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Renoprotective activity of aliskiren, a renin inhibitor in cyclosporine A induced hypertensive nephropathy in dTG mice
Pharmacological Reports 66 (2014) 62–67
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Original research article
Renoprotective activity of aliskiren, a renin inhibitor in cyclosporine A induced hypertensive nephropathy in dTG mice Megha S. Saraswat a, Veeranjaneyulu Addepalli a,*, Mukul Jain b, Vishwanath D. Pawar b, Rakesh B. Patel b a b
SPP SPTM, SVKM’s NMIMS, Mumbai, Maharashtra, India Department of Pharmacology, Zydus Research Centre, Ahmedabad, Gujarat, India
A R T I C L E I N F O
Article history: Received 12 June 2012 Received in revised form 29 June 2013 Accepted 2 August 2013 Available online 31 January 2014 Keywords: Hypertensive nephropathy Aliskiren Cyclosporine A TNF-a IL-6 TGF-b1 KIM-1
A B S T R A C T
Background: Hypertensive nephropathy is moving up the charts to number 2 after diabetic nephropathy in terms of diagnostic frequency cited as causing end stage renal disease (ESRD). Method: Hypertensive nephropathy was produced in mildly hypertensive C57BL/6-(hREN)/(hAGT) double transgenic (dTG) mice with 20 mg/kg of cyclosporine A (CsA) administered subcutaneously (sc) daily for 28 days. CsA dose 20 mg/kg was selected for the study as this dose offered significant alteration in blood pressure, biochemical parameters and moderate nephropathy in kidney. Effect of aliskiren oral treatment twice daily consequently for 28 days at 10 mg/kg body weight was evaluated against CsA induced hypertensive nephropathy. Systolic blood pressure (SBP) was measured by non invasive tail cuff method. Kidney function test (blood urea nitrogen, serum creatinine, urea and uric acid) and kidney injury biomarker (tumor necrosis factor-alpha (TNF-a) and interlekin-6) level was assessed in serum, TNF-a, IL-6, transforming growth factor-beta1 (TGF-b1) and kidney injury molecule-1 (KIM-1) was assayed in kidney homogenate. Urinary KIM-1 levels were assessed as an early biomarker of nephropathy. Result: Significant hypertensive nephropathy and increase in serum levels of biomarkers was observed in CsA treated animals when compared with Control group. Aliskiren treatment elicited significant renoprotection by preventing the increase in blood pressure and levels of serum biomarkers and also reduced the nephropathic alterations in the kidney histoarchitecture. Conclusion: A correlation between pharmacological, biochemical and histological findings has been established in mouse model. The present findings have indicated the renoprotective activity of aliskiren in CsA induced hypertensive nephropathy, which may be due to its antihypertensive, anti-inflammatory as well as anti-apoptopic action. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Hypertension is a major cardiovascular risk factor associated with significant morbidity and mortality worldwide and will increase in importance as a public health problem by 2020 [22]. Hypertensive nephropathy, a consequence of chronic high blood pressure is moving up the charts after diabetic nephropathy in terms of diagnostic frequency cited as causing end stage renal disease (ESRD) [16]. Complications often associated with hypertensive nephropathy include glomerular damage resulting in inflammatory responses and compromised kidney function that seems to be superimposed on the intrinsic phenotype of the underlying renal disease [3]. Findings from the recent investigations of experimental animal models have suggested that
* Corresponding author. E-mail address: [email protected] (V. Addepalli).
chemically induced nephropathy share many features which are common to human nephropathy and therefore it was explored in the present study. Cyclosporine A (CsA) is an immunosuppressive drug commonly used in organ transplantation, but its use is complicated by the development of hypertension in 40–100% of patients [31,33]. CsA is also associated with the development of microvascular and tubulointerstitial disease in the kidney, consisting of hyalinosis of the afferent arteriole in association with tubular atrophy and ‘‘striped’’ interstitial fibrosis [2,23]. The pathogenesis of the renal injury is thought to be mediated by renal vasoconstriction and ischemia [26], and this appears to result from direct effects of CsA as well as by the ability of CsA to alter intrarenal vasoactive mediators such as renin, endothelin-1, and nitric oxide (NO) [30]. Therefore, in light with these findings CsA was employed for inducing hypertensive nephropathy in the present study. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.08.005
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Cyclosporine Oral Solution USP, 100 mg/ml, Panacea Biotech Ltd., New Delhi, India) was administered once daily as a subcutaneous injection of 20 mg/kg for 28 days. The doses were finalized on the basis of pilot studies carried out at ZRC, Ahmedabad (results not shown). Olive oil used to prepare cyclosporine A dilutions was procured from local market and administered daily at a dose volume of 2 ml/kg subcutaneously.
