlPANR-568PAN+R-568Mean cell surface (μm2)3043±2211497±179aP<0.05 vs control.4582±438aP<0.05 vs control.,bP<0.05 vs PAN.2739±317bP<0.05 vs PAN.Mean actin per μm2 (AU)949±331110±35aP<0.05 vs control.1102±49aP<0.05 vs control.1126±46aP<0.05 vs control.Mean actin per cell (AU × 103)2537±2311484±168aP<0.05 vs control.4769±571aP<0.05 vs control.,bP<0.05 vs PAN.2585±258bP<0.05 vs PAN.Abbreviations: AU, arbitrary units; PAN, puromycin aminonucleoside.Podocytes were treated with puromycin (30 μg/ml) and R-568 (10 nmol/l) for 72 h. See Materials and Methods for details.a P<0.05 vs control.b P<0.05 vs PAN. Open table in a new tab Abbreviations: AU, arbitrary units; PAN, puromycin aminonucleoside. Podocytes were treated with puromycin (30 μg/ml) and R-568 (10 nmol/l) for 72 h. See Materials and Methods for details. Cytochalasin D exposure (2 μmol/l) for 48 h caused cell retraction and disruption of podocyte actin filaments; co-incubation with R-568 prevented cytoskeletal damage (data not shown). To investigate whether R-568 also improves actin skeleton reassembly, we treated cytochalasin D preincubated podocytes with R-568. Addition of R-568 after 48 h of incubation with cytochalasin D significantly accelerated actin recovery compared with cytochalasin D-pretreated podocytes that were incubated only with medium after cytochalasin exposure (Figure 8, Table 3). The actin cytoskeleton was largely restored in both groups after 72 h.Table 3Recovery of podocyte actin skeleton from CD treatmentControl (48 h)CD (48 h)24 h after CD24 h after CD+R-56848 h after CD48 h after CD +R-568Mean cell surface (μm2)2891±2901265±108aP<0.05 vs control.1498±105aP<0.05 vs control.4704±329aP<0.05 vs control.,bP<0.05 vs cytochalasin D.,cP<0.05 vs 24-h recovery.3409±228bP<0.05 vs cytochalasin D.,cP<0.05 vs 24-h recovery.,dP<0.05 vs 24-h recovery + R-568.3080±337bP<0.05 vs cytochalasin D.,cP<0.05 vs 24-h recovery.,dP<0.05 vs 24-h recovery + R-568.Mean actin per μm2 (AU)928±38224±14aP<0.05 vs control.311±22aP<0.05 vs control.,bP<0.05 vs cytochalasin D.593±19aP<0.05 vs control.,bP<0.05 vs cytochalasin D.,cP<0.05 vs 24-h recovery.795±36aP<0.05 vs control.,bP<0.05 vs cytochalasin D.,cP<0.05 vs 24-h recovery.,dP<0.05 vs 24-h recovery + R-568.1141±51aP<0.05 vs control.,bP<0.05 vs cytochalasin D.,cP<0.05 vs 24-h recovery.,dP<0.05 vs 24-h recovery + R-568.,eP<0.05 vs 48-h recovery.Mean actin per cell (AU × 103)2688±328284±28aP<0.05 vs control.469±44a,b2813±218b,c2697±202b,c3708±543b,cAbbreviations: AU, arbitrary units; CD, cytochalasin D.After treatment with the polymerization inhibitor cytochalasin D (2 μmol/l) for 48 h, podocytes were incubated with medium and with medium containing R-568 (10 nmol/l). The actin cytoskeleton recovered faster in podocytes post-incubated with R-568 than in podocytes treated with medium alone.a P<0.05 vs control.b P<0.05 vs cytochalasin D.c P<0.05 vs 24-h recovery.d P<0.05 vs 24-h recovery + R-568.e P<0.05 vs 48-h recovery. Open table in a new tab Abbreviations: AU, arbitrary units; CD, cytochalasin D. After treatment with the polymerization inhibitor cytochalasin D (2 μmol/l) for 48 h, podocytes were incubated with medium and with medium containing R-568 (10 nmol/l). The actin cytoskeleton recovered faster in podocytes post-incubated with R-568 than in podocytes treated with medium alone. A single PAN injection induced severe proteinuria within 6 days (3.426±352 vs 73±3.7 g/mol creatinine in solvent-treated controls, P<0.0001). Pre-administration of R-568 prevented the development of proteinuria (339±116 g/mol creatinine, P<0.0001 vs PAN alone). The protective effect persisted through day 9 (Figure 9); proteinuria disappeared within 24 days. Partial prevention of proteinuria was achieved when R-568 was given 2 and 4 days after PAN injection (1.457±339 and 1.486±75 g/mol creatinine vs 4.474±303 g/mol creatinine without R-568 rescue; P<0.05 vs PAN alone). In a subsequent experiment, Sprague–Dawley rats underwent three sequential PAN injections at 4-week intervals, with or without concomitant injections of R-568. Nine days after the third PAN injection four PAN-treated animals had died, whereas all animals in the other groups survived. The surviving animals displayed reduced weight gain, proteinuria, hypoalbuminemia, hypercholesterolemia, and a markedly reduced GFR (Table 4). Co-administration of R-568 significantly lowered PAN-induced