Calcium channel inhibition accelerates polycystic kidney disease progression in the Cy/+ rat

In polycystic kidney disease, abnormal epithelial cell proliferation is the main factor leading to cyst formation and kidney enlargement. Cyclic AMP (cAMP) is mitogenic in cystic but antimitogenic in normal human kidney cells, which is due to reduced steady-state intracellular calcium levels in cystic compared to the normal cells. Inhibition of intracellular calcium entry with channel blockers, such as verapamil, induced cAMP-dependent cell proliferation in normal renal cells. To determine if calcium channel blockers have a similar effect on cell proliferation in vivo, Cy/+ rats, a model of dominant polycystic kidney disease, were treated with verapamil. Kidney weight and cyst index were elevated in verapamil-treated Cy/+ rats. This was associated with increased cell proliferation and apoptosis, elevated expression, and phosphorylation of B-Raf with stimulation of the mitogen-activated protein kinase MEK/ERK (mitogen-activated protein kinase kinase/extracellular-regulated kinase) pathway. Verapamil had no effect on kidney morphology or B-Raf stimulation in wild-type rats. We conclude that treatment of Cy/+ rats with calcium channel blockers increases activity of the B-Raf/MEK/ERK pathway accelerating cyst growth in the presence of endogenous cAMP, thus exacerbating renal cystic disease. In polycystic kidney disease, abnormal epithelial cell proliferation is the main factor leading to cyst formation and kidney enlargement. Cyclic AMP (cAMP) is mitogenic in cystic but antimitogenic in normal human kidney cells, which is due to reduced steady-state intracellular calcium levels in cystic compared to the normal cells. Inhibition of intracellular calcium entry with channel blockers, such as verapamil, induced cAMP-dependent cell proliferation in normal renal cells. To determine if calcium channel blockers have a similar effect on cell proliferation in vivo, Cy/+ rats, a model of dominant polycystic kidney disease, were treated with verapamil. Kidney weight and cyst index were elevated in verapamil-treated Cy/+ rats. This was associated with increased cell proliferation and apoptosis, elevated expression, and phosphorylation of B-Raf with stimulation of the mitogen-activated protein kinase MEK/ERK (mitogen-activated protein kinase kinase/extracellular-regulated kinase) pathway. Verapamil had no effect on kidney morphology or B-Raf stimulation in wild-type rats. We conclude that treatment of Cy/+ rats with calcium channel blockers increases activity of the B-Raf/MEK/ERK pathway accelerating cyst growth in the presence of endogenous cAMP, thus exacerbating renal cystic disease. Autosomal-dominant polycystic kidney disease (ADPKD) is a common hereditary renal disorder caused by mutations in PKD1 or PKD2.1.Boletta A. Germino G.G. Role of polycystins in renal tubulogenesis.Trends Cell Biol. 2003; 13: 484-492Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 2.Arnaout M.A. Molecular genetics and pathogenesis of autosomal dominant polycystic kidney disease.Annu Rev Med. 2001; 52: 93-123Crossref PubMed Scopus (85) Google Scholar, 3.Igarashi P. Somlo S. Genetics and pathogenesis of polycystic kidney disease.J Am Soc Nephrol. 2002; 13: 2384-2398Crossref PubMed Scopus (422) Google Scholar, 4.Mochizuki T. Wu G. Hayashi T. et al.PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1103) Google Scholar, 5.The European Polycystic Kidney Disease Consortium. The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16.Cell. 1994; 78: 725Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 6.Burn T.C. Connors T.D. Dackowski W.R. et al.Analysis of the genomic sequence for the autosomal dominant polycystic kidney disease (PKD1) gene predicts the presence of a leucine-rich repeat. The American PKD1 Consortium (APKD1 Consortium).Hum Mol Genet. 