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T-Type calcium channel blockage ameliorates proteinuria and renal extracellular matrix accumulation in experimental diabetic rats
J Nephrol Hong Kong Journal of 2001;3(1):27-32. Nephrology 2001;3(1):27-32.
J MA, et al
O R I G I N A L A R T I C L E
T-Type calcium channel blockage ameliorates proteinuria and renal extracellular matrix accumulation in experimental diabetic rats Ji MA, Yong GU, Ru-Zhong ZHANG, Hai-Chun YANG, Shan-Yan LIN Division of Nephrology, Fu Dan University Medical Center (former Shanghai Medical University) Hua Shan Hospital, Shanghai, China.
Abstract Calcium channels have been shown to play an important role in the pathogenesis of diabetic nephropathy. The purpose of this study is to investigate the effect of T-type calcium channel blockers in uninephrectomized diabetic rats. Two weeks after uninephrectomy, streptozotocin (65 mg/kg) was given to male Sprague-Dawley rats to induce diabetes mellitus. In the succeeding days, four groups of animals were randomized to receive 30 mg/kg mibefradil (Mib, T-type calcium channel blocker), 30 mg/kg amlodipine (Aml, L-type calcium channel blocker), 10 mg/kg cilazapril (Cil, angiotensin converting enzyme inhibitor) or 25 mg/kg Mib + 8 mg/kg Cil (M+C), which were delivered daily by gavage (n = 6-8 for each group). At the fourth week, 24-hour urine, blood and kidney samples were collected for examinations. Renal morphometric analyses and assay for type IV collagen and laminin were performed. Untreated diabetic rats (DM) exhibited overt hyperglycemia, proteinuria and albuminuria when compared with uninephrectomized (UNx) control (17.67 ±4.9 vs 2.97 ±0.72 µg/24 hours, p < 0.05), while Cil and M+C treated rats had significantly reduced urinary protein excretion (Cil: 5.53 ±1.45, M+C: 3.87 ±0.88 vs DM: 17.67 ± 4.9 µg/24 hours; p < 0.05, respectively). Mibefradil alone also ameliorated albuminuria in DM rats (7.69 ±1.87 vs 17.67 ±4.9 µg/24 hours, p < 0.05). Histopathological study revealed significantly less glomerular matrix, laminin, type IV collagen and proximal tubule cells (PTC) hypertrophy in the Cil and M+C groups when compared with the DM group (Collagen IV: DM 7.3 ±0.65 vs Mib 4.82 ±0.78, Cil 4.11 ±0.74, M+C 2. 64 ±0.66, UNx 3.71 ±0.99, p < 0.05). Albuminuria, glomerular matrix accumulation and collagen IV deposition were not changed in the Aml treated group. There was, however, significantly lower creatinine clearance and laminin volume in the Aml group. In conclusion, our study demonstrated that T-type calcium channel blockage reduced albuminuria and renal extracellular matrix accumulation in experimental diabetic rats, suggesting an important role for T-type calcium channel in the progression of diabetic nephropathy. Additional benefit can be achieved by combination with an angiotensin converting enzyme inhibitor. The mechanism of action, however, requires further investigation. Key words: Amlodipine, Calcium channel blocker (CCB), Diabetic nephropathy, Mibefradil
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Correspondence: Dr. Shan-Yan LIN, Dr. Ji MA, 12 Wulumuqi Middle Rd., Division of Nephrology, Hua-Shan Hospital, Shanghai 200040, China. Fax: (8621) 6248 8638, E-mail: [email protected], [email protected] Hong Kong Journal of Nephrology, April 2001
27
T-type calcium channel and diabetic nephropathy
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INTRODUCTION
Measurement of biochemical parameters
Calcium channel blockers (CCB) are effective antihypertensive agents commonly used in patients with diabetic mellitus. Unlike angiotensin converting enzyme inhibitor (ACEi), its renoprotective effect in diabetic patients remains unsettled (1). It has recently been shown that T-type calcium channel is involved in glomerular hypertrophy, mitogenesis and macromolecular trafficking (2), implicating a critical role in the progression of chronic renal diseases. In this study, we examined the renoprotective effects of T-type CCB, mibefradil (Ro405967), in streptozotocin-induced diabetic rats, and compared them with those of L-type CCB amlodipine, ACEi cilazapril as well as the combination of mibefradil and cilazapril.
Blood glucose, urinary protein, serum and urine creatinine were measured in the clinical laboratory by an automatic biochemical analyzer (Hitachi 7170, Japan). Creatinine clearance (Ccr) was calculated according to the formula: Ccr = urine creatinine x 24 hours urine volume/serum creatinine. Urinary albumin level was determined by radioimmunoassay (China Atomic Science Institution). Plasma angiotensin converting enzyme activity was measured by ultraviolet spectrophotometry.
