T-type calcium channel blockade as a therapeutic strategy against renal injury in rats with subtotal nephrectomy
2008; Elsevier BV; Volume: 73; Issue: 7 Linguagem: Inglês
10.1038/sj.ki.5002793
ISSN1523-1755
AutoresNaoki Sugano, Shu Wakino, Takeshi Kanda, Satoru Tatematsu, K. Homma, Kyoko Yoshioka, Kazuhiro Hasegawa, Yoshikazu Hara, Yasuko Suetsugu, Takeo Yoshizawa, Yoshikazu Hara, Yasunori Utsunomiya, Goro Tokudome, T. Hosoya, T Saruta, Kôichi Hayashi,
Tópico(s)Ion channel regulation and function
ResumoT-type calcium channel blockers have been previously shown to protect glomeruli from hypertension by regulating renal arteriolar tone. To examine whether blockade of these channels has a role in protection against tubulointerstitial damage, we used a stereo-selective T-type calcium channel blocker R(-)-efonidipine and studied its effect on the progression of this type of renal injury in spontaneously hypertensive rats that had undergone subtotal nephrectomy. Treatment with racemic efonidipine for 7 weeks significantly reduced systolic blood pressure and proteinuria. The R(-)-enantiomer, however, had no effect on blood pressure but significantly reduced proteinuria compared to vehicle-treated rats. Both agents blunted the increase in tubulointerstitial fibrosis, renal expression of α-smooth muscle actin and vimentin along with transforming growth factor-β (TGF-β)-induced renal Rho-kinase activity seen in the control group. Subtotal nephrectomy enhanced renal T-type calcium channel α1G subunit expression mimicked in angiotensin II-stimulated mesangial cells or TGF-β-stimulated proximal tubular cells. Our study shows that T-type calcium channel blockade has renal protective actions that depend not only on hemodynamic effects but also pertain to Rho-kinase activity, tubulointerstitial fibrosis, and epithelial–mesenchymal transitions. T-type calcium channel blockers have been previously shown to protect glomeruli from hypertension by regulating renal arteriolar tone. To examine whether blockade of these channels has a role in protection against tubulointerstitial damage, we used a stereo-selective T-type calcium channel blocker R(-)-efonidipine and studied its effect on the progression of this type of renal injury in spontaneously hypertensive rats that had undergone subtotal nephrectomy. Treatment with racemic efonidipine for 7 weeks significantly reduced systolic blood pressure and proteinuria. The R(-)-enantiomer, however, had no effect on blood pressure but significantly reduced proteinuria compared to vehicle-treated rats. Both agents blunted the increase in tubulointerstitial fibrosis, renal expression of α-smooth muscle actin and vimentin along with transforming growth factor-β (TGF-β)-induced renal Rho-kinase activity seen in the control group. Subtotal nephrectomy enhanced renal T-type calcium channel α1G subunit expression mimicked in angiotensin II-stimulated mesangial cells or TGF-β-stimulated proximal tubular cells. Our study shows that T-type calcium channel blockade has renal protective actions that depend not only on hemodynamic effects but also pertain to Rho-kinase activity, tubulointerstitial fibrosis, and epithelial–mesenchymal transitions. Voltage-dependent Ca2+ channels are categorized into two subfamilies by their electrophysiological properties: a high voltage-activated Ca2+ channel family, including P-/Q-, L-, N-, and R-type Ca2+ channels, and low-voltage-activated Ca2+ channels, which correspond to T-type Ca2+ channels (TCCs).1Catterall W.A. Structure and regulation of voltage-gated Ca2+ channels.Annu Rev Cell Dev Biol. 2000; 16: 521-555Crossref PubMed Scopus (1855) Google Scholar Although L-type Ca2+ channel blockers (L-CCBs), including nifedipine, are widely prescribed for the treatment of hypertension, there is some concern that L-CCBs might aggravate renal damage.2Hayashi K. Ozawa Y. Fujiwara K. et al.Role of actions of calcium antagonists on efferent arterioles—with special references to glomerular hypertension.Am J Nephrol. 2003; 23: 229-244Crossref PubMed Scopus (93) Google Scholar As L-type Ca2+ channels (LCCs) prevail predominantly in the afferent arteriole but are sparsely expressed in the efferent arteriole,3Loutzenhiser R. Hayashi K. Epstein M. Divergent effects of KCl-induced depolarization on afferent and efferent arterioles.Am J Physiol. 1989; 257: F561-F564PubMed Google Scholar,4Honda M. Hayashi K. Matsuda H. et al.Divergent renal vasodilator action of L- and T-type calcium antagonists in vivo.J Hypertens. 2001; 19: 2031-2037Crossref PubMed Scopus (54) Google Scholar L-CCBs would dilate the afferent arteriole preferentially and thus might accelerate glomerular hypertension if the hypotensive effect is insufficient. In contrast, as TCCs are expressed in both afferent and efferent arterioles,5Hansen P.B. Jensen B.L. Andreasen D. et al.Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels.Circ Res. 2001; 89: 630-638Crossref PubMed Scopus (179) Google Scholar it is reasonably conjectured that TCC blockers (T-CCBs) protect glomeruli from systemic hypertension by regulating renal arteriolar tone.4Honda M. Hayashi K. Matsuda H. et al.Divergent renal vasodilator action of L- and T-type calcium antagonists in vivo.J Hypertens. 2001; 19: 2031-2037Crossref PubMed Scopus (54) Google Scholar, 6Feng M.G. Navar L.G. Angiotensin II-mediated constriction of afferent and efferent arterioles involves T-type Ca2+ channel activation.Am J Nephrol. 2004; 24: 641-648Crossref PubMed Scopus (28) Google Scholar, 7Hayashi K. Wakino S. Sugano N. et al.Ca2+ channel subtypes and pharmacology in the kidney.Circ Res. 2007; 100: 342-353Crossref PubMed Scopus (191) Google Scholar, 8Ozawa Y. Hayashi K. Nagahama T. et al.Effect of T-type selective calcium antagonist on renal microcirculation.Hypertension. 2001; 38: 343-347Crossref PubMed Scopus (63) Google Scholar We previously reported that efonidipine, a CCB with a blocking activity on both TCCs and LCCs,9Masuda Y. Tanaka S. Efonidipine hydrochloride: a new calcium antagonist.Cardiovasc Drug Rev. 1994; 12: 123-135Crossref Scopus (30) Google Scholar ameliorated proteinuria more potently than nifedipine in subtotally nephrectomized spontaneously hypertensive rats (SHR) despite similar blood pressure-lowering effects.10Fujiwara K. Kanno Y. Hayashi K. et al.Renal protective effects of efonidipine in partially nephrectomized spontaneously hypertensive rats.Clin Exp Hypertens. 1998; 20: 295-312Crossref PubMed Scopus (28) Google Scholar Although this result suggests that the T-CCB improves the progression of renal injury, it remains to be determined whether this effect of efonidipine is mediated by the specific blockade of TCCs because efonidipine has substantial effects on LCCs as well. Furthermore, the role of the TCC in mediating the tubulointerstitial changes in the development of renal injury has not been examined. Several lines of investigations have witnessed that the Rho/Rho-kinase pathway is activated in progressive renal injury.11Kanda T. Wakino S. Hayashi K. et al.Effect of fasudil on Rho-kinase and nephropathy in subtotally nephrectomized spontaneously hypertensive rats.Kidney Int. 2003; 64: 2009-2019Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Nishikimi T. Akimoto K. Wang X. et al.Fasudil, a Rho-kinase inhibitor, attenuates glomerulosclerosis in Dahl salt-sensitive rats.J Hypertens. 2004; 22: 1787-1796Crossref PubMed Scopus (89) Google Scholar, 13Sun G.P. Kohno M. Guo P. et al.Involvements of Rho-kinase and TGF-β pathways in aldosterone-induced renal injury.J Am Soc Nephrol. 2006; 17: 2193-2201Crossref PubMed Scopus (97) Google Scholar Small-molecule G protein, Rho, and its effector Rho-kinase contribute significantly to the vascular contractile response as well as various cellular functions, including cell adhesion/migration and proliferation.14Narumiya S. The small GTPase Rho: cellular functions and signal transduction.J Biochem. 1996; 120: 215-228Crossref PubMed Scopus (354) Google Scholar, 15Amano M. Chihara K. Kimura K. et al.Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase.Science. 1997; 275: 1308-1311Crossref PubMed Scopus (931) Google Scholar, 16Seasholtz T.M. Majumdar M. Kaplan D.D. et al.Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration.Circ Res. 1999; 84: 1186-1193Crossref PubMed Scopus (239) Google Scholar We have recently demonstrated that the Rho/Rho-kinase pathway participates in the regulation of renal afferent and efferent arteriolar tone and glomerular filtration.17Nakamura A. Hayashi K. Ozawa Y. et al.Vessel- and vasoconstrictor-dependent role of Rho/Rho-kinase in renal microvascular tone.J Vasc Res. 2003; 40: 244-251Crossref PubMed Scopus (56) Google Scholar Furthermore, the long-term Rho-kinase inhibition by fasudil reduces proteinuria in the rat remnant kidney model.11Kanda T. Wakino S. Hayashi K. et al.Effect of fasudil on Rho-kinase and nephropathy in subtotally nephrectomized spontaneously hypertensive rats.Kidney Int. 2003; 64: 2009-2019Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar In this regard, it has been reported that increases in intracellular Ca2+ concentration are associated with the activation of Rho-kinase in several types of vascular beds.18Sakurada S. Takuwa N. Sugimoto N. et al.Ca2+-dependent activation of Rho and Rho kinase in membrane depolarization-induced and receptor stimulation-induced vascular smooth muscle contraction.Circ Res. 2003; 93: 548-556Crossref PubMed Scopus (247) Google Scholar,19Liu C. Zuo J. Pertens E. et al.Regulation of Rho/ROCK signaling in airway smooth muscle by membrane potential and [Ca2+]i.Am J Physiol Lung Cell Mol Physiol. 