1995; 4: 575-582Crossref PubMed Scopus (233) Google Scholar Aberrant growth of renal epithelial cells leads to the formation of fluid-filled cysts that destroy surrounding tissue and disrupt renal function. Polycystin-1 (PC-1) and polycystin-2 (PC-2), the products of PKD1 and PKD2, respectively, are multifunctional proteins implicated in the regulation of cell proliferation via multiple signaling pathways. PC-1 and PC-2 form a Ca2+-permeable cation channel7.Hanaoka K. Qian F. 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The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia.J Am Soc Nephrol. 2002; 13: 2508-2516Crossref PubMed Scopus (689) Google Scholar It remains unclear how functional loss of PC-1 or PC-2 disrupts intracellular Ca2+ regulation and gives rise to an abnormal cell proliferation phenotype. Recent evidence has shown that epithelial cells isolated from human ADPKD cysts have an aberrant intracellular Ca2+ response to ciliary sensing of fluid flow12.Nauli S.M. Rossetti S. Kolb R.J. et al.Loss of polycystin-1 in human cyst-lining epithelia leads to ciliary dysfunction.J Am Soc Nephrol. 2006; 17: 1015-1025Crossref PubMed Scopus (135) Google Scholar and reduced intracellular Ca2+ levels compared to normal renal cells.13.Yamaguchi T. Hempson S.J. Reif G.A. et al.Calcium restores a normal proliferation phenotype in human polycystic kidney disease epithelial cells.J Am Soc Nephrol. 2006; 17: 178-187Crossref PubMed Scopus (195) Google Scholar Thus, renal cell hyperplasia in ADPKD may be a result of dysfunctional intracellular Ca2+ metabolism. Cyclic AMP (cAMP) agonists, including arginine vasopressin, accelerate the proliferation of cyst-derived cells through activation protein kinase A and subsequent stimulation of the B-Raf/mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK/ERK) pathway.13.Yamaguchi T. Hempson S.J. Reif G.A. et al.Calcium restores a normal proliferation phenotype in human polycystic kidney disease epithelial cells.J Am Soc Nephrol. 2006; 17: 178-187Crossref PubMed Scopus (195) Google Scholar, 14.Yamaguchi T. Nagao S. 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Qian Q. et al.Effective treatment of an orthologous model of autosomal dominant polycystic kidney disease.Nat Med. 2004; 10: 363-364Crossref PubMed Scopus (358) Google Scholar, 19.Wang X. Gattone II, V. Harris P.C. et al.Effectiveness of vasopressin V2 receptor antagonists OPC-31260 and OPC-41061 on polycystic kidney disease development in the PCK rat.J Am Soc Nephrol. 2005; 16: 846-851Crossref PubMed Scopus (240) Google Scholar By contrast, cAMP does not activate ERK or proliferation of normal renal cells. This phenotypic difference in the proliferative response to cAMP appears to be secondary to differences in basal intracellular Ca2+ levels. Sustained reduction of intracellular Ca2+ with Ca2+ channel blockers (CCBs), including verapamil and nifedipine (L-type CCBs), predisposed cells cultured from normal human kidneys to cAMP-dependent activation of the B-Raf/MEK/ERK pathway and increased cell proliferation, mimicking the ADPKD cell phenotype.15.Yamaguchi T. Wallace D.P. Magenheimer B.S. et al.Calcium restriction allows cAMP activation of the B-Raf/ERK pathway, switching cells to a cAMP-dependent growth-stimulated phenotype.J Biol Chem. 2004; 279: 40419-40430Crossref PubMed Scopus (256) Google Scholar Treatment of ADPKD cells with Ca2+ entry blockers amplified cAMP-dependent ERK activation and proliferation, suggesting that further reduction in intracellular Ca2+ may accelerate cyst growth.13.Yamaguchi T. Hempson S.J. Reif G.A. et al.Calcium restores a normal proliferation phenotype in human polycystic kidney disease epithelial cells.J Am Soc Nephrol. 