SUBJECTS AND METHODS Experimental animals Thirty male Sprague-Dawley rats weighing 180 to 220 g underwent uninephrectomy after being anesthetized using intraperitoneal chloral hydrate. Two weeks after the operation, streptozotocin (STZ, Sigma) 65 mg/kg was injected intraperitoneally to induce diabetes mellitus. Diabetes was defined as heavy glycosuria (3+ to 4+) and severe hyperglycemia (> 16.7 mmol/L), which usually appeared 48 hours after STZ administration. Another six uninephrectomized rats were injected with isovolumic buffered citrate solution and served as uninephrectomized non-diabetic control (UNx). The diabetic rats were randomly allocated to receive the following treatments in the succeeding 4 weeks: mibefradil (Mib, 30 mg/kg/ day; n = 6), amlodipine (Aml, 30 mg/kg/day; n = 6), cilazapril (Cil, 10 mg/kg/day; n = 8), combined mibefradil and cilazapril (M+C, Mib 25 mg/kg/day, Cil 8 mg/kg/ day; n =7) and vehicle (DM, n = 7). All drugs or vehicle were delivered daily by gavage from the day after being confirmed as diabetic. Animals were kept in a pathogenfree environment with free access to water and standard rat chow. Blood pressure was measured weekly by tail-cuff method. After the 4-week treatment period, the animals were kept in metabolic cages for 24-hour urine collection. Blood samples were collected through the right carotid artery. The animals were then sacrificed and the left kidneys were removed, weighed, and fixed overnight in 4% buffered formaldehyde. 28
Histopathological analysis Renal tissue was embedded in paraffin, sectioned into 4 µm thickness and screened by periodic acid-Schiff (PAS) and periodic acid silver methenamine (PASM) staining. Relative area of glomerular matrix and height of renal proximal tubular epithelial cells were measured by an image analysis system (LEIKA, Germany). The cumulative area of glomeruli and intraglomerular matrix in 20 consecutive non-overlapping cortical fields on PASM stained sections were measured under the magnification of 20 x objective. The relative area of glomerular matrix was defined as the ratio of the sum of intraglomerular matrix to that of glomeruli. Cross sections of proximal tubules in 10 consecutive non-overlapping cortical fields under magnification of 10 x objective were selected to determine the outer and inner diameters of the tubules, the discrepancy between which was taken as the height of proximal tubular epithelial cell. Matrix components were demonstrated by immunohistochemical staining. After routine dewax and rehydration, the sections were treated by 0.1% buffered trypsin at 37 oC for 20 minutes for antigen retrieval. Endogenous hydrogen peroxidase was quenched by 3% H2O2/methanol. After blocking in 10% goat serum, the sections were incubated with rabbit anti-rat collagen IV and laminin polyclonal antibodies (a gift from the Department of Pathology, Shanghai Medical University) at room temperature for 30 minutes. The sections were then treated with goat-anti-rabbit IgG conjugated with horseradish peroxidase and developed by 3,3’-diaminobenzidine (DAKO) solution. Under magnification of 10 x objective, 20 consecutive nonoverlapping cortical fields were analyzed morphometrically for intraglomerular collagen IV and laminin by LEIKA image analysis system.
Hong Kong J Nephrol 2001;3(1):27-32.
J MA, et al
Statistical analysis
Data were presented as mean ±standard error. Data from the six groups were analyzed by analysis of variance (ANOVA) and the level of significance was defined as p < 0.05.
with DM, the decreases in urinary albumin excretion rate in rats treated with Mib, Cil and their combination (M+C) were significant (p < 0.05). The difference in albuminuria between Mib and Cil was not significant (p > 0.05) (Table 1).
RESULTS
Creatinine clearance
Blood pressure Results of the mean arterial pressure (MAP) measured by tail-cuff method were listed in table 1. Though no difference in MAP was found in Mib, Aml, Cil and M+C, all these four groups had a significant decrease in MAP compared with DM or UNx (all p < 0.05).
There was elevated Ccr in all diabetic rats compared with the UNx control (all p < 0.05). When compared with the DM, it was decreased in all treated groups (all p < 0.05). Ccr was significantly lower in the Cil and M+C groups than that in the Mib and Aml groups (all p < 0.05) (Table 1).
Ratio of kidney weight to body weight Blood glucose level All STZ-injected rats had substantially elevated blood glucose (all p < 0.05). Interestingly, it was lower in the Cil and M+C group compared with the DM group (both p < 0.05).
Ratio of kidney weight to body weight (KW/BW) in each group were: Mib 9.2 ±1.2, Aml 9.1 ±0.7, Cil 7.9 ± 0.5, M+C 7.8 ±0.5, DM 9.6 ±0.9, UNx 6.8 ±0.7 x 10 -3. Significant difference was only detected between the DM and the UNx group (p < 0.05).
Proteinuria and albuminuria
Histopathological analyses
All diabetic rats except in the M+C group showed significantly higher urinary protein and albumin excretion rate compared with the UNx (all p < 0.05). The proteinuria, however, was decreased in all treated groups (all p < 0.05), and even less in M+C and Cil than that of Mib and Aml (all p < 0.05). Moreover, compared
The results of morphometric examination for relative glomerular matrix and height of proximal tubular cells (PTC) were shown in table 2. The relative glomerular matrix area was significantly higher in all diabetic groups than that in UNx control (all p < 0.05). Compared with that in UNx, PTC height was also increased in DM, Aml
Table 1. General conditions and biochemical parameters in each group.