Pressure (JNC7) establishes that the goal of antihypertensive therapy is to reduce cardiovascular and renal morbidity and mortality [5]. Since Ang II is reported to play a significant role in the progression of chronic kidney disease (CKD), its pharmacological inhibition with angiotensin converting enzyme inhibitors (ACEIs) or angiotensin II receptor antagonists (ARBs) significantly reduces markers of renal injury and can even attenuate the progression of glomerular and tubulointerstitial fibrosis. However, the observation that treatment with ACEI or ARBs alone does not completely inhibit the progression of CKD raises the possibility that other pathways besides the formation of Ang II may be involved in CKD progression. By attenuating feedback inhibition of renin release, ACEIs and ARBs effects lead to an increase in plasma renin concentration and activity, rendering incomplete inhibition of the RAS system [1,21]. Moreover, inhibition of ACE causes an increase in angiotensin I, which is then available for conversion to angiotensin II by ACE-independent pathways not blocked by ACEIs, namely cathepsins and tonins [9,13,34]. Consequently, despite adequate blood pressure control, angiotensin II levels increase aldosterone levels to certain degrees, rendering these inflammatory molecules free to play an active role in tissue remodeling and scarring. Henceforth, it is tempting and reasonable to assess the effects of a different pharmacologic strategy that blocks the RAS upstream completely. Thus there is an unmet need to develop new strategy to target hypertension mediated end organ damage along with improved efficacy and safety. Aliskiren is a first-in-class oral renin inhibitor, developed by Novartis in conjunction with the biotech company Speedel [35]. Aliskiren is the first known representative of a new class of nonpeptide orally active renin inhibitors that block the RAS at its rate-limiting step and induce a net reduction in plasma renin activity and angiotensin II and aldosterone levels [11,15]. Aliskiren with established antihypertensive action elicits a notorious renoprotective effect such that, aliskiren can further decrease the risk of renal disease progression [32]. Therefore, the present work focuses to evaluate the ameliorating effect of administering aliskiren, a renin inhibitor, on further disease progression in cyclosporine A induced hypertensive nephropathy.
After 25 days, systolic blood pressure (SBP) was measured using non invasive blood pressure monitor (Columbus Instruments, USA) by the tail cuff method wherein the average of the two days SBP was recorded and considered.
Materials and methods
Assessment of kidney injury biomarkers
Animals
At the end of the treatment period mice were housed in metabolic cages for 24 h for urine collection which was then frozen at 80 8C till analysis. The urine samples were then employed for the estimation of KIM-1. After 24 h period, the blood was obtained from the retroorbital plexus of mice from all the groups after being lightly anesthetized with ether [29]. The blood was allowed to flow into clean dry centrifuge tube and left to stand for 30 min before centrifugation to avoid hemolysis. Serum was separated by centrifugation for 5 min at 4000 rpm and stored at 20 8C until analysis. The clear supernatant, serum was separated and collected into a dry clean tube for the biochemical tests: BUN and serum levels of creatinine, urea, uric acid and the ELISA test for TNF-alpha and IL-6. Animals were then sacrificed by cervical dislocation and the kidneys were quickly isolated, washed with saline, blotted dry on filter paper. One kidney of each group was snap frozen in dry ice for preparation of Kidney Homogenates wherein kidneys were thawed in 2 ml sterile phosphate buffered saline (PBS) and homogenized three times for 15 s with a Brinkmann homogenizer (Brinkmann Instruments, Mississauga, Ontario, Canada). Kidney homogenates were then subjected to three freeze-thaw cycles at 70 8C for 4 h and 37 8C for 15 min. Supernatants were separated from tissue residues by centrifugation twice at 12,000 g for 10 min at 4 8C and then employed for the ELISA estimation of TNF-alpha, IL-6, TGF-beta1 and KIM-1.
Thirty-six healthy female C57BL/6-(hREN)/(hAGT) double transgenic (dTG) mice were used throughout the experiment with initial body weight of 20–25 g. Animals were bred at animal breeding facility of Zydus Research Centre (ZRC), Ahmedabad. Institutional Animal Ethical Committee approved the experimental protocol as per the guidance of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. Animals were maintained in the specific pathogen-free animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC). Mice in groups of 6 of similar sex were housed in clean, sterilized solid floor polycarbonate individually ventilated (IVC) cages. Animals were kept in an environmentally controlled room designed to maintain a temperature of 22 3 8C and a relative humidity of 30–70% with at least 14 fresh air changes per hour. Drugs and chemicals Aliskiren (Tekturna1, 300 mg Tablet, Novartis, USA) was procured and it was administered twice daily as an oral dose of 10 mg/kg for 28 days. Cyclosporine A (PanimunBioral1,
Diagnostic kits Mouse tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6) estimations were performed using commercially available sandwich ELISA kits (R&D, Minneapolis, MN, USA). The detection limit of each kit is 1000 pg/ml for both IL-6 and TNF-alpha. Mouse kidney injury molecule-1 (KIM-1) and transforming growth factor beta-1 (TGF b-1) estimations were performed using commercially available competitive ELISA kits (MyBiosource, USA). The detection limit of each kit is 25 ng/ml for KIM-1 and 1000 pg/ml for TGF b-1. Kidney Function Tests (blood urea nitrogen (BUN), creatinine, urea, uric acid) estimation kits were obtained and analysis was performed using Roche/Hitachi Cobas – C system. Experimental design Thirty-six mice were divided into three groups as follows: Group 1: Control group (subcutaneously received 2 ml/kg olive oil daily for 28 days); Group 2: CsA group (subcutaneously received 20 mg/kg of CsA daily for 28 days); Group 3: Aliskiren group (orally received 10 mg/kg of aliskiren twice daily for 28 days). Assessment of blood pressure
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Histopathology The second kidney from each group was stored into 10% paraformaldehyde solution and embedded in wax. After dewaxing, 4 mm sections were processed and stained with Hematoxylin and Eosin (H&E).