2006; 17: 178-187Crossref PubMed Scopus (195) Google Scholar To investigate the effect of CCBs on growth of renal cysts, verapamil was administered twice daily for 7 weeks to Han:SPRD Cy/+ rats. This classical rodent model of polycystic kidney disease (PKD) transmits the disease in an autosomal-dominant pattern and thus resembles human ADPKD.20.Kranzlin B. Schieren G. Gretz N. Azotemia and extrarenal manifestations in old female Han:SPRD (cy/+) rats.Kidney Int. 1997; 51: 1160-1169Abstract Full Text PDF PubMed Scopus (18) Google Scholar This model has been used to investigate factors that modulate the progression of renal cystic disease.21.Cowley Jr, B.D. Gudapaty S. Kraybill A.L. et al.Autosomal-dominant polycystic kidney disease in the rat.Kidney Int. 1993; 43: 522-534Abstract Full Text PDF PubMed Scopus (196) Google Scholar Previously, Cowley et al.22.Cowley Jr, B.D. Grantham J.J. Muessel M.J. et al.Modification of disease progression in rats with inherited polycystic kidney disease.Am J Kidney Dis. 1996; 27: 865-879Abstract Full Text PDF PubMed Scopus (45) Google Scholar showed that cyst progression could be accelerated by growth-promoting stimuli, including NH4Cl, K+ restriction, and increased dietary protein. An advantage of this model is the sexual dimorphism in the progression of Cy/+ renal disease. Male rats have a more rapid disease progression, whereas the disease in females is relatively mild. Thus, drug treatment can be investigated in mild and severe forms of PKD using the same animal model. In this study, we determined if verapamil, a CCB that reduces Ca2+ in cultured renal cells, enhances cell proliferation and cyst enlargement in Cy/+ rats. Cy/+ and +/+ female and male rats were treated with verapamil from age 5 to 12 weeks, and renal morphology, cellular proliferation and apoptosis, and renal activity of the B-Raf/MEK/ERK pathway were determined. Blood pressure (BP) of 12-week-old Cy/+ rats was significantly higher than that of +/+ rats (174±6 vs 152±4 mm Hg, P<0.001) (Figure 1). Treatment with 20 mg kg−1 verapamil twice daily decreased BP of the Cy/+ rats to 142±9 mm Hg (P<0.001, compared to control-treated Cy/+ rats). This demonstrates that verapamil dosing with this concentration was sufficient to normalize BP in Cy/+ rats without inducing hypotension in +/+ animals (144±3 mm Hg). At 12 weeks, serum urea nitrogen (SUN) was relatively normal in control-treated female Cy/+ rats compared to control-treated +/+ rats (Figure 2). Verapamil treatment for 7 weeks increased SUN in female Cy/+ rats from 32.4±1.8 to 42.6±3.8 mg per 100 ml (P<0.01). This effect of verapamil on SUN was less pronounced in male Cy/+ rats on a percentage basis, which already have elevated SUN levels above 80 mg per 100 ml, indicating an advanced stage of renal insufficiency at 12 weeks. As such, SUN levels were higher in Cy/+ males following verapamil treatment (83.6±5.6 mg per 100 ml for the control-treated vs 98.1±8.2 mg per 100 ml for verapamil-treated male rats). Verapamil had no effect on SUN levels in male or female +/+ rats. Body weight of sex-matched Cy/+ and +/+ littermates did not differ and was unaffected by verapamil treatment (Figure 3). By contrast, kidney weight (% body weight) was 2- and 2.9-fold greater in the Cy/+ female and male rats, respectively, compared to +/+ rats. Verapamil