Experimental group
n
Mibefradil Amlodipine Cilazapril Mibefradil+Cilazapril Uninephrectomized diabetes Uninephrectomized control
6 6 8 7 7 6
MAP Serum ACE activity Blood glucose (mmHg) (nM/min/mL) (mmol/L) 140.4 ±13.4 25.6 ±1.71 110.3 ±5.81 109.8 ±3.51 160.3 ±12.0 23.6 ±2.41 1 1 107.2 ±3.1 45.6 ±8.7 20.1 ±2.81,2 1 1 113.1 ±4.7 34.5 ±4.5 20.5 ±0.91,2 139.6 ±5.2 180.9 ±21.6 22.4 ±1.81 130.4 ±6.6 220.6 ±32.4 6.8 ±0.7
Proteinuria (mg/24 h) 27.2 ±9.71,2,3 30.6 ±4.81,2,3 21.8 ±3.21,2 18.1 ±4.82 50.7 ±11.51 14.8 ±4.8
Albuminuria (µg/24 h) 7.69 ±1.871,2,3 15.11 ±4.031,3 5.53 ±1.451,2 3.87 ±0.881,2 17.67 ±4.901 2.97 ±0.72
Creatinine clearance (mL/min) 2.53 ±0.331,2,3 2.20 ±0.171,2,3 1.81 ±0.271,2 1.88 ±0.281,2 3.44 ±0.441 1.25 ±0.17
1
p < 0.05 compared with uninephrectomized control. p < 0.05 compared with uninephrectomized diabetes. 3 p < 0.05 compared with mibefradil+cilazapril. 2
Table 2. Relative glomerular matrix area, height of PTC, and glomerular type IV collagen and laminin deposition.
Experimental group Mibefradil Amlodipine Cilazapril Mibefradil+Cilazapril Uninephrectomized diabetes Uninephrectomized control
n 6 6 8 7 7 6
Glomerular matrix 0.301 ±0.0251,3 0.307 ±0.0261,3 0.223 ±0.0301,2,3 0.191 ±0.0181,2 0.322 ±0.0351 0.179 ±0.021
PTC height (µm) 4.99 ±0.271 5.20 ±0.211 3.89 ±0.242 3.55 ±0.202 6.07 ±0.381 4.11 ±0.18
Type IV collagen 4.82 ±0.782,3 6.54 ±0.751,3 4.11 ±0.742,3 2.64 ±0.062 7.30 ±0.651 3.71 ±0.99
Laminin 5.06 ±0.981,2 5.35 ±0.671,2 3.87 ±1.022 3.12 ±0.062 7.99 ±0.881 3.70 ±0.49
1
p < 0.05 compared with uninephrectomized control. p < 0.05 compared with uninephrectomized diabetes. 3 p < 0.05 compared with mibefradil+cilazapril. 2
29
T-type calcium channel and diabetic nephropathy
and Mib (all p < 0.05), while there was no difference for Cil and M+C (p > 0.05). The expression of glomerular type IV collagen and laminin were listed in table 2. Positively stained area for type IV collagen was more extensive in DM and Aml groups than that in UNx. Furthermore, glomerular type IV collagen deposition in M+C was less when compared with that in Mib and Cil (both p < 0.05) (Fig. 1). Similarly, the positively stained area of glomerular laminin in DM, Aml and Mib was larger than that in UNx (all p < 0.05). Compared with that in DM, it was smaller in all treated groups (all p < 0.05) (Fig. 2).
DISCUSSION Intraglomerular hypertension and hyperfiltration is an important mechanism for progression of diabetic nephropathy. Extracellular matrix (ECM) accumulation caused by proteinuria and a variety of growth factors could lead to glomerulosclerosis. Beside intensive glycemic control, interventions targeting systemic and intraglomerular hypertension, proteinuria and extracellular matrix accumulation are also being actively investigated (3). Calcium channels are classified into L-, T-, P- and Ntype according to their activation characteristics. Conventional calcium channel blockers are all of the Ltype, which can further be divided into three groups: dihydropyridine (e.g., nifedipine, amlodipine, felodipine, nicardipine, etc.), phenylalkylamines (e.g., verapamil), and benzothiazepines (e.g., diltiazem). Despite their potent antihypertensive effect, the protective effects for progressive renal deterioration are still controversial. Several studies have demonstrated a more potent antiproteinuria effect by non-dihydropyridine compared to dihydropyridine CCB. The beneficial effects on glomerular sclerosis and proteinuria, however, are still inferior to those of ACEi (1). A body of evidence from in vivo and in vitro studies showed that T-type calcium channel plays an important role in some pathophysiological changes of cardiovascular diseases. Blockage of the channel results in blood pressure reduction and attenuation of pathological cellular growth. T-type calcium channel also mediates vascular smooth muscle cell contraction (4). Blockage of T-type calcium channel by Ro 40-5967 (mibefradil) could inhibit the proliferation and hypertrophy of smooth muscle cell while L-type CCB e.g. verapamil, amlodipine and felodipine are devoid of such effect (5). Moreover, in DOCA salt-induced (6) and spontaneous (7) hypertensive rats, mibefradil significantly ameliorates proteinuria and glomerular changes, indicating the role of T-type calcium channel in kidney diseases. These effects of mibefradil are related mostly to its effects on the reduction of 30
intracellular calcium level via inhibition of calcium influx (9). They may also be mediated via inhibition of renin secretion (8), inhibitory actions on protein kinase K activity (10) and platelet aggregation (9). Improvement of endothelial function (11), deactivation of sympathetic nerve (12), decrease of vascular permeability (13) may also be involved. In this study, we found that blockage of T-type calcium channel by mibefradil attenuated proteinuria and glomerular hyperfiltration, i.e. elevated Ccr, in early stage STZ-diabetic rats. Histological study also showed significantly less glomerular matrix deposition compared with controls. These data suggested a role for T-type calcium channel in early diabetic nephropathy and are in accord with the study of Cao et al, in which low-dose mibefradil ameliorates hypertrophic changes of mesenteric artery in STZ-diabetic rats without lowering of systemic blood pressure (14). As