Table 1 Functional alterations in dTG C57 CsA challenged mice. BUN, serum creatinine, serum urea and serum uric acid were used as indicators of renal function. Results are expressed as mean SEM. N = 6 for each treatment group.
BUN (mg/dl) Serum creatinine (mg/dl) Serum urea (mg/dl) Serum uric acid (mg/dl)
Statistical analysis All data were expressed as mean standard error mean (SEM). All data were compared by one-way analysis of variance (ANOVA). Each test was followed by the Bonferroni’s multiple comparison test. p values less than 0.05 (p < 0.05) were considered statistically significant. Result Effect on blood pressure Systolic blood pressure (SBP) in mice was measured and observed that mice challenged with CsA (20 mg/kg, sc) showed significant elevation of blood pressure as compared to Control group. Aliskiren (10 mg/kg, po, bid) treated CsA challenged animals showed significant prevention in blood pressure as compared to CsA group. These results suggested that aliskiren could antagonize the hypertensive effect of CsA in dTG C57 mice (Fig. 1). Effect on renal function In order to evaluate renal functional alterations in mice, kidney function tests were performed in the serum at study termination. These estimations indicated deterioration of the renal function indicating kidney disorder wherein CsA significantly increased serum biomarker concentration (BUN, serum creatinine, serum urea and serum uric acid). These results suggested significant decline in renal function of CsA challenged mice. Chronic treatment with aliskiren significantly prevented the anomalous rise in concentrations of serum biomarkers as compared to CsA group. These, results suggested significant improvement in the renal function with aliskiren treatment as compared to CsA group and directed toward it renoprotective action (Table 1).
****
Tail Cuff Systolic BP (mmHg)
120
110
####
100
90
*
Control
CsA
Aliskiren
20.60 0.75 0.22 0.02 44.20 1.9 1.98 0.19
43.00 5.18*** 0.30 0.01** 92.00 1.2*** 2.95 0.17**
22.33 0.56## 0.22 0.02## 44.67 2.39### 1.80 0.15###
and # indicates statistically significant difference within groups. p < 0.001 as compared to Control and ##p < 0.01, ###p < 0.001.
**
p < 0.01,
***
Effect on cytokine levels The changes in the cytokine levels (TNF-a, IL-6 and TGFb-1) were estimated in the experimental groups with the respective ELISA methods. CsA group significantly raised the TNF-a level both in serum as well as the kidney tissue homogenate. Moreover, CsA increased the IL-6 concentration in the serum and kidney tissue and tissue TGF b-1 levels were also raised. Chronic treatment with aliskiren significantly prevented the increase in TNF-a (Fig. 2A and B) and IL-6 (Fig. 2C and D) levels both in the serum as well as the kidney homogenates as compared to CsA group. Secondly, aliskiren could also significantly prevent the increase in levels of TGF b-1 in the kidney homogenates (Fig. 2E). Taken together these results became indicative of the anti-inflammatory and property of aliskiren that could substantiate its role in the prevention of hypertension induced renal damage. Effect on newer kidney injury biomarker Estimation of the newer kidney injury biomarker i.e. Kidney Injury Molecule-1 (KIM-1) was performed with the help of competitive ELISA method. At 28 day after CsA administration, there was significant increase in the urinary and tissue KIM-1 concentration. However, aliskiren prevented the rise in KIM-1 concentrations in mouse urine as well as kidney tissue homogenates and thereby indicated renoprotective action (Fig. 3). Histopathological alterations Sections of mouse kidney stained with Hematoxylin and Eosin (H&E) were examined by light microscopy. Nephropathic changes in kidney architecture in different experimental groups were observed. Glomerulosclerosis, tubular dilatation and atrophy, interstitial cell infilteration and interstitial fibrosis, hyalinization of the glomerular foci, proteinaceous material in the tubules, vacuolar degeneration of tubular epithelium, hyperplasic tubules, Necrosis, etc. were observed to indicate nephropathic changes. Animals challenged with CsA showed significant nephropathic alterations as described compared to that of Control group (Fig. 4A and B). Aliskiren treated animals did not elicit such nephropathic changes and were found to be similar to normal (Fig. 4C). Hence, aliskiren could prevent the kidney from undergoing nephropathic changes as those seen in CsA group. Discussion
80 Control
CsA
Aliskiren
Fig. 1. Effect of drug treatment on systolic blood pressure (SBP) after 25 days. All values are expressed as mean SEM (N = 10). **** indicates p < 0.0001 as compared to Control group, #### indicates p < 0.0001 as compared to CsA group. Data was analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test.