2005; 289: L574-L582Crossref PubMed Scopus (58) Google Scholar Furthermore, in mesangial cells, efonidipine, but not nifedipine, suppresses nuclear factor-κB expression,20Hayashi M. Yamaji Y. Nakazato Y. et al.The effects of calcium channel blockers on nuclear factor kappa B activation in the mesangium cells.Hypertens Res. 2000; 23: 521-525Crossref PubMed Scopus (39) Google Scholar which is also a target for the Rho/Rho-kinase pathway.21Ruperez M. Sanchez-Lopez E. Blanco-Colio L.M. et al.The Rho-kinase pathway regulates angiotensin II-induced renal damage.Kidney Int. 2005; 68: S39-S45Abstract Full Text Full Text PDF Scopus (44) Google Scholar No studies have been conducted, however, examining the role of the TCC-mediated signaling pathway in the activation of Rho-kinase and the subsequent impact on renal injury. Efonidipine is the raceme of R(-)-enantiomer and S(+)-enantiomer, and these isomers have been recently isolated. The R(-)-enantiomer of efonidipine has been demonstrated to block the T-type Ca2+ current almost exclusively in native myocardial cells, Xenopus oocytes, and BHK cells, whereas the S(+)-enantiomer blocks both T-type and L-type Ca2+ current.22Tanaka H. Komikado C. Shimada H. et al.The R(-)-enantiomer of efonidipine blocks T-type but not L-type calcium current in guinea pig ventricular myocardium.J Pharmacol Sci. 2004; 96: 499-501Crossref PubMed Scopus (31) Google Scholar,23Furukawa T. Miura R. Honda M. et al.Identification of R(-)-isomer of efonidipine as a selective blocker of T-type Ca2+ channels.Br J Pharmacol. 2004; 143: 1050-1057Crossref PubMed Scopus (54) Google Scholar Herein, with the use of the R(-)-enantiomer of efonidipine, we investigated the role of the TCC in mediating the development of renal injury in the remnant kidney model. Furthermore, whether the TCC activated the Rho/Rho-kinase pathway in the deterioration process was assessed. At the end of the study, serum creatinine was elevated in subtotally nephrectomized SHR (SHR-Nx, 0.66±0.12 mg per 100 ml, n=13) compared with sham-operated rats (sham, 0.19±0.01 mg per 100 ml, n=8, P<0.01), and was reduced in subtotally nephrectomized SHR given efonidipine (SHR-Nx+Efo, 0.49±0.03 mg per 100 ml, n=10, P<0.05) and subtotally nephrectomized SHR given R(-)-efonidipine (SHR-Nx+R, 0.49±0.02 mg per 100 ml, n=12, P 0.5). After 4 weeks of subtotal nephrectomy, systolic blood pressure (SBP) in the SHR-Nx group was elevated, compared with sham (P<0.01), and remained higher throughout the study period (Figure 1a). Treatment with efonidipine markedly suppressed the elevated SBP at week 4 (P 0.5 vs SHR-Nx). Daily urinary protein excretion in SHR-Nx was elevated at week 4 (P<0.01 vs sham), reaching 96±13 mg day−1 at week 8 (P<0.01 vs sham; Figure 1b). Treatment with efonidipine blunted the increase in proteinuria at week 4 (P<0.05 vs SHR-Nx) and week 8 (P<0.01 vs SHR-Nx). Similarly, in SHR-Nx+R, the increase in proteinuria was suppressed at week 4 (P<0.05 vs SHR-Nx), at week 6 (P<0.05), and at week 8 (P<0.01). In SHR-Nx, several glomeruli revealed pathological manifestation, including mesangial expansion (Figure 2a). These alterations were ameliorated by both efonidipine and R(-)-efonidipine. Glomerular size was increased in SHR-Nx, compared with sham (P<0.01; Figure 2b). This change was restored partially by treatment with efonidipine and R(-)-efonidipine (P<0.01 vs SHR-Nx). Masson-trichrome staining revealed increased levels of renal interstitial fibrosis and tubular casts in kidneys from SHR-Nx (Figure 2c). Treatment with efonidipine and R(-)-efonidipine markedly suppressed the changes observed in SHR-Nx (P<0.01). When the tubulointerstitial changes were semiquantified, it was evident that both agents potently inhibited the tubulointerstitial fibrosis (Figure 2d). We examined whether TCC expression was altered in the remnant kidney. Thus, immunohistochemical analysis revealed that the expression of α1G, an α1 subunit of the TCC, was enhanced widely in the kidney, especially in renal arterioles and glomerular areas, from SHR-Nx (Figure 3a). In analogy, the expression of α1G was upregulated in the renal cortical tissues of SHR-Nx, with a 3.2-fold increase compared with sham (P<0.05; Figure 3b). To elucidate the role of humoral factors in mediating the upregulation of α1G expression in renal injury, angiotensin II (Ang II) and transforming growth factor-β (TGF-β) were administered in cultured mesangial cells and human kidney proximal tubular epithelial cells (PTECs), respectively. Thus, Ang II (100 nmol l−1) upregulated α1G expression in mesangial cells, with a 1.5-fold increase compared with control (n=4, P<0.05; Figure 3c). Similarly, treatment with TGF-β (5 ng ml−1) caused a 1.8-fold increase in α1G expression in PTECs (Figure 3d). Immunohistochemical analysis showed an enhanced expression of TGF-β in the remnant kidney from SHR-Nx (P<0.01 vs sham; Figure 4a and b). Furthermore, the upregulated TGF-β expression was suppressed by treatment with R(-)-efonidipine (SHR-Nx+R, P<0.01 vs SHR-Nx), but not by efonidipine (SHR-Nx+Efo). Previously, we demonstrated that the renal cortical tissue from SHR-Nx manifested enhanced Rho-kinase activity, which was responsible in part for the progression of renal injury.11Kanda T. Wakino S. Hayashi K. et al.Effect of fasudil on Rho-kinase and nephropathy in subtotally nephrectomized spontaneously hypertensive rats.Kidney Int. 2003; 64: 2009-2019Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar We therefore investigated whether treatment with efonidipine and R(-)-efonidipine affected the Rho/Rho-kinase pathway in this experimental rat. Thus, the phosphorylation level of MYPT1, which indicated Rho-kinase activity, was enhanced in the renal cortical tissue from SHR-Nx (P<0.01 vs sham) (Figure 5a). Treatment with efonidipine and R(-)-efonidipine inhibited the increased Rho-kinase activity, with similar suppression in SHR-Nx+Efo and SHR-Nx+R (P<0.01 vs SHR-Nx). We further evaluated the effect of TGF-β on Rho-kinase activity and the role of TCCs in TGF-β-mediated Rho-kinase activation in PTECs. Treatment with TGF-β elicited substantial activation of Rho-kinase (as assessed by MYPT-1 phosphorylation) in PTECs (P<0.05; Figure 5b). Furthermore, this upregulation was completely prevented by both efonidipine and R(-)-efonidipine (P<0.01 vs TGF-β). In the renal cortical interstitial area, the expression of α-smooth muscle actin (α-SMA) was increased in SHR-Nx (P<0.01 vs sham; Figure 6a and b). Treatment with efonidipine and R(-)-efonidipine completely suppressed the enhanced expression of α-SMA (P<0.05 vs SHR-Nx). The expression of vimentin was also enhanced in the tubulointerstitial area in SHR-Nx (P<0.01 vs sham; Figure 6c and d). The enhanced expression of vimentin was markedly downregulated in SHR-Nx+Efo and SHR-Nx+R (P<0.01 vs SHR-Nx). No significant difference in vimentin expression levels was observed between SHR-Nx+Efo and SHR-Nx+R. A large amount of evidence has been accumulated that lowering systemic blood pressure constitutes a pivotal determinant of renal protection in the treatment of hypertension. Renal protection is also afforded by the correction of glomerular hypertension with specific agents that elicit vasodilation of the postglomerular (i.e., efferent) arteriole. Our previous study demonstrated that efonidipine improved renal injury much more than nifedipine in subtotally nephrectomized SHR, although both CCBs equally lowered SBP.10Fujiwara K. Kanno Y. Hayashi K. et al.Renal protective effects of efonidipine in partially nephrectomized spontaneously hypertensive rats.Clin Exp Hypertens. 1998; 20: 295-312Crossref PubMed Scopus (28) Google Scholar These data suggest that the renal protective effect of efonidipine results from its salutary action on glomerular hemodynamics independent of SBP, based on the finding that efonidipine elicits dilation of the efferent as well as the afferent arteriole. Nevertheless, it has not been determined whether the renal protective action of efonidipine depends on the inhibition of TCC activity, because efonidipine has the ability to block both TCCs and LCCs. This study has demonstrated that efonidipine improves proteinuria and glomerular hypertrophy and reduces SBP in subtotally nephrectomized SHR. As subtotal nephrectomy predisposes the afferent arteriole to vasodilation24Kimura K. Tojo A. Nanba S. et al.Morphometric analysis of arteriolar diameters in experimental nephropathies: application of microvascular casts.Virchows Arch A Pathol Anat Histopathol. 1990; 417: 319-323Crossref PubMed Scopus (20) Google Scholar and the effect of CCBs on the afferent arteriolar tone may not be apparent in chronic renal disease, it can be speculated that the glomerular capillary pressure is determined by the systemic blood pressure and the efferent arteriolar tone. Thus, the beneficial action of efonidipine on glomerular hemodynamics may depend on a marked reduction in blood pressure and the efferent arteriolar dilation.2Hayashi K. Ozawa Y. Fujiwara K. et al.Role of actions of calcium antagonists on efferent arterioles—with special references to glomerular hypertension.Am J Nephrol. 2003; 23: 229-244Crossref PubMed Scopus (93) Google Scholar, 4Honda M. Hayashi K. Matsuda H. et al.Divergent renal vasodilator action of L- and T-type calcium antagonists in vivo.J Hypertens. 2001; 19: 2031-2037Crossref PubMed Scopus (54) Google Scholar, 7Hayashi K. Wakino S. Sugano N. et al.Ca2+ channel subtypes and pharmacology in the kidney.Circ Res. 2007; 100: 342-353Crossref PubMed Scopus (191) Google Scholar, 10Fujiwara K. Kanno Y. Hayashi K. et al.Renal protective effects of efonidipine in partially nephrectomized spontaneously hypertensive rats.Clin Exp Hypertens. 