mibefradil may also affect the L-type calcium channel, the possible involvement of L-type calcium channel by mibefradil should be considered. The modest effect of amlodipine on proteinuria and glomerular matrix deposition, however, speaks to the contrary. Diabetic nephropathy is caused by hyperglycemia, and the therapeutic effect of ACEi is partly medicated by its insulin sensitizing mechanism (15). The current study, as well as our previous investigations using the same model (16), also demonstrated that ACEi reduced blood glucose level. Intervention on neither T-type nor L-type calcium channel had such effect, suggesting that improvement of insulin metabolism is not involved in the therapeutic mechanisms of CCB. With comparable systemic blood pressure reduction, combination therapy with T-type CCB mibefradil and ACEi cilazapril were more effective than mibefradil alone in reducing proteinuria and glomerular ECM and type IV collagen deposition. As for glomerular matrix volume and positive-stained type IV collagen area, cilazapril was less effective than that of combination therapy. These overall results indicate that the therapeutic effect of mibefradil on diabetic nephropathy is not dependent on the renin-angiotensin system (RAS) inhibition. This hypothesis is also supported by the results of our previous study, in which mibefradil could cause a similar degree of proteinuria and glomerular ECM reduction in spontaneous hypertensive rats with STZinduced diabetes, whilst there were no obvious changes in plasma angiotensin II and renal angiotensin type 1 receptor (AT 1) expression level (Chinese Journal of Nephrology, in press).
Hong Kong J Nephrol 2001;3(1):27-32.
J MA, et al
Figure 1. Immunohistological detection for glomerular deposition of type IV collagen in differently treated diabetic rats after uninephrectomy ( x 400 magnification). Mib = mibefradil; Aml = amlodipine; Cil = cilazapril; M+C = combined treatment with mibefradil and cilazapril; DM = uninephrectomized diabetes; UNx = uninephrectomized control.
Figure 2. Immunohistological detection for glomerular deposition of laminin in differently treated diabetic rats after uninephrectomy ( x 400 magnification). Mib = mibefradil; Aml = amlodipine, Cil = cilazapril; M+C = combined treatment with mibefradil and cilazapril; DM = uninephrectomized diabetes; UNx = uninephrectomized control.
31
T-type calcium channel and diabetic nephropathy
In summary, blockage of T-type calcium channel could ameliorate proteinuria and glomerular extracellular matrix accumulation in rats with early diabetic nephropathy. The more potent effect of T-type over Ltype CCB suggests a more important role of the former in the pathogenesis of early diabetic nephropathy. Further study is necessary to examine the mechanism of action of T-type CCB.
a s e l e c t i v e c a l c i u m T- c h a n n e l b l o c k e r, i n s t r o k e - p r o n e spontaneously hypertensive rats. J Cardiovasc Pharmacol 1996; 27:686-694. 8. Wagner C, Kramer BK, Hinder M, Kieninger M, Kurtz A. T-type and L-type calcium channel blockers exert opposite effects on renin secretion and renin gene expression in conscious rats. Br J Pharmacol 1998;124(3):579-585. 9. Hahn M, Tkachuk VA, Bochkov VN, Cheglakov IB, Clozel J-P. Blockade of receptor-operated calcium channels by mibefradil (Ro 40-5967): effects on intracellular calcium and platelet aggregation.
Acknowledgment This project was supported by China National Science Foundation (39470335).
Cardiovasc Drugs Therapy 1995;9:815-821. 10. Hermsmeyer K, Miyagawa K. Protein kinase C mechanism enhances vascular muscle relaxation by the Ca 2+ antagonist, Ro 40-5967. J Vasc Res 1996;33:71-77. 11. Boulanger CM, Desta B, Clozel J-P, Vanhoutte DM. Chronic
REFERENCES 1. Kloke HJ, Branten AJ, Huysmans FT, Wetzels JF. Antihypertensive treatment of patients with proteinuric renal diseases: Risks or benefits of calcium channel blockers? Kidney Int 1998;53:15591573. 2. Clozel J-P, Ertel EA, Ertel SI. Discovery and main pharmacological properties of mibefradil (Ro 40-5967), the first selective T-type calcium channel blocker. J Hypertens 1997;15(Suppl 5):S17-S25. 3. Parving HH, Tarnow L, Rossing P. Renal protection in diabetes: an emerging role for calcium antagonists. J Hypertens 1996;14(Suppl 4):S21-S25. 4. Luscher TF, Clozel J-P, Noll G. Pharmacology of the calcium antagonist mibefradil. J Hypertens 1997;15(Suppl 5):S11-S18. 5. Schmitt R, Clozel J-P, Iberg N, Buhler FR. Mibefradil prevents
treatment with the Ca2+ channel inhibitor Ro 40-5967 potentiates endothelium-dependent relaxations in the aorta of the hypertensive salt sensitive Dahl rat. Blood Press 1994;3:193-196. 12. Buhler FR. Cardiovascular care with the new T-type calcium channel antagonist: possible role of attendant sympathetic nervous system inhibition. J Hypertens 1997;15(Suppl 5):S3-S7. 13. Lacolley P, Poitevin P, Koen R, Levy BI. Different effects of calcium antagonists on fluid filtration of large arteries and albumin permeability in spontaneously hypertensive rats. J Hypertens 1998; 16(3):349-355. 14. Cao Z, Hulthen UL, Allen TJ, Cooper ME. Angiotensin converting enzyme inhibition and calcium antagonism attenuate streptozotocindiabetes-associated mesenteric vascular hypertrophy independently of their hypotensive action. J Hypertens 1998;16(6): 793-799.