Many chronic renal diseases evolve progressively with few outward clinical symptoms until the onset of renal dysfunction and, often, structural damage. An appropriate therapeutic agent is therefore one that effectively treats existing as well as developing renal injury. In order to address this critical issue, a mouse model that developed renal lesions similar to clinical nephropathic condition was employed in the present study. Documented reports have indicated that human renin (hREN) and human angiotensi-
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B
*
25
Serum TNF-apha (pg/ml)
TNF-apha in mouse kidney tissue (pg/mg of protein)
A
20 15 10
*
5 0 Control
CsA
Aliskiren
***
1200
#
1000 800 600 400 200 0 Control
CsA
Aliskiren
D
Serum IL-6 (pg/ml)
30
IL-6 in mouse Kidney tissue (pg/mg of protein)
C
1400
*
20
10
# 0 Control
CsA
1200
**
1000
Aliskiren
800 600
#
400 200 0
Control
CsA
Aliskiren
TGF Beta-1 in mouse Kidney Tissue (pg/mg of protein)
E 200
***
150 100
### 50 0 Control
CsA
Aliskiren
Fig. 2. Effect of drug treatment on cytokine levels. (A) Effect on TNF-a level in mouse serum, (B) effect on TNF-a level in mouse kidney tissue homogenate, (C) effect on IL-6 level in mouse serum, (D) effect on IL-6 level in mouse kidney tissue homogenate, (E) effect on TGF-b1 level in mouse kidney tissue homogenate. All values are expressed as mean SEM, N = 6. * and # indicates statistically significant difference within groups. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to Control and #p < 0.05, ##p < 0.01, ### p < 0.001.
hypertensive nephropathic response was verified. The following findings explain, at least partly, aliskiren counteraction of CSA induced hypertensive response wherein aliskiren significantly prevented the rise in systolic blood pressure induced due to chronic CsA administration. These findings established convincing evidence that aliskiren offsets the hypertensive effect of CSA via ameliorating significant RAS derangements caused by CSA. We reasoned that concurrent exposure to aliskiren would mitigate or at least minimize the hypertensive action of CSA and potentially related nephropathic manifestations. This assumption was ascertained in the current study because the hypertensive effect of CSA
nogen (hAGT) gene – harboring double transgenic (dTG) rats develop hypertension and severe hypertension-related end-organ damage due to local Ang II formation in the heart, kidney, and vasculature [10,19]. The current study is the first to report on the potential protective effect of the orally acting direct renin inhibitor drug aliskiren against the hypertensive and deleterious nephropathic effects of chronic CsA administration in dTG mice. Initially, we verified that CsA did in fact induce renal dysfunction in dTG C57 mice. We examined a range of well-established patho-physiological features associated with CsA nephropathy. Further, concurrent aliskiren administration virtually abolished the CsA induced
B
Urine KIM-1 (ng/ml)
6
* #
4
2
0 Control
CsA
Aliskiren
KIM-1 in mouse Kidney Tissue (ng/mg of protein)
A
2.0
***
1.5 1.0 0.5
### 0.0 Control
CsA
Aliskiren
Fig. 3. Effect of drug treatment on kidney injury molecule-1. (A) KIM-1 level in mouse serum and (B) KIM-1 level in mouse kidney tissue homogenate. All values are expressed as mean SEM, N = 6. * and # indicates statistically significant difference within groups. *p < 0.05, ***p < 0.001 as compared to Control and #p < 0.05, ###p < 0.001.