1998; 20: 295-312Crossref PubMed Scopus (28) Google Scholar In contrast, R(-)-efonidipine, a specific T-CCB, fails to lower blood pressure but ameliorates proteinuria and glomerular hypertrophy to the same extent as efonidipine (Figures 1 and 2). Because R(-)-efonidipine possesses more potent inhibitory activity on TCCs than efonidipine,23Furukawa T. Miura R. Honda M. et al.Identification of R(-)-isomer of efonidipine as a selective blocker of T-type Ca2+ channels.Br J Pharmacol. 2004; 143: 1050-1057Crossref PubMed Scopus (54) Google Scholar efferent arteriolar dilation could play a major role in preventing glomerular hypertrophy. In concert, our findings clearly indicate that specific blockade of TCCs offers renal protective action independent of systemic blood pressure. Alternatively, the T-CCB may directly prevent the pathological process of renal injury. This study demonstrates that the renal tissue from SHR-Nx manifests enhanced expression of α1G subunits of the TCC (Figure 3a and b). Furthermore, in cultured mesangial cells, the expression of α1G is upregulated by Ang II (Figure 3c). Ang II constitutes a pivotal vasoactive substance involved in the progression of renal injury,25Sanchez-Lozada L.G. Tapia E. Johnson R.J. et al.Glomerular hemodynamic changes associated with arteriolar lesions and tubulointerstitial inflammation.Kidney Int. 2003; 64: S9-S14Abstract Full Text Full Text PDF Scopus (62) Google Scholar and the renal Ang II content is increased in renal injury induced by five-sixths nephrectomized rats.26Mackie F.E. Meyer T.W. Campbell D.J. Effects of antihypertensive therapy on intrarenal angiotensin and bradykinin levels in experimental renal insufficiency.Kidney Int. 2002; 61: 555-563Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar Moreover, TGF-β, which is enhanced and is responsible for the development of renal injury,27Bottinger E.P. TGF-β in renal injury and disease.Semin Nephrol. 2007; 27: 309-320Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar upregulates α1G expression (Figure 3d). Our current observation therefore implies that the blockade of TCCs would provide exaggerated mitigatory action on the progression of glomerular injury. Furthermore, it has been reported that mibefradil, a mixed TCC and LCC blocker, improves proteinuria and glomerular damage in DOCA-salt rats whereas amlodipine fails to blunt the progression of renal injury.28Baylis C. Qiu C. Engels K. Comparison of L-type and mixed L- and T-type calcium channel blockers on kidney injury caused by deoxycorticosterone-salt hypertension in rats.Am J Kidney Dis. 2001; 38: 1292-1297Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Of note, both amlodipine and mibefradil exert similar antihypertensive actions and decrease glomerular capillary pressure to the same level. It appears therefore that the T-CCB confers greater benefit distinct from that provided by the L-CCB. In concert, available evidence indicates that the TCC per se regulates glomerular pathological process, which does not absolutely require the effect on systemic blood pressure or glomerular capillary pressure for the renal action of the TCC. The expression of the TCC has been reported in several pathological conditions. It has been demonstrated that TCCs are expressed in the fetal myocardium29Cribbs L.L. Martin B.L. Schroder E.A. et al.Identification of the T-type calcium channel (Ca(v)3.1d) in developing mouse heart.Circ Res. 2001; 88: 403-407Crossref PubMed Scopus (83) Google Scholar and in the condition of morbid cardiac muscle, such as cardiac hypertrophy30Nuss H.B. Houser S.R. T-type Ca2+ current is expressed in hypertrophied adult feline left ventricular myocytes.Circ Res. 1993; 73: 777-782Crossref PubMed Scopus (268) Google Scholar and heart failure.31Izumi T. Kihara Y. Sarai N. et al.Reinduction of T-type calcium channels by endothelin-1 in failing hearts in vivo and in adult rat ventricular myocytes in vitro.Circulation. 2003; 108: 2530-2535Crossref PubMed Scopus (55) Google Scholar In our model of subtotally nephrectomized SHR, the expression level of α1G subunits of the TCC is markedly upregulated (Figure 3a and b). These results support the idea that the T-CCB has the potential to block the pathological processes, and suggest the role of the TCC in ‘atavism’ of damaged organs. In the morbid state, the suppression of the TCC might be exceedingly important for arresting the process that builds up vicious circle. Indeed, Ang II is reported to increase T-type Ca2+ current and TCC expression in cardiac cells.32Ferron L. Capuano V. Ruchon Y. et al.Angiotensin II signaling pathways mediate expression of cardiac T-type calcium channels.Circ Res. 2003; 93: 1241-1248Crossref PubMed Scopus (74) Google Scholar We therefore propose that exaggerated TCC expression plays a substantial role in Ang II-associated organ damage. Further studies are required to elucidate the role of the TCC in Ang II-associated organ remodeling and cellular phenotypic changes. Although it is well established that TGF-β plays a