neointima formation after vascular injury in rats. Possible role of
15. Wu Y, Lin S, Zhou J. Regulation of renal cortex cell membranes
the blockade of the T-type voltage-operated calcium channel.
insulin receptor in diabetic rats by ACEI. Chin J Endocrinol Metab
Arterioscler Thromb Vasc Biol 1995;15:1161-1165.
1999;15(2):86-89.
6. Menard J, Karam H, Veniant M, Heudes D, Bruneval P, Clozel J-P.
16. Zhang R, Hua X, Ma J, Gu Y, Zhou J, Lin S. No relation of
Effects of calcium blockade on end-organ damage in experimental
carbohydrate and lipid metabolism in ameliorating of renal function
hypertension. J Hypertens 1997;15(Suppl 3):S19-S30.
by T-type and L-type calcium channel blockage on diabetic rats.
7. Vacher E, Richer C, Fornes P, Clozel J-P, Giudicelli J-F. Mibefradil,
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Chin J Hypertens 1999;7(2):172-175.
J MA, et al
O R I G I N A L A R T I C L E
T-Type calcium channel blockage ameliorates proteinuria and renal extracellular matrix accumulation in experimental diabetic rats Ji MA, Yong GU, Ru-Zhong ZHANG, Hai-Chun YANG, Shan-Yan LIN Division of Nephrology, Fu Dan University Medical Center (former Shanghai Medical University) Hua Shan Hospital, Shanghai, China.
Abstract Calcium channels have been shown to play an important role in the pathogenesis of diabetic nephropathy. The purpose of this study is to investigate the effect of T-type calcium channel blockers in uninephrectomized diabetic rats. Two weeks after uninephrectomy, streptozotocin (65 mg/kg) was given to male Sprague-Dawley rats to induce diabetes mellitus. In the succeeding days, four groups of animals were randomized to receive 30 mg/kg mibefradil (Mib, T-type calcium channel blocker), 30 mg/kg amlodipine (Aml, L-type calcium channel blocker), 10 mg/kg cilazapril (Cil, angiotensin converting enzyme inhibitor) or 25 mg/kg Mib + 8 mg/kg Cil (M+C), which were delivered daily by gavage (n = 6-8 for each group). At the fourth week, 24-hour urine, blood and kidney samples were collected for examinations. Renal morphometric analyses and assay for type IV collagen and laminin were performed. Untreated diabetic rats (DM) exhibited overt hyperglycemia, proteinuria and albuminuria when compared with uninephrectomized (UNx) control (17.67 ±4.9 vs 2.97 ±0.72 µg/24 hours, p < 0.05), while Cil and M+C treated rats had significantly reduced urinary protein excretion (Cil: 5.53 ±1.45, M+C: 3.87 ±0.88 vs DM: 17.67 ± 4.9 µg/24 hours; p < 0.05, respectively). Mibefradil alone also ameliorated albuminuria in DM rats (7.69 ±1.87 vs 17.67 ±4.9 µg/24 hours, p < 0.05). Histopathological study revealed significantly less glomerular matrix, laminin, type IV collagen and proximal tubule cells (PTC) hypertrophy in the Cil and M+C groups when compared with the DM group (Collagen IV: DM 7.3 ±0.65 vs Mib 4.82 ±0.78, Cil 4.11 ±0.74, M+C 2. 64 ±0.66, UNx 3.71 ±0.99, p < 0.05). Albuminuria, glomerular matrix accumulation and collagen IV deposition were not changed in the Aml treated group. There was, however, significantly lower creatinine clearance and laminin volume in the Aml group. In conclusion, our study demonstrated that T-type calcium channel blockage reduced albuminuria and renal extracellular matrix accumulation in experimental diabetic rats, suggesting an important role for T-type calcium channel in the progression of diabetic nephropathy. Additional benefit can be achieved by combination with an angiotensin converting enzyme inhibitor. The mechanism of action, however, requires further investigation. Key words: Amlodipine, Calcium channel blocker (CCB), Diabetic nephropathy, Mibefradil
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Correspondence: Dr. Shan-Yan LIN, Dr. Ji MA, 12 Wulumuqi Middle Rd., Division of Nephrology, Hua-Shan Hospital, Shanghai 200040, China. Fax: (8621) 6248 8638, E-mail: [email protected], [email protected] Hong Kong Journal of Nephrology, April 2001
27
T-type calcium channel and diabetic nephropathy
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INTRODUCTION
Measurement of biochemical parameters
Calcium channel blockers (CCB) are effective antihypertensive agents commonly used in patients with diabetic mellitus. Unlike angiotensin converting enzyme inhibitor (ACEi), its renoprotective effect in diabetic patients remains unsettled (1). It has recently been shown that T-type calcium channel is involved in glomerular hypertrophy, mitogenesis and macromolecular trafficking (2), implicating a critical role in the progression of chronic renal diseases. In this study, we examined the renoprotective effects of T-type CCB, mibefradil (Ro405967), in streptozotocin-induced diabetic rats, and compared them with those of L-type CCB amlodipine, ACEi cilazapril as well as the combination of mibefradil and cilazapril.