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Fig. 4. Histopathological alterations in kidney tissue. (A) Control group showed overall no abnormalities except few instances of mild tubular dilatation and presence of proteinaceous material in the tubule were observed, (B) CsA Group eilicited presence of proteinaceous material in the tubules, foci of lymphocytic infiltration (FLI), dilatation, basophilic tubules, hyalinization of the glomerular foci, vacuolation, and necrotic changes and (C) Aliskiren group elicited overall improvement as compared to CsA group.
disappeared when mice were treated simultaneously with aliskiren. In order to evaluate renal functional alterations in mice, kidney function tests were performed. Dieterle et al. have demonstrated that serum creatinine in conjunction with other kidney function parameters are excellent indicators of renal dysfunction [7]. In the present study, significantly elevated levels of creatinine, urea and uric acid in mice serum and increased BUN levels were evident after 28 day administration of CsA which became indicative of its nephrotoxic response. However, concurrent treatment with aliskiren elicited significant prevention of the renal dysfunction caused due to chronic CsA administration. This substantiated the protective role of aliskiren in CsA induced renal dysfunction. Data in animal models suggest that the inflammatory response during kidney injury is dysregulated. The effect of kidney injury on the production and elimination of proinflammatory cytokines may be a key mechanism by which patients with kidney injury have increased distant organ dysfunction and increased mortality. Since cytokine production also increases in the kidney, renal cytokine production may contribute to renal injury and cause the increase in serum cytokines. Circulating cytokines may then contribute to extra renal organ injury. Hence, for the purposes of this study putative nephrotoxicity markers were designated as those identified in the literature as having a significant role in nephrotoxicity in a number of in vitro and in vivo models of renal disease. These included cytokines like TNF-a, IL-6 and TGF-b1. In patients with chronic renal failure not on dialysis, serum IL-6 and TNF-alpha are increased, suggesting that impaired kidney function results in increased cytokine levels. Additionally, increasing levels of serum IL-6 are significantly correlated with decreasing levels of glomerular filtration rate, further suggesting that impaired kidney function may affect cytokine clearance. Although filtration and excretion cytokines may occur, current evidence suggests that cytokines are not primarily cleared via filtration and excretion, but may be filtered, reabsorbed and metabolized by the proximal tubule [12]. ESRD patients have lower urinary IL-6 receptor excretion than controls [20]. Recently links between the IL-6 system and the residual renal function, showing an association between sIL-6R and the progression rate of renal function in the predialysis phase, as well as an association between changes in glomerular filtration rate and changes in IL-6 during peritoneal dialysis (PD) treatment were described [25]. From the present study it is inferred that mice challenged with CsA elicit significant rise in the levels of TNF-alpha and IL-6 both in the serum as well as in the kidney homogenates which demonstrates signs of inflammation and the deterioration of renal function that can be associated with a significant increase in serum cytokine levels [6]. The profibrotic cytokine TGF-b1 is a major contributor to
tubulointerstitial fibrosis and nephropathy [17]. Increased TGF-
b1 levels after CsA treatment suggests a role for TGF-b1 in the current model, and this is in agreement with previous studies [18,27,28]. Moreover, despite some initially promising results in experimental models of CsA nephropathy [8,17], TGF-b1 blockade has not yet translated into an effective therapeutic strategy in human patients. However, concomitant treatment with aliskiren significantly prevented the inflammatory responses of CsA that led to deleterious nephrotoxic reaction. The effect of aliskiren on the cytokine levels, as observed from the present study indicated its protective action as an antiinflammatory over and above its antihypertensive activity. This property of aliskiren can be attributed to its renin blocking action that in turn prevented the detrimental effects of angiotensin II which is reported to play a key role in the progression of CsA induced nephrotoxicity [4,24] Another evidence that suggested the protective role of aliskiren against CsA induced nephrotoxicity was the evaluation of its effect on newer kidney injury biomarker KIM-1. KIM-1 has recently been identified as the first non-myeloid phosphatidylderine receptor that confers a phagocytic phenotype on injured epithelial cells both in vivo and in vitro [14]. Urinary KIM-1 is a non-invasive, rapid, sensitive and reproducible biomarker for the early detection of kidney injury. In the present study, a sandwich KIM-1 ELISA test was used. Initially, we verified the role of KIM-1 in the pathophysiology of CsA induced nephropathy wherein it was evident that CsA challenge for 28 days illustrated significant increase in the urinary as well as kidney tissue KIM-1 concentration. Following this, it was observed that concurrent treatment with aliskiren significantly prevented the rise in the level of KIM-1 both in the urine and the mouse kidney homogenate. This further substantiated the renoprotective action of aliskiren against CsA induced nephrotoxicity. Nephropathic changes in kidney architecture in different experimental groups were observed where sections of mouse kidney stained with H&E were examined by light microscopy. Glomerulosclerosis, tubular dilatation and atrophy, interstitial cell infilteration and interstitial fibrosis, hyalinization of the glomerular foci, proteinaceous material in the tubules, vacuolar degeneration of tubular epithelium, hyperplasic tubules, necrosis, etc. were observed to indicate nephropathic changes. Groups challenged with CsA showed significant nephropathic alterations as described. However, aliskiren treated animals did not elicit nephropathic changes and showed improved architecture similar to those observed in the Control group. Hence, histomorphologically also it could be indicated that aliskiren indeed is a protective agent against CsA induced hypertensive nephropathy. This research has identified a number of potential markers of CsA hypertensive nephropathy. Further investigation will determine if these indicators translate to a clinical setting, and to what extent they may be useful as therapeutic targets.