increased kidney weight 35% in the Cy/+ females and 50% in Cy/+ males compared to control-treated Cy/+ rats. Measurement of the cystic area in representative histological kidney sections revealed that verapamil increased cystic surface area from 13.0±1.0 to 31.7±0.7% of total area (P<0.001) in female kidneys and from 29.7±1.9 to 52.0±3.5% (P<0.001) in Cy/+ male kidneys (Figure 4). Importantly, verapamil treatment failed to induce renal cyst formation in normal rats (data not shown) and had no effect on kidney weight (Figure 3).Figure 4Effect of verapamil (VP) on renal cyst development in female and male Cy/+ rats. Representative kidney sections obtained from (a) female or (b) male Cy/+ rats treated with either CONT or VP. (c) Cross-sectional surface area of cysts, represented as % of total area (mean±s.e.), from representative sections of Cy/+ kidneys was measured by morphometric analysis. Comparisons between CONT and VP showed that VP increased cyst area 140% in the female kidneys and 75% in Cy/+ male kidneys, *P<0.001.View Large Image Figure ViewerDownload (PPT) Intracellular cAMP is a central mitogenic factor for the progression of renal disease. We found that urinary cAMP levels were fourfold higher in the Cy/+ male rats than in normal rats (1540 vs 6289 pmol per 24 h, n=2 per group), suggesting that renal cAMP production was elevated in Cy/+ kidneys consistent with several other rodent models of PKD.17.Gattone II, V.H. Wang X. Harris P.C. et al.Inhibition of renal cystic disease development and progression by a vasopressin V2 receptor antagonist.Nat Med. 2003; 9: 1323-1326Crossref PubMed Scopus (483) Google Scholar, 18.Torres V.E. Wang X. Qian Q. et al.Effective treatment of an orthologous model of autosomal dominant polycystic kidney disease.Nat Med. 2004; 10: 363-364Crossref PubMed Scopus (358) Google Scholar, 23.Yamaguchi T. Nagao S. Kasahara M. et al.Renal accumulation and excretion of cyclic adenosine monophosphate in a murine model of slowly progressive polycystic kidney disease.Am J Kidney Dis. 1997; 30: 703-709Abstract Full Text PDF PubMed Scopus (103) Google Scholar Immunohistochemical analysis revealed that control-treated Cy/+ kidneys had significantly higher levels of proliferating cell nuclear antigen (PCNA)-positive cells than control-treated +/+ kidneys (Figure 5). Verapamil increased the number of PCNA-positive cells in female Cy/+ kidneys from 20.6±4.2 to 39.3±1.2% (P<0.01) and in male Cy/+ kidneys from 24.3±1.3 to 30.0±1.0% (P<0.001). By contrast, there were few PCNA-positive cells in +/+ kidneys and verapamil had no effect on the number of proliferating cells. In female Cy/+ rats, verapamil treatment increased the number of PCNA-positive epithelial cells in normal and dilated renal tubules, and cysts compared to control-treated animals (Table 1). A similar trend was observed in the male Cy/+ kidneys. Verapamil treatment had no effect on the proliferation of interstitial cells.Table 1Effect of VP on PCNA-positive cells in Cy/+ kidneysEpithelial cellsTreatmentNNormal tubulesDilated tubulesCystsInterstitial cellsFemale Cy/+ CONT37.8±0.88.9±1.19.1±2.718.2±5.4 VP313.7±1.7*P<0.0516.2±2.0*P<0.0521.6±1.5*P<0.0514.0±3.4Male Cy/+ CONT314.2±0.615.9±3.617.3±0.216.1±4.5 VP320.2±4.823.8±2.723.6±2.0*P<0.0510.3±2.3CONT, control; PCNA, proliferating cell nuclear antigen; VP, verapamil.Summary of the effect of VP treatment on PCNA-positive epithelial cells of normal tubules (diameter ≤50 μm), dilated tubules (50 μm100 μm); and interstitial cells in Cy/+ male and female kidneys. The data are % PCNA-positive cells (mean±s.e.) counted from 900 to 1400 cells per thin section of