crucial role in the progression of renal fibrosis, the mechanism whereby TCCs affect renal tubulointerstitial fibrotic process remains undetermined. In this study, we observed enhanced expression levels of TGF-β, particularly in the interstitial area of kidneys from SHR-Nx, and the increased TGF-β expression was prevented by a specific TCC-B, R(-)-efonidipine, and tended to be suppressed by efonidipine (Figure 4a and b). These observations suggest that TCCs participate in the induction of TGF-β and the subsequent renal fibrosis. In this regard, amlodipine, a CCB with L-type and N-type calcium channel blocking activity, is reported to stimulate TGF-β production in human mononuclear cells.33Kaynar K. Ulusoy S. Ovali E. et al.TGF-β and TNF-α producing effects of losartan and amlodipine on human mononuclear cell culture.Nephrology. 2005; 10: 478-482Crossref PubMed Scopus (12) Google Scholar Thus, the failure of efonidipine to suppress renal TGF-β expression might be related to variable degrees of blocking activity on the TCC and LCC. This study demonstrates that Rho-kinase is activated in SHR-Nx and is suppressed by efonidipine and R(-)-efonidipine (Figure 5a). In this regard, we have recently shown that Ang II-induced activation of Rho-kinase is inhibited by efonidipine and mibefradil but not by nifedipine in vascular smooth muscle cells (unpublished observation). Furthermore, R(-)-efonidipine blunts the Ang II-induced enhancement in GTP-Rho. As Ang II is responsible in large part for TGF-β stimulation and the aggravation and/or initiation of renal injury, TCCs may mediate or modulate the activation of Rho/Rho-kinase in renal injury. Of note, a growing body of evidence has been accumulated that the Rho/Rho-kinase pathway participates in the control of glomerular hemodynamics17Nakamura A. Hayashi K. Ozawa Y. et al.Vessel- and vasoconstrictor-dependent role of Rho/Rho-kinase in renal microvascular tone.J Vasc Res. 2003; 40: 244-251Crossref PubMed Scopus (56) Google Scholar and the pathological process of renal disease.11Kanda T. Wakino S. Hayashi K. et al.Effect of fasudil on Rho-kinase and nephropathy in subtotally nephrectomized spontaneously hypertensive rats.Kidney Int. 2003; 64: 2009-2019Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar,12Nishikimi T. Akimoto K. Wang X. et al.Fasudil, a Rho-kinase inhibitor, attenuates glomerulosclerosis in Dahl salt-sensitive rats.J Hypertens. 2004; 22: 1787-1796Crossref PubMed Scopus (89) Google Scholar We have recently demonstrated that fasudil, a Rho-kinase inhibitor, improves renal injury in subtotally nephrectomized SHR.11Kanda T. Wakino S. Hayashi K. et al.Effect of fasudil on Rho-kinase and nephropathy in subtotally nephrectomized spontaneously hypertensive rats.Kidney Int. 2003; 64: 2009-2019Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar Moreover, the Rho/Rho-kinase pathway is involved in the development of tubulointerstitial fibrosis.34Moriyama T. Nagatoya K. The Rho–ROCK system as a new therapeutic target for preventing interstitial fibrosis.Drug News Perspect. 2004; 17: 29-34Crossref PubMed Scopus (39) Google Scholar Finally, this study shows that TGF-β stimulates Rho-kinase activity in PTECs and this effect is inhibited by T-CCBs (Figure 5b). Previous studies demonstrated that Rho-kinase plays a substantial role not only in inducing TGF-β12Nishikimi T. Akimoto K. Wang X. et al.Fasudil, a Rho-kinase inhibitor, attenuates glomerulosclerosis in Dahl salt-sensitive rats.J Hypertens. 2004; 22: 1787-1796Crossref PubMed Scopus (89) Google Scholar, 13Sun G.P. Kohno M. Guo P. et al.Involvements of Rho-kinase and TGF-β pathways in aldosterone-induced renal injury.J Am Soc Nephrol. 2006; 17: 2193-2201Crossref PubMed Scopus (97) Google Scholar, 35Ozawa Y. Kobori H. Cruicial role of Rho–nuclear factor-κB axis in angiotensin II-induced renal injury.Am J Physiol. 2007; 293: F100-F109Crossref PubMed Scopus (41) Google Scholar but also in mediating the renal tubular action of TGF-β.36Tian Y.C. Fraser D. Attisano L. et al.TGF-β-mediated alterations of renal proximal tubular cell phenotype.Am J Physiol. 2003; 285: F130-F142Crossref PubMed Scopus (95) Google Scholar It is thus conjectured that T-CCBs ameliorate renal tubulointerstitial fibrosis, and the suppression of the Rho/Rho-kinase pathway by T-CCBs is responsible in part for the improvement of this pathological process. Of note, this study shows that R(-)-efonidipine ameliorates renal injury with no appreciable effect on blood pressure. As Rho-kinase is one of the important factors causing hypertension, the suppression of the Rho/Rho-kinase pathway by T-CCBs might efficiently correct hypertension in this experiment. In our previous study using the rat remnant kidney model, however, fasudil reduced proteinuria, but failed to induce a significant decrease in blood pressure.11Kanda T. Wakino S. Hayashi K. et al.Effect of fasudil on Rho-kinase and nephropathy in subtotally nephrectomized spontaneously hypertensive rats.Kidney Int. 2003; 64: 2009-2019Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar Another study also demonstrates that fasudil significantly improves renal function, proteinuria, and histological findings, without changing blood pressure in Dahl salt-sensitive rats.12Nishikimi T. Akimoto K. Wang X. et al.Fasudil, a Rho-kinase inhibitor, attenuates glomerulosclerosis in Dahl salt-sensitive rats.J Hypertens. 