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[15] Jeunemaitre X, Menard J, Nussberger J, Guyenne TT, Brunner HR, Corvol P. Plasma angiotensins, renin, and blood pressure during acute renin inhibition by CGP 38 560 A in hypertensive patients. Am J Hypertens 1989;2:819–27. [16] Klag MJ, Whelton PK, Randall BL, Neaton JD, Brancati FL, Ford CE, et al. Blood pressure and end-stage renal disease in men. N Engl J Med 1996;334:13–8. [17] Ling H, Li X, Jha S, Wang W, Karetskaya L, Pratt B, et al. Therapeutic role of TGFbeta-neutralizing antibody in mouse cyclosporin A nephropathy: morphologic improvement associated with functional preservation. J Am Soc Nephrol 2003;14:377–88. [18] Lloberas N, Torras J, Alperovich G, Cruzado JM, Gimenez-Bonafe P, HerreroFresneda I, et al. Different renal toxicity profiles in the association of cyclosporine and tacrolimus with sirolimus in rats. Nephrol Dial Transplant 2008;23:3111–9. [19] Luft FC, Winberger MH. Antihypertensive therapy with aliskiren. Kidney Int 2008;73:679–83. [20] Memoli B, Postiglione L, Cianciaruso B, Bisesti V, Cimmaruta C, Marzano L, et al. Role of different dialysis membranes in the release of interleukin-6 soluble receptor in uremic patients. Kidney Int 2000;58:417–24. [21] Mooser V, Nussberger J, Jullierat L. Reactive hyperreninemia is a major determinant of plasma angiotensin II during ACE inhibition. J Cardiovasc Pharmacol 1990;15:276–82. [22] Murray CJ, Lopez AD. Alternative projections of mortality and disability by cause 1990–2020: global burden of disease study. Lancet 1997;349:1498– 504. [23] Myers BD. Cyclosporine nephrotoxicity. Kidney Int 1986;30:964–74. [24] Olyaei AJ, de Mattos AM, Bennett WM. Nephrotoxicity of immunosuppressive drugs: new insight and preventive strategies. Curr Opin Crit Care 2001;7:384–9. [25] Pecoits-Filho R, Heimbu¨rger O, Lindholm B, Ba´ra´ny B, Stenvinkel P. Interleukin-6 system is associated with renal function in end-stage renal disease. Blood Purif 2002;20:511. [26] Roullet JB, Xue H, McCarron DA, Holcomb S, Bennett W. Vascular mechanisms of cyclosporin-induced hypertension in the rat. J Clin Invest 1994;93:2244– 50. [27] Shihab FS, Bennett WM, Yi H, Andoh TF. Effect of cyclosporine and sirolimus on the expression of connective tissue growth factor in rat experimental chronic nephrotoxicity. Am J Nephrol 2006;26:400–7. [28] Shihab FS, Bennett WM, Yi H, Choi SO, Andoh TF. Mycophenolate mofetil ameliorates arteriolopathy and decreases transforming growth factor-beta1 in chronic cyclosporine nephrotoxicity. Am J Transplant 2003;3:1550–9. [29] Sorg DA, Buckner B. A simple method of obtaining venous blood from small animals. Proc Sec Exp Med 1964;115:1131–2. [30] Takenaka T, Hashimoto Y, Epstein M. Diminished acetylcholine-induced vasodilation in renal microvessels of cyclosporine-treated rats. J Am Soc Nephrol 1992;3:42–50. [31] Thompson ME, Shapiro AP, Johnsen AM. New onset of hypertension following cardiac transplantation: a preliminary report and analysis. Transplant Proc 1983;15(Suppl. 1):2573–7. [32] Trimarchi H, Orias M. Aliskiren and the kidney: beyond hypertension. Nephrology Reviews 2009;1:e1–4. [33] Weidle PJ, Vlasses PH. Systemic hypertension associated with cyclosporin: a review. Drug Intell Clin Pharm 1988;22:443–51. [34] Wolny A, Clozel JP, Rein J, Mory P, Vogt P, Turino M, et al. Functional and biochemical analysis of angiotensin II-forming pathways in the human heart. Circ Res 1997;80:219–27. [35] Yokokawa F, Maibaum J. Recent advances in the discovery of non-peptidic direct renin inhibitors as antihypertensives: new patent applications in years 2000–2008. Expert Opin Ther Patents 2008;18:581–602.