Blood glucose, urinary protein, serum and urine creatinine were measured in the clinical laboratory by an automatic biochemical analyzer (Hitachi 7170, Japan). Creatinine clearance (Ccr) was calculated according to the formula: Ccr = urine creatinine x 24 hours urine volume/serum creatinine. Urinary albumin level was determined by radioimmunoassay (China Atomic Science Institution). Plasma angiotensin converting enzyme activity was measured by ultraviolet spectrophotometry.
SUBJECTS AND METHODS Experimental animals Thirty male Sprague-Dawley rats weighing 180 to 220 g underwent uninephrectomy after being anesthetized using intraperitoneal chloral hydrate. Two weeks after the operation, streptozotocin (STZ, Sigma) 65 mg/kg was injected intraperitoneally to induce diabetes mellitus. Diabetes was defined as heavy glycosuria (3+ to 4+) and severe hyperglycemia (> 16.7 mmol/L), which usually appeared 48 hours after STZ administration. Another six uninephrectomized rats were injected with isovolumic buffered citrate solution and served as uninephrectomized non-diabetic control (UNx). The diabetic rats were randomly allocated to receive the following treatments in the succeeding 4 weeks: mibefradil (Mib, 30 mg/kg/ day; n = 6), amlodipine (Aml, 30 mg/kg/day; n = 6), cilazapril (Cil, 10 mg/kg/day; n = 8), combined mibefradil and cilazapril (M+C, Mib 25 mg/kg/day, Cil 8 mg/kg/ day; n =7) and vehicle (DM, n = 7). All drugs or vehicle were delivered daily by gavage from the day after being confirmed as diabetic. Animals were kept in a pathogenfree environment with free access to water and standard rat chow. Blood pressure was measured weekly by tail-cuff method. After the 4-week treatment period, the animals were kept in metabolic cages for 24-hour urine collection. Blood samples were collected through the right carotid artery. The animals were then sacrificed and the left kidneys were removed, weighed, and fixed overnight in 4% buffered formaldehyde. 28
Histopathological analysis Renal tissue was embedded in paraffin, sectioned into 4 µm thickness and screened by periodic acid-Schiff (PAS) and periodic acid silver methenamine (PASM) staining. Relative area of glomerular matrix and height of renal proximal tubular epithelial cells were measured by an image analysis system (LEIKA, Germany). The cumulative area of glomeruli and intraglomerular matrix in 20 consecutive non-overlapping cortical fields on PASM stained sections were measured under the magnification of 20 x objective. The relative area of glomerular matrix was defined as the ratio of the sum of intraglomerular matrix to that of glomeruli. Cross sections of proximal tubules in 10 consecutive non-overlapping cortical fields under magnification of 10 x objective were selected to determine the outer and inner diameters of the tubules, the discrepancy between which was taken as the height of proximal tubular epithelial cell. Matrix components were demonstrated by immunohistochemical staining. After routine dewax and rehydration, the sections were treated by 0.1% buffered trypsin at 37 oC for 20 minutes for antigen retrieval. Endogenous hydrogen peroxidase was quenched by 3% H2O2/methanol. After blocking in 10% goat serum, the sections were incubated with rabbit anti-rat collagen IV and laminin polyclonal antibodies (a gift from the Department of Pathology, Shanghai Medical University) at room temperature for 30 minutes. The sections were then treated with goat-anti-rabbit IgG conjugated with horseradish peroxidase and developed by 3,3’-diaminobenzidine (DAKO) solution. Under magnification of 10 x objective, 20 consecutive nonoverlapping cortical fields were analyzed morphometrically for intraglomerular collagen IV and laminin by LEIKA image analysis system.
Hong Kong J Nephrol 2001;3(1):27-32.
J MA, et al
Statistical analysis
Data were presented as mean ±standard error. Data from the six groups were analyzed by analysis of variance (ANOVA) and the level of significance was defined as p < 0.05.
with DM, the decreases in urinary albumin excretion rate in rats treated with Mib, Cil and their combination (M+C) were significant (p < 0.05). The difference in albuminuria between Mib and Cil was not significant (p > 0.05) (Table 1).
RESULTS
Creatinine clearance
Blood pressure Results of the mean arterial pressure (MAP) measured by tail-cuff method were listed in table 1. Though no difference in MAP was found in Mib, Aml, Cil and M+C, all these four groups had a significant decrease in MAP compared with DM or UNx (all p < 0.05).
There was elevated Ccr in all diabetic rats compared with the UNx control (all p < 0.05). When compared with the DM, it was decreased in all treated groups (all p < 0.05). Ccr was significantly lower in the Cil and M+C groups than that in the Mib and Aml groups (all p < 0.05) (Table 1).
Ratio of kidney weight to body weight Blood glucose level All STZ-injected rats had substantially elevated blood glucose (all p < 0.05). Interestingly, it was lower in the Cil and M+C group compared with the DM group (both p < 0.05).