2004; 22: 1787-1796Crossref PubMed Scopus (89) Google Scholar Thus, the inhibition of TCCs and Rho-kinase may offer renal protection, which may not exclusively require a reduction in systemic blood pressure. The role of epithelial–mesenchymal transition (EMT) in renal fibrosis merits comments. This study demonstrates that subtotal nephrectomy induces renal tubulointerstitial fibrosis (Figure 2) and upregulates the expression of α-SMA and vimentin (Figure 6). Furthermore, these changes are inhibited by treatment with efonidipine and R(-)-efonidipine. Once epithelial cells acquire mesenchymal phenotypic characteristics, the expression levels of α-SMA and vimentin are increased,37Bariety J. Hill G.S. Mandet C. et al.Glomerular epithelial–mesenchymal transdifferentiation in pauci-immune crescentic glomerulonephritis.Nephrol Dial Transplant. 2003; 18: 1777-1784Crossref PubMed Scopus (73) Google Scholar,38Rastaldi M.P. Ferrario F. Giardino L. et al.Epithelial–mesenchymal transition of tubular epithelial cells in human renal biopsies.Kidney Int. 2002; 62: 137-146Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar which implies enhanced mesenchymal transdifferentiation of tubular epithelial cells39Manotham K. Tanaka T. Matsumoto M. et al.Transdifferentiation of cultured tubular cells induced by hypoxia.Kidney Int. 2004; 65: 871-880Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar and the subsequent tubulointerstitial fibrosis. To the extent that activation of Rho-GTPase serves as an important mechanism for EMT,40Patel S. Takagi K. Suzuki J. et al.RhoGTPase activation is a key step in renal epithelial mesenchymal transdifferentiation.J Am Soc Nephrol. 2005; 16: 1977-1984Crossref PubMed Scopus (73) Google Scholar the potent inhibitory action of T-CCBs on Rho-kinase would halt the process of EMT and alleviate renal tubulointerstitial fibrosis. Of interest, treatment with efonidipine or R(-)-efonidipine mitigates but does not completely abolish the activated Rho-kinase observed in SHR-Nx, whereas it nearly totally suppresses the expression of α-SMA and vimentin. It is surmised therefore that other mechanisms contribute to the inhibition of EMT and fibrosis, including the suppression of nuclear factor-κB20Hayashi M. Yamaji Y. Nakazato Y. et al.The effects of calcium channel blockers on nuclear factor kappa B activation in the mesangium cells.Hypertens Res. 2000; 23: 521-525Crossref PubMed Scopus (39) Google Scholar and cell proliferation.41Rodman D.M. Reese K. Harral J. et al.Low-voltage-activated (T-type) calcium channels control proliferation of human pulmonary artery myocytes.Circ Res. 2005; 96: 864-872Crossref PubMed Scopus (107) Google Scholar In summary, the blockade of the TCC exerts potent renal protective action, reflected by the suppression of glomerular hypertrophy and tubulointerstitial fibrosis in the model of chronic kidney disease. The beneficial action does not absolutely require a reduction in systemic blood pressure, but is mediated by multiple mechanisms, including inhibition of EMT, in which activation of the Rho/Rho-kinase pathway is involved (Figure 7). Finally, as TCCs are upregulated in the chronic renal injury model, T-CCBs may constitute a novel therapeutic tool for the treatment of chronic kidney disease. Six-week-old male SHR (n=43) (Japan SLC Inc., Shizuoka, Japan) were placed in individual metabolic cages and were initially fed a standard rat chow (15 g day−1; Nippon Clea, Tokyo, Japan) containing 0.3% NaCl, 0.74% potassium, and 20.6% protein, and were given water ad libitum. Subtotal nephrectomy was performed by removal of two-thirds of the left kidney (week 0), followed by total right nephrectomy (week 1) under ether anesthesia. Rats were then randomly assigned to four groups: group 1, sham-operated rats (sham, n=8); group 2, subtotally nephrectomized rats (SHR-Nx, n=13); group 3, SHR-Nx given efonidipine (Efo, 100 mg per kg per day, n=10; Nissan Chemical Industries, Tokyo, Japan), and group 4, SHR-Nx given R(-)-efonidipine (R, 100 mg per kg per day, n=12; Nissan Chemical Industries). SBP, measured by the tail-cuff method, and daily urinary protein excretion were evaluated every other week. At week 8, rats in each group were decapitated in the morning under the fasting condition. Blood was collected, and serum total protein and creatinine were measured by an autoanalyzer. The