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Renoprotective activity of aliskiren, a renin inhibitor in cyclosporine A induced hypertensive nephropathy in dTG mice Megha S. Saraswat a, Veeranjaneyulu Addepalli a,*, Mukul Jain b, Vishwanath D. Pawar b, Rakesh B. Patel b a b
SPP SPTM, SVKM’s NMIMS, Mumbai, Maharashtra, India Department of Pharmacology, Zydus Research Centre, Ahmedabad, Gujarat, India
A R T I C L E I N F O
Article history: Received 12 June 2012 Received in revised form 29 June 2013 Accepted 2 August 2013 Available online 31 January 2014 Keywords: Hypertensive nephropathy Aliskiren Cyclosporine A TNF-a IL-6 TGF-b1 KIM-1
A B S T R A C T
Background: Hypertensive nephropathy is moving up the charts to number 2 after diabetic nephropathy in terms of diagnostic frequency cited as causing end stage renal disease (ESRD). Method: Hypertensive nephropathy was produced in mildly hypertensive C57BL/6-(hREN)/(hAGT) double transgenic (dTG) mice with 20 mg/kg of cyclosporine A (CsA) administered subcutaneously (sc) daily for 28 days. CsA dose 20 mg/kg was selected for the study as this dose offered significant alteration in blood pressure, biochemical parameters and moderate nephropathy in kidney. Effect of aliskiren oral treatment twice daily consequently for 28 days at 10 mg/kg body weight was evaluated against CsA induced hypertensive nephropathy. Systolic blood pressure (SBP) was measured by non invasive tail cuff method. Kidney function test (blood urea nitrogen, serum creatinine, urea and uric acid) and kidney injury biomarker (tumor necrosis factor-alpha (TNF-a) and interlekin-6) level was assessed in serum, TNF-a, IL-6, transforming growth factor-beta1 (TGF-b1) and kidney injury molecule-1 (KIM-1) was assayed in kidney homogenate. Urinary KIM-1 levels were assessed as an early biomarker of nephropathy. Result: Significant hypertensive nephropathy and increase in serum levels of biomarkers was observed in CsA treated animals when compared with Control group. Aliskiren treatment elicited significant renoprotection by preventing the increase in blood pressure and levels of serum biomarkers and also reduced the nephropathic alterations in the kidney histoarchitecture. Conclusion: A correlation between pharmacological, biochemical and histological findings has been established in mouse model. The present findings have indicated the renoprotective activity of aliskiren in CsA induced hypertensive nephropathy, which may be due to its antihypertensive, anti-inflammatory as well as anti-apoptopic action. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Hypertension is a major cardiovascular risk factor associated with significant morbidity and mortality worldwide and will increase in importance as a public health problem by 2020 [22]. Hypertensive nephropathy, a consequence of chronic high blood pressure is moving up the charts after diabetic nephropathy in terms of diagnostic frequency cited as causing end stage renal disease (ESRD) [16]. Complications often associated with hypertensive nephropathy include glomerular damage resulting in inflammatory responses and compromised kidney function that seems to be superimposed on the intrinsic phenotype of the underlying renal disease [3]. Findings from the recent investigations of experimental animal models have suggested that
* Corresponding author. E-mail address: [email protected] (V. Addepalli).
chemically induced nephropathy share many features which are common to human nephropathy and therefore it was explored in the present study. Cyclosporine A (CsA) is an immunosuppressive drug commonly used in organ transplantation, but its use is complicated by the development of hypertension in 40–100% of patients [31,33]. CsA is also associated with the development of microvascular and tubulointerstitial disease in the kidney, consisting of hyalinosis of the afferent arteriole in association with tubular atrophy and ‘‘striped’’ interstitial fibrosis [2,23]. The pathogenesis of the renal injury is thought to be mediated by renal vasoconstriction and ischemia [26], and this appears to result from direct effects of CsA as well as by the ability of CsA to alter intrarenal vasoactive mediators such as renin, endothelin-1, and nitric oxide (NO) [30]. Therefore, in light with these findings CsA was employed for inducing hypertensive nephropathy in the present study. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.08.005
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Cyclosporine Oral Solution USP, 100 mg/ml, Panacea Biotech Ltd., New Delhi, India) was administered once daily as a subcutaneous injection of 20 mg/kg for 28 days. The doses were finalized on the basis of pilot studies carried out at ZRC, Ahmedabad (results not shown). Olive oil used to prepare cyclosporine A dilutions was procured from local market and administered daily at a dose volume of 2 ml/kg subcutaneously.