proteinuria and GFR loss, improved weight gain, and tended to attenuate hypoalbuminemia.Table 4R-568 mitigates puromycin aminonucleoside (PAN)-induced nephrotic syndrome and GFR lossBody weight gain (g)S-Ca (mmol/l)S-albumin (g/l)S-creatinine (mg/dl)S-cholesterol (mg/dl)Creatinine clearance (ml/h)U-protein (g/mol Cr)S-PTH (pg/ml)Solvent343±82.3±0.136±1.50.28±0.0149±4211±22102±7168±33R-568348±1372.42±0.0438±1.20.32±0.0153±4203±3876±4183±66PAN242±11aP<0.05 vs solvent.,bP<0.05 vs R-568.2.35±0.0726±1.0aP<0.05 vs solvent.,bP<0.05 vs R-568.0.75±0.16aP<0.05 vs solvent.,bP<0.05 vs R-568.560±85aP<0.05 vs solvent.,bP<0.05 vs R-568.29±7aP<0.05 vs solvent.,bP<0.05 vs R-568.5719±596aP<0.05 vs solvent.,bP<0.05 vs R-568.1740±534aP<0.05 vs solvent.,bP<0.05 vs R-568.PAN+R-568291±16aP<0.05 vs solvent.,bP<0.05 vs R-568.,cP<0.05 vs PAN.2.52±0.0730±2.4bP<0.05 vs R-568.0.50±0.06aP<0.05 vs solvent.,bP<0.05 vs R-568.323±58aP<0.05 vs solvent.,bP<0.05 vs R-568.,cP<0.05 vs PAN.112±24cP<0.05 vs PAN.3247±760aP<0.05 vs solvent.,bP<0.05 vs R-568.,cP<0.05 vs PAN.1173±880aP<0.05 vs solvent.,bP<0.05 vs R-568.Abbreviations: GFR, glomerular filtration rate; PTH, parathyroid hormone; S-PTH, serum parathyroid hormone.Serum and urine biochemistry in rats receiving repeated injections of solvent, R-568, PAN or both, R-568 and PAN. See Materials and Methods for details.a P<0.05 vs solvent.b P<0.05 vs R-568.c P<0.05 vs PAN. Open table in a new tab Abbreviations: GFR, glomerular filtration rate; PTH, parathyroid hormone; S-PTH, serum parathyroid hormone. Serum and urine biochemistry in rats receiving repeated injections of solvent, R-568, PAN or both, R-568 and PAN. See Materials and Methods for details. Histomorphometric assessment demonstrated significantly fewer FSGS lesions in animals with PAN/R-568 co-administration (glomerulosclerosis index 1.50±0.2 vs 2.47±0.4 with PAN alone, P<0.05). The glomerular podocyte number, visualized by Wilms tumor-1 staining, was significantly reduced in PAN-treated rats (number of podocytes per glomerulus section 4.55±0.64) as compared with controls (7.9±0.62, P=0.01), but conserved in PAN and R-568 co-treated rats (7.61±0.91, P=0.05; vs PAN alone, Figure 10). R-568 treatment alone had no effect on podocyte number (7.07±1.16, NS vs control). Ten days after R-568 treatment, glomerular pBAD abundance was not different between groups (PAN: 128±32% and PAN +R-568: 140±33% as compared with untreated rats, all P=NS). Phalloidin staining indicated increased glomerular actin content with R-568 treatment. Glomerular and tubular CaSR abundance was not influenced by R-568 treatment, but was significantly increased in PAN-treated rats (R-568: 104±7%, PAN: 255±37%, PAN +R-568: 253±26% vs CaSR/β-actin ratio in solvent-treated controls, all P<0.05; (Figure 11a and b).Figure 11Calcium-sensing receptor (CaSR) expression in rat kidney. Rats received three intraperitoneal injections of R-568 (20 mg/kg body weight) or puromycin aminonucleoside (PAN, 150 mg/kg) or PAN and R-568 at monthly intervals. Immunohistochemistry (a) and western immunoblotting (b) demonstrate increased glomerular and tubular CaSR expression in response to PAN.View Large Image Figure ViewerDownload (PPT) The growing prevalence of progressive chronic kidney disease, a condition associated with high morbidity, excessive mortality, and enormous costs to health systems worldwide, creates a need for effective preventive strategies. To date, concepts of pharmacological nephroprotection are largely limited to tight blood pressure control and antiproteinuric intervention using antagonists of the renin–angiotensin system.16.Hirsch S. Preventing end-stage renal disease: flexible strategies to overcome obstacles.Curr Opin Nephrol Hypertens. 2006; 15: 473-480PubMed Google Scholar In numerous kidney disorders such as diabetic nephropathy, the most common cause of chronic renal failure in adults, a central pathophysiological role is attributed to the podocyte. Podocyte dysfunction and loss lead to glomerular proteinuria, a marker and key mediator of progressive renal damage. We set out to investigate the hypothesis that calcimimetic compounds, in addition to their PTH-suppressing and their recently detected blood pressure-lowering effect,15.Odenwald T. Nakagawa K. Hadtstein C. et al.Acute blood pressure effects and chronic hypotensive action of calcimimetics in uremic rats.J Am Soc Nephrol. 