Ratio of kidney weight to body weight (KW/BW) in each group were: Mib 9.2 ±1.2, Aml 9.1 ±0.7, Cil 7.9 ± 0.5, M+C 7.8 ±0.5, DM 9.6 ±0.9, UNx 6.8 ±0.7 x 10 -3. Significant difference was only detected between the DM and the UNx group (p < 0.05).
Proteinuria and albuminuria
Histopathological analyses
All diabetic rats except in the M+C group showed significantly higher urinary protein and albumin excretion rate compared with the UNx (all p < 0.05). The proteinuria, however, was decreased in all treated groups (all p < 0.05), and even less in M+C and Cil than that of Mib and Aml (all p < 0.05). Moreover, compared
The results of morphometric examination for relative glomerular matrix and height of proximal tubular cells (PTC) were shown in table 2. The relative glomerular matrix area was significantly higher in all diabetic groups than that in UNx control (all p < 0.05). Compared with that in UNx, PTC height was also increased in DM, Aml
Table 1. General conditions and biochemical parameters in each group.
Experimental group
n
Mibefradil Amlodipine Cilazapril Mibefradil+Cilazapril Uninephrectomized diabetes Uninephrectomized control
6 6 8 7 7 6
MAP Serum ACE activity Blood glucose (mmHg) (nM/min/mL) (mmol/L) 140.4 ±13.4 25.6 ±1.71 110.3 ±5.81 109.8 ±3.51 160.3 ±12.0 23.6 ±2.41 1 1 107.2 ±3.1 45.6 ±8.7 20.1 ±2.81,2 1 1 113.1 ±4.7 34.5 ±4.5 20.5 ±0.91,2 139.6 ±5.2 180.9 ±21.6 22.4 ±1.81 130.4 ±6.6 220.6 ±32.4 6.8 ±0.7
Proteinuria (mg/24 h) 27.2 ±9.71,2,3 30.6 ±4.81,2,3 21.8 ±3.21,2 18.1 ±4.82 50.7 ±11.51 14.8 ±4.8
Albuminuria (µg/24 h) 7.69 ±1.871,2,3 15.11 ±4.031,3 5.53 ±1.451,2 3.87 ±0.881,2 17.67 ±4.901 2.97 ±0.72
Creatinine clearance (mL/min) 2.53 ±0.331,2,3 2.20 ±0.171,2,3 1.81 ±0.271,2 1.88 ±0.281,2 3.44 ±0.441 1.25 ±0.17
1
p < 0.05 compared with uninephrectomized control. p < 0.05 compared with uninephrectomized diabetes. 3 p < 0.05 compared with mibefradil+cilazapril. 2
Table 2. Relative glomerular matrix area, height of PTC, and glomerular type IV collagen and laminin deposition.
Experimental group Mibefradil Amlodipine Cilazapril Mibefradil+Cilazapril Uninephrectomized diabetes Uninephrectomized control
n 6 6 8 7 7 6
Glomerular matrix 0.301 ±0.0251,3 0.307 ±0.0261,3 0.223 ±0.0301,2,3 0.191 ±0.0181,2 0.322 ±0.0351 0.179 ±0.021
PTC height (µm) 4.99 ±0.271 5.20 ±0.211 3.89 ±0.242 3.55 ±0.202 6.07 ±0.381 4.11 ±0.18
Type IV collagen 4.82 ±0.782,3 6.54 ±0.751,3 4.11 ±0.742,3 2.64 ±0.062 7.30 ±0.651 3.71 ±0.99
Laminin 5.06 ±0.981,2 5.35 ±0.671,2 3.87 ±1.022 3.12 ±0.062 7.99 ±0.881 3.70 ±0.49
1
p < 0.05 compared with uninephrectomized control. p < 0.05 compared with uninephrectomized diabetes. 3 p < 0.05 compared with mibefradil+cilazapril. 2
29
T-type calcium channel and diabetic nephropathy
and Mib (all p < 0.05), while there was no difference for Cil and M+C (p > 0.05). The expression of glomerular type IV collagen and laminin were listed in table 2. Positively stained area for type IV collagen was more extensive in DM and Aml groups than that in UNx. Furthermore, glomerular type IV collagen deposition in M+C was less when compared with that in Mib and Cil (both p < 0.05) (Fig. 1). Similarly, the positively stained area of glomerular laminin in DM, Aml and Mib was larger than that in UNx (all p < 0.05). Compared with that in DM, it was smaller in all treated groups (all p < 0.05) (Fig. 2).