kidneys were harvested for further analysis. All experiments were performed in accordance with the animal experimentation guidelines of Keio University School of Medicine. Each paraffin section of rat kidneys was stained with periodic acid-Schiff and Masson-trichrome stain. Glomerular changes were assessed as the size of mesangial and vascular areas at the level of vascular pole using a computer-aided manipulator (microscope, Leica DM4000B; camera, Leica DFC300FX; software, Leica IM500). Tubulointerstitial fibrotic areas stained in light blue with Masson-trichrome stain were picked on the digital images using a computer-aided manipulator, and were graded as follows: 0, none; +1, 50%. The scores were determined in each section selected at random and more than 15 fields were examined under × 400 magnification. Morphological evaluation was conducted by two independent observers in a blinded fashion. Immunohistochemical analysis for renal fibrosis was performed using antibodies against α-SMA (Dako Cytomation, Glostrup, Denmark) and vimentin (Dako). α-SMA-positive cells, vimentin-positive cells, and TGF-β-positive cells were counted and graded as follows: 0, none; +1, 50% of α-SMA- or vimentin-positive area. We further examined whether the expression of the TCC was altered in kidneys from SHR-Nx. Immunohistochemical evaluation was conducted using an antibody against α1G subunits of the TCC (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Mesangial cells were prepared from 3-week-old male Sprague–Dawley rats, using the mesh sieving technique. Briefly, the renal cortical tissue was removed, washed with Hank's balanced salt solution (Nissui pharmaceutical Co, Ltd, Tokyo, Japan), and passed through 212 and 150 μm mesh sieves. Glomeruli were finally recovered after sieving through a 53 μm mesh filter. The cells were cultured in D-MEM/F-12 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), 10 μl ml−1 ITS+culture supplement (Becton Dickinson, Frankin Lakes, NJ, USA), 100 U ml−1 penicillin (Invitrogen), 100 mg ml−1 streptomycin (Invitrogen), and 200 mmol l−1L-glutamine (Invitrogen). Early passaged (passages 4–8) mesangial cells were grown to 60–70% confluence and made quiescent by serum starvation for 24 h. Ang II (100 nmol l−1; Sigma, St Louis, MO, USA) was added and the cells were harvested after 24 h of Ang II treatment. PTECs (Cambrex Bio Science Walkersville Inc., Walkersville, MD, USA) were cultured in renal epithelial cell basal medium (Cambrex Bio Science Walkersville Inc.) supplemented with 0.5 μg ml−1 hydrocortisone, 10 ng ml−1 human recombinant epidermal growth factor, 5 μg ml−1 insulin, 0.5 μg ml−1 epinephrine, 6.5 ng ml−1 triiodothyronine, 10 μg ml−1 transferrin, 0.5 ml GA-1000, 0.5% fetal bovine serum, 100 U ml−1 penicillin (Invitrogen), 100 mg ml−1 streptomycin (Invitrogen), and 200 mmol l−1L-glutamine (Invitrogen). Each cell was grown at 37°C in 5% CO2 and the medium was changed every other day. After starvation, recombinant human TGF-β1 (R&D Systems, Minneapolis, MN, USA) was added before the treatment of efonidipine or R(-)-efonidipine, and the cells were harvested after 24 h of TGF-β treatment. Renal cortical tissues, mesangial cells, and PTECs were lysed and fornicated in solubilization buffer. Immunoblotting was performed as described previously,42Wakino S. Kintscher U. Liu Z. et al.Peroxisome proliferator-activated receptor gamma ligands inhibit mitogenic induction of p21 (Cip1) by modulating the protein kinase C delta pathway in vascular smooth muscle cells.J Biol Chem. 2001; 276: 47650-47657Crossref PubMed Scopus (70) Google Scholar using specific antibodies against phospho-MYPT1 (Upstate Biochemistry, Lake Placid, NY, USA) and α1G (Alomone, Jerusalem, Israel). Immunoreactive bands were detected using an ECL detection kit (Amersham Biosciences, Uppsala, Sweden). Immunoblots were analyzed by computer-aided planimetry using the Scion Image software (Scion Corp., Fredrick, MD, USA). In detail, images were digitized and captured with a CCD camera connected to a personal computer, and scanned into Photoshop (Adobe Systems, San Jose, CA, USA). A binary overlay was created automatically, and the threshold of the binary overlays was adjusted by comparing image to optimal match of each spot. The value of the threshold setting was kept constant. Data were expressed as the mean±s.e.m. Data were analyzed by one- or two-way analysis of variance as appropriate, followed by Newman–Keuls post hoc test. Histological results were analyzed by Kruskal–Wallis nonparametric test. P<0.05 was considered statistically significant. We thank Mrs Tomoko Hayakawa and Ms Hitomi Nishine for the technical support of our experimentation. This work was supported by the Scientific Research Fund of the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 16590716).
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