2006; 17: 655-662Crossref PubMed Scopus (58) Google Scholar might exert direct podocyte protective actions. A biological basis for this hypothesis was provided by the detection of CaSR expression in rat glomeruli17.Piecha G. Kokeny G. Nakagawa K. et al.Calcimimetic R-568 or calcitriol: equally beneficial on progression of renal damage in subtotally nephrectomized rats.Am J Physiol Renal Physiol. 2008; 294: F748-F757Crossref PubMed Scopus (46) Google Scholar and, as demonstrated here, in murine and human podocytes. The protein is found both in the cytoplasm and at the cell membrane of foot processes in the immediate vicinity of the slit diaphragm. In podocytes, membrane-bound CaSR does not seem to be enriched in ‘lipid raft’ domains. Localization to these multifunctional membrane regions has been reported in parathyroid and osteoblast-like cells, but not in endothelial cells.18.Jung S.Y. Kwak J.O. Kim H.W. et al.Calcium sensing receptor forms complex with and is up-regulated by caveolin-1 in cultured human osteosarcoma (Saos-2) cells.Exp Mol Med. 2005; 37: 91-100Crossref PubMed Scopus (41) Google Scholar, 19.Weston A.H. Absi M. Ward D.T. et al.Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: studies with calindol and Calhex 231.Circ Res. 2005; 97: 391-398Crossref PubMed Scopus (124) Google Scholar, 20.Kifor O. Diaz R. Butters R. et al.The calcium-sensing receptor is localized in caveolin-rich plasma membrane domains of bovine parathyroid cells.J Biol Chem. 1998; 273: 21708-21713Crossref PubMed Scopus (119) Google Scholar Allosteric binding of R-568 to the CaSR activates several specific intracellular signaling cascades and results in an array of cellular responses, such as reduced parathyroid hormone secretion from the parathyroid glands and calcium reabsorption in the kidney. Less attention has thus far been given to other principal cell functions such as proliferation, differentiation, and apoptosis.1.Molostvov G. Fletcher S. Bland R. et al.Extracellular calcium-sensing receptor mediated signalling is involved in human vascular smooth muscle cell proliferation and apoptosis.Cell Physiol Biochem. 2008; 22: 413-422Crossref PubMed Scopus (46) Google Scholar, 2.Whitfield J.F. The calcium-sensing receptor–a driver of colon cell differentiation.Curr Pharm Biotechnol. 2009; 10: 311-316Crossref PubMed Scopus (19) Google Scholar, 3.Huang C. Sindic A. Hill C.E. et al.Interaction of the Ca2+-sensing receptor with the inwardly rectifying potassium channels Kir4.1 and Kir4.2 results in inhibition of channel function.Am J Physiol Renal Physiol. 2007; 292: F1073-F1081Crossref PubMed Scopus (66) Google Scholar, 4.Lin K.I. Chattopadhyay N. Bai M. et al.Elevated extracellular calcium can prevent apoptosis via the calcium-sensing receptor.Biochem Biophys Res Commun. 1998; 249: 325-331Crossref PubMed Scopus (87) Google Scholar, 5.Hurtel-Lemaire A.S. Mentaverri R. Caudrillier A. et al.The calcium-sensing receptor is involved in strontium ranelate-induced osteoclast apoptosis. New insights into the associated signaling pathways.J Biol Chem. 2009; 284: 575-584Crossref PubMed Scopus (181) Google Scholar In the kidney, podocyte loss is a critical determinant underlying the development of glomerulosclerosis and progressive renal failure. PAN exposure is an established model applicable in vivo and in vitro to produce podocyte damage. PAN causes oxidative stress and accelerates proliferation and apoptosis of podocytes, which precedes glomerulosclerosis.21.D’Agati V.D. Podocyte injury in focal segmental glomerulosclerosis: lessons from animal models (a play in five acts).Kidney Int. 2008; 73: 399-406Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar Our microarray data suggest a nephroprotective effect of R-568 opposite to PAN, as R-568 induces antioxidative mechanisms, enhances cell cycle control, and suppresses apoptosis in podocyte cell culture. The antiapoptotic effect of R-568 is supported by its activation of pro-survival signaling, such" @default.
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• W2070960669 date "2011-09-01" @default.
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• W2070960669 title "Stimulation of the calcium-sensing receptor stabilizes the podocyte cytoskeleton, improves cell survival, and reduces toxin-induced glomerulosclerosis" @default.
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