cortex. VP (20 mg kg−1 given twice daily) caused a significant increase in PCNA-positive cells in normal and dilated tubules, and cysts within the female Cy/+ kidneys. In male Cy/+ kidneys, a significant increase in PCNA-positive cells was demonstrated only in the cysts; however, the number of cells in normal and dilated tubules that stained positive for PCNA was greater in VP-treated compared to CONT-treated kidneys. There was no effect of VP on interstitial cells in female or male Cy/+ kidneys. A comparison of the effect of VP treatment on PCNA-positive epithelial cells (tubules and cysts) of male and female Cy/+ and +/+ kidneys is shown in Figure 5.* P<0.05* P<0.05 Open table in a new tab CONT, control; PCNA, proliferating cell nuclear antigen; VP, verapamil. Summary of the effect of VP treatment on PCNA-positive epithelial cells of normal tubules (diameter ≤50 μm), dilated tubules (50 μm100 μm); and interstitial cells in Cy/+ male and female kidneys. The data are % PCNA-positive cells (mean±s.e.) counted from 900 to 1400 cells per thin section of cortex. VP (20 mg kg−1 given twice daily) caused a significant increase in PCNA-positive cells in normal and dilated tubules, and cysts within the female Cy/+ kidneys. In male Cy/+ kidneys, a significant increase in PCNA-positive cells was demonstrated only in the cysts; however, the number of cells in normal and dilated tubules that stained positive for PCNA was greater in VP-treated compared to CONT-treated kidneys. There was no effect of VP on interstitial cells in female or male Cy/+ kidneys. A comparison of the effect of VP treatment on PCNA-positive epithelial cells (tubules and cysts) of male and female Cy/+ and +/+ kidneys is shown in Figure 5. Previously, we found that ERK activity generally reflected the mass of cystic epithelia within Cy/+ kidneys.24.Nagao S. Kusaka M. Nishii K. et al.Androgen receptor pathway in rats with autosomal dominant polycystic kidney disease.J Am Soc Nephrol. 2005; 16: 2052-2062Crossref PubMed Scopus (25) Google Scholar To determine if verapamil treatment increased the activity of the B-Raf/MEK/ERK pathway, we measured levels of B-Raf, ERK, and phosphorylated form of B-Raf (P-BRaf) and ERK (P-ERK) in normal and cystic kidneys by immunoblot analysis (Figure 6, Table 2). There was no consistent difference between normal and Cy/+ female kidneys in the expression of the 95 kDa isoform of B-Raf and ERK; and the level of P-ERK was only slightly increased in female Cy/+ kidneys consistent with the mild cystic phenotype.24.Nagao S. Kusaka M. Nishii K. et al.Androgen receptor pathway in rats with autosomal dominant polycystic kidney disease.J Am Soc Nephrol. 2005; 16: 2052-2062Crossref PubMed Scopus (25) Google Scholar Treatment with verapamil increased the expression of B-Raf and increased the levels of P-BRaf/B-Raf and P-ERK/ERK in Cy/+ female kidneys. By contrast, renal B-Raf expression and the activity of the B-Raf/MEK/ERK pathway were already elevated in male Cy/+ rats. There were higher levels of P-BRaf and P-ERK in the male Cy/+ kidneys treated with verapamil; however, a statistically significant effect of verapamil on P-BRaf and P-ERK could not be demonstrated because of the already elevated levels. By contrast, verapamil treatment had no effect on B-Raf, ERK, P-BRaf, or P-ERK levels in +/+ kidneys. We propose that renal cystic disease is related to the activity of the B-Raf/MEK/ERK pathway in the Cy/+ rats, and CCB increases the activity of B-Raf/MEK/ERK pathway and PKD severity. This effect of verapamil was best demonstrated in female Cy/+ rats, which have a milder disease phenotype. Male Cy/+ kidneys