DISCUSSION Intraglomerular hypertension and hyperfiltration is an important mechanism for progression of diabetic nephropathy. Extracellular matrix (ECM) accumulation caused by proteinuria and a variety of growth factors could lead to glomerulosclerosis. Beside intensive glycemic control, interventions targeting systemic and intraglomerular hypertension, proteinuria and extracellular matrix accumulation are also being actively investigated (3). Calcium channels are classified into L-, T-, P- and Ntype according to their activation characteristics. Conventional calcium channel blockers are all of the Ltype, which can further be divided into three groups: dihydropyridine (e.g., nifedipine, amlodipine, felodipine, nicardipine, etc.), phenylalkylamines (e.g., verapamil), and benzothiazepines (e.g., diltiazem). Despite their potent antihypertensive effect, the protective effects for progressive renal deterioration are still controversial. Several studies have demonstrated a more potent antiproteinuria effect by non-dihydropyridine compared to dihydropyridine CCB. The beneficial effects on glomerular sclerosis and proteinuria, however, are still inferior to those of ACEi (1). A body of evidence from in vivo and in vitro studies showed that T-type calcium channel plays an important role in some pathophysiological changes of cardiovascular diseases. Blockage of the channel results in blood pressure reduction and attenuation of pathological cellular growth. T-type calcium channel also mediates vascular smooth muscle cell contraction (4). Blockage of T-type calcium channel by Ro 40-5967 (mibefradil) could inhibit the proliferation and hypertrophy of smooth muscle cell while L-type CCB e.g. verapamil, amlodipine and felodipine are devoid of such effect (5). Moreover, in DOCA salt-induced (6) and spontaneous (7) hypertensive rats, mibefradil significantly ameliorates proteinuria and glomerular changes, indicating the role of T-type calcium channel in kidney diseases. These effects of mibefradil are related mostly to its effects on the reduction of 30
intracellular calcium level via inhibition of calcium influx (9). They may also be mediated via inhibition of renin secretion (8), inhibitory actions on protein kinase K activity (10) and platelet aggregation (9). Improvement of endothelial function (11), deactivation of sympathetic nerve (12), decrease of vascular permeability (13) may also be involved. In this study, we found that blockage of T-type calcium channel by mibefradil attenuated proteinuria and glomerular hyperfiltration, i.e. elevated Ccr, in early stage STZ-diabetic rats. Histological study also showed significantly less glomerular matrix deposition compared with controls. These data suggested a role for T-type calcium channel in early diabetic nephropathy and are in accord with the study of Cao et al, in which low-dose mibefradil ameliorates hypertrophic changes of mesenteric artery in STZ-diabetic rats without lowering of systemic blood pressure (14). As mibefradil may also affect the L-type calcium channel, the possible involvement of L-type calcium channel by mibefradil should be considered. The modest effect of amlodipine on proteinuria and glomerular matrix deposition, however, speaks to the contrary. Diabetic nephropathy is caused by hyperglycemia, and the therapeutic effect of ACEi is partly medicated by its insulin sensitizing mechanism (15). The current study, as well as our previous investigations using the same model (16), also demonstrated that ACEi reduced blood glucose level. Intervention on neither T-type nor L-type calcium channel had such effect, suggesting that improvement of insulin metabolism is not involved in the therapeutic mechanisms of CCB. With comparable systemic blood pressure reduction, combination therapy with T-type CCB mibefradil and ACEi cilazapril were more effective than mibefradil alone in reducing proteinuria and glomerular ECM and type IV collagen deposition. As for glomerular matrix volume and positive-stained type IV collagen area, cilazapril was less effective than that of combination therapy. These overall results indicate that the therapeutic effect of mibefradil on diabetic nephropathy is not dependent on the renin-angiotensin system (RAS) inhibition. This hypothesis is also supported by the results of our previous study, in which mibefradil could cause a similar degree of proteinuria and glomerular ECM reduction in spontaneous hypertensive rats with STZinduced diabetes, whilst there were no obvious changes in plasma angiotensin II and renal angiotensin type 1 receptor (AT 1) expression level (Chinese Journal of Nephrology, in press).
Hong Kong J Nephrol 2001;3(1):27-32.
J MA, et al
Figure 1. Immunohistological detection for glomerular deposition of type IV collagen in differently treated diabetic rats after uninephrectomy ( x 400 magnification). Mib = mibefradil; Aml = amlodipine; Cil = cilazapril; M+C = combined treatment with mibefradil and cilazapril; DM = uninephrectomized diabetes; UNx = uninephrectomized control.
Figure 2. Immunohistological detection for glomerular deposition of laminin in differently treated diabetic rats after uninephrectomy ( x 400 magnification). Mib = mibefradil; Aml = amlodipine, Cil = cilazapril; M+C = combined treatment with mibefradil and cilazapril; DM = uninephrectomized diabetes; UNx = uninephrectomized control.
31
T-type calcium channel and diabetic nephropathy
In summary, blockage of T-type calcium channel could ameliorate proteinuria and glomerular extracellular matrix accumulation in rats with early diabetic nephropathy. The more potent effect of T-type over Ltype CCB suggests a more important role of the former in the pathogenesis of early diabetic nephropathy. Further study is necessary to examine the mechanism of action of T-type CCB.
a s e l e c t i v e c a l c i u m T- c h a n n e l b l o c k e r, i n s t r o k e - p r o n e spontaneously hypertensive rats. J Cardiovasc Pharmacol 1996; 27:686-694. 8. Wagner C, Kramer BK, Hinder M, Kieninger M, Kurtz A. T-type and L-type calcium channel blockers exert opposite effects on renin secretion and renin gene expression in conscious rats. Br J Pharmacol 1998;124(3):579-585. 9. Hahn M, Tkachuk VA, Bochkov VN, Cheglakov IB, Clozel J-P. Blockade of receptor-operated calcium channels by mibefradil (Ro 40-5967): effects on intracellular calcium and platelet aggregation.
Acknowledgment This project was supported by China National Science Foundation (39470335).
Cardiovasc Drugs Therapy 1995;9:815-821. 10. Hermsmeyer K, Miyagawa K. Protein kinase C mechanism enhances vascular muscle relaxation by the Ca 2+ antagonist, Ro 40-5967. J Vasc Res 1996;33:71-77. 11. Boulanger CM, Desta B, Clozel J-P, Vanhoutte DM. Chronic
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