Pressure (JNC7) establishes that the goal of antihypertensive therapy is to reduce cardiovascular and renal morbidity and mortality [5]. Since Ang II is reported to play a significant role in the progression of chronic kidney disease (CKD), its pharmacological inhibition with angiotensin converting enzyme inhibitors (ACEIs) or angiotensin II receptor antagonists (ARBs) significantly reduces markers of renal injury and can even attenuate the progression of glomerular and tubulointerstitial fibrosis. However, the observation that treatment with ACEI or ARBs alone does not completely inhibit the progression of CKD raises the possibility that other pathways besides the formation of Ang II may be involved in CKD progression. By attenuating feedback inhibition of renin release, ACEIs and ARBs effects lead to an increase in plasma renin concentration and activity, rendering incomplete inhibition of the RAS system [1,21]. Moreover, inhibition of ACE causes an increase in angiotensin I, which is then available for conversion to angiotensin II by ACE-independent pathways not blocked by ACEIs, namely cathepsins and tonins [9,13,34]. Consequently, despite adequate blood pressure control, angiotensin II levels increase aldosterone levels to certain degrees, rendering these inflammatory molecules free to play an active role in tissue remodeling and scarring. Henceforth, it is tempting and reasonable to assess the effects of a different pharmacologic strategy that blocks the RAS upstream completely. Thus there is an unmet need to develop new strategy to target hypertension mediated end organ damage along with improved efficacy and safety. Aliskiren is a first-in-class oral renin inhibitor, developed by Novartis in conjunction with the biotech company Speedel [35]. Aliskiren is the first known representative of a new class of nonpeptide orally active renin inhibitors that block the RAS at its rate-limiting step and induce a net reduction in plasma renin activity and angiotensin II and aldosterone levels [11,15]. Aliskiren with established antihypertensive action elicits a notorious renoprotective effect such that, aliskiren can further decrease the risk of renal disease progression [32]. Therefore, the present work focuses to evaluate the ameliorating effect of administering aliskiren, a renin inhibitor, on further disease progression in cyclosporine A induced hypertensive nephropathy.
After 25 days, systolic blood pressure (SBP) was measured using non invasive blood pressure monitor (Columbus Instruments, USA) by the tail cuff method wherein the average of the two days SBP was recorded and considered.
Materials and methods
Assessment of kidney injury biomarkers
Animals
At the end of the treatment period mice were housed in metabolic cages for 24 h for urine collection which was then frozen at 80 8C till analysis. The urine samples were then employed for the estimation of KIM-1. After 24 h period, the blood was obtained from the retroorbital plexus of mice from all the groups after being lightly anesthetized with ether [29]. The blood was allowed to flow into clean dry centrifuge tube and left to stand for 30 min before centrifugation to avoid hemolysis. Serum was separated by centrifugation for 5 min at 4000 rpm and stored at 20 8C until analysis. The clear supernatant, serum was separated and collected into a dry clean tube for the biochemical tests: BUN and serum levels of creatinine, urea, uric acid and the ELISA test for TNF-alpha and IL-6. Animals were then sacrificed by cervical dislocation and the kidneys were quickly isolated, washed with saline, blotted dry on filter paper. One kidney of each group was snap frozen in dry ice for preparation of Kidney Homogenates wherein kidneys were thawed in 2 ml sterile phosphate buffered saline (PBS) and homogenized three times for 15 s with a Brinkmann homogenizer (Brinkmann Instruments, Mississauga, Ontario, Canada). Kidney homogenates were then subjected to three freeze-thaw cycles at 70 8C for 4 h and 37 8C for 15 min. Supernatants were separated from tissue residues by centrifugation twice at 12,000 g for 10 min at 4 8C and then employed for the ELISA estimation of TNF-alpha, IL-6, TGF-beta1 and KIM-1.
Thirty-six healthy female C57BL/6-(hREN)/(hAGT) double transgenic (dTG) mice were used throughout the experiment with initial body weight of 20–25 g. Animals were bred at animal breeding facility of Zydus Research Centre (ZRC), Ahmedabad. Institutional Animal Ethical Committee approved the experimental protocol as per the guidance of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. Animals were maintained in the specific pathogen-free animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AALAC). Mice in groups of 6 of similar sex were housed in clean, sterilized solid floor polycarbonate individually ventilated (IVC) cages. Animals were kept in an environmentally controlled room designed to maintain a temperature of 22 3 8C and a relative humidity of 30–70% with at least 14 fresh air changes per hour. Drugs and chemicals Aliskiren (Tekturna1, 300 mg Tablet, Novartis, USA) was procured and it was administered twice daily as an oral dose of 10 mg/kg for 28 days. Cyclosporine A (PanimunBioral1,
Diagnostic kits Mouse tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6) estimations were performed using commercially available sandwich ELISA kits (R&D, Minneapolis, MN, USA). The detection limit of each kit is 1000 pg/ml for both IL-6 and TNF-alpha. Mouse kidney injury molecule-1 (KIM-1) and transforming growth factor beta-1 (TGF b-1) estimations were performed using commercially available competitive ELISA kits (MyBiosource, USA). The detection limit of each kit is 25 ng/ml for KIM-1 and 1000 pg/ml for TGF b-1. Kidney Function Tests (blood urea nitrogen (BUN), creatinine, urea, uric acid) estimation kits were obtained and analysis was performed