already have elevated B-Raf expression and ERK activity and advanced renal cystic disease.Table 2Effect of VP on the activity of the B-Raf/MEK/ERK pathway in Cy/+ and +/+ kidneysGenotypeTreatmentNP-BRaf/B-RafB-RafP-ERK/ERKERKFemale +/+CONT51.00±0.001.00±0.001.00±0.001.00±0.00 +/+VP51.15±0.110.76±0.171.04±0.080.98±0.11 Cy/+CONT51.81±0.07‡P<0.0011.16±0.052.07±0.211.16±0.10 Cy/+VP52.16±0.17‡P<0.001, **P<0.051.41±0.11*P<0.053.75±0.63‡P<0.001, #P<0.011.03±0.05Male +/+CONT51.00±0.001.00±0.001.00±0.001.00±0.00 +/+VP51.00±0.080.94±0.031.62±0.500.98±0.08 Cy/+CONT51.88±0.23†P<0.011.65±0.24*P<0.054.77±0.58†P<0.011.13±0.14 Cy/+VP51.96±0.27†P<0.011.86±0.27*P<0.056.50±0.98‡P<0.0011.09±0.12CONT, control; ERK, extracellular-regulated kinase; MEK, mitogen-activated protein kinase kinase; VP, verapamil.Summary of the effect of VP treatment on renal expression and activity of B-Raf (95 kDa isoform), and ERK in normal (+/+) and Cy/+ female and male rats. Expression levels for ERK and B-Raf were normalized to glyceraldehyde-3-phosphate dehydrogenase, a loading control. Antibodies were used to detect the phosphorylation of S446 of B-Raf (P-BRaf) and Y204 of ERK (P-ERK) and relative activity levels were expressed as P-BRaf/B-Raf and P-ERK/ERK, respectively. It has been reported that S446 (previously S445) of B-Raf is constitutively phosphorylated in proliferating cells and tumor cell lines.25.Dumaz N. Marais R. Integrating signals between cAMP and the RAS/RAF/MEK/ERK signalling pathways. Based on the anniversary prize of the Gesellschaft fur Biochemie und Molekularbiologie Lecture delivered on 5 July 2003 at the Special FEBS Meeting in Brussels.FEBS J. 2005; 272: 3491-3504Crossref PubMed Scopus (241) Google Scholar However, it has been shown recently that phosphorylation at this site is negatively regulated by Rheb, and that S446 phosphorylation promotes binding of B-Raf to H-Ras.26.Karbowniczek M. Robertson G.P. Henske E.P. Rheb inhibits C-raf activity and B-raf/C-raf heterodimerization.J Biol Chem. 2006; 281: 25447-25456Crossref PubMed Scopus (67) Google Scholar Comparison to +/+ kidneys; comparison between CONT and VP treatment in either Cy/+ or +/+ kidneys.* P<0.05† P<0.01‡ P<0.001** P<0.05# P<0.01 Open table in a new tab CONT, control; ERK, extracellular-regulated kinase; MEK, mitogen-activated protein kinase kinase; VP, verapamil. Summary of the effect of VP treatment on renal expression and activity of B-Raf (95 kDa isoform), and ERK in normal (+/+) and Cy/+ female and male rats. Expression levels for ERK and B-Raf were normalized to glyceraldehyde-3-phosphate dehydrogenase, a loading control. Antibodies were used to detect the phosphorylation of S446 of B-Raf (P-BRaf) and Y204 of ERK (P-ERK) and relative activity levels were expressed as P-BRaf/B-Raf and P-ERK/ERK, respectively. It has been reported that S446 (previously S445) of B-Raf is constitutively phosphorylated in proliferating cells and tumor cell lines.25.Dumaz N. Marais R. Integrating signals between cAMP and the RAS/RAF/MEK/ERK signalling pathways. Based on the anniversary prize of the Gesellschaft fur Biochemie und Molekularbiologie Lecture delivered on 5 July 2003 at the Special FEBS Meeting in Brussels.FEBS J. 2005; 272: 3491-3504Crossref PubMed Scopus (241) Google Scholar However, it has been shown recently that phosphorylation at this site is negatively regulated by Rheb, and that S446 phosphorylation promotes binding of B-Raf to H-Ras.26.Karbowniczek M. Robertson G.P. Henske E.P. Rheb inhibits C-raf activity and B-raf/C-raf heterodimerization.J Biol Chem. 2006; 281: 25447-25456Crossref PubMed Scopus (67) Google Scholar Comparison to +/+ kidneys; comparison between CONT and VP treatment in either Cy/+ or +/+ kidneys. Cellular hyperplasia and apoptosis are both hallmarks of hereditary cystic kidney diseases. To determine if verapamil treatment altered the number of apoptotic cells, we used a TdT-mediated dUTP nick end labeling assay to count the number of apoptotic cells in Cy/+ rats treated with either verapamil or vehicle control. Consistent with previous reports,27.Tao Y. Kim J. Stanley M. et al.Pathways of caspase-mediated apoptosis in autosomal-dominant polycystic kidney disease (ADPKD).Kidney Int. 2005; 67: 909-919Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar the number of apoptotic cells (% of total cells counted per field) in the kidneys of Cy/+ rats was significantly higher than in +/+ kidneys (Table 3). Verapamil treatment increased the % apoptotic cells in Cy/+ female and male kidneys 70 and 87%, respectively. Apoptotic cells in dilated tubules and cysts were significantly increased in verapamil-treated Cy/+ rats. By contrast, apoptotic levels were unaffected in normal tubule cells or interstitial cells.Table 3Effect of VP on apoptosis in Cy/+ and +/+ kidneysGenotypeTreatmentNApoptotic index (%)Female +/+CONT30.4±0.2 +/+VP30.0±0.0 Cy/+CONT34.0±0.8*P<0.05 Cy/+VP36.8±1.2†P<0.05Male +/+CONT30.6±1.3 +/+VP30.6±0.3 Cy/+CONT33.8±0.2†P<0.05 Cy/+VP37.1±0.2**P<0.01VP, verapamil.The number of apoptotic cells was counted from approximately 700 cells per section of Cy/+ or +/+ kidneys. Apoptotic cells were detected by the ApopTag Peroxidase In situ Apoptosis Detection Kit (Chemicon International). Female and male Cy/+ kidneys had elevated levels of apoptosis compared to +/+ rats. Treatment with VP increased the number of apoptotic cells in the Cy/+ female and male kidneys, but did not affect the level of apoptosis in +/+ rat kidneys. Apoptotic cells were increased in dilated tubules plus cysts of VP-treated Cy/+ female (2.6±0.4 vs 7.2±0.9, P<0.02) and male (3.4±0.4 vs 8.2±1.1, P<0.02) kidneys, whereas normal tubule cells and interstitial cells were unaffected by VP treatment.* P<0.05† P<0.05** P<0.01 Open table in a new tab VP, verapamil. The number of apoptotic cells was counted from approximately 700 cells per section of Cy/+ or +/+ kidneys. Apoptotic cells were detected by the ApopTag Peroxidase In situ Apoptosis Detection Kit (Chemicon International). Female and male Cy/+ kidneys had elevated levels of apoptosis compared to +/+ rats. Treatment with VP increased the number of apoptotic cells in the Cy/+ female and male kidneys, but did not affect the level of apoptosis in +/+ rat kidneys. Apoptotic cells were increased in dilated tubules plus cysts of VP-treated Cy/+ female (2.6±0.4 vs 7.2±0.9, P<0.02) and male (3.4±0.4 vs 8.2±1.1, P<0.02) kidneys, whereas normal tubule cells and interstitial cells were unaffected by VP treatment. Intracellular Ca2+ signaling in epithelial cells is important for regulation of a variety of cellular functions, including cell volume regulation, ion and fluid transport, differentiation and cell proliferation.28.Leipziger J. Nitschke R. Greger R. Regulation of the intracellular calcium concentration in epithelial cells.Kidney Blood Press Res. 1996; 19: 148-150Crossref PubMed Scopus (7) Google Scholar Various types of Ca2+ channels have been identified in renal tubule cells, including non-selective cation channels,29.Hunter M. Stretch-activated channels in the basolateral membrane of single proximal cells of frog kidney.Pflugers Arch. 1990; 416: 448-453Crossref PubMed Scopus (39) Google Scholar,30.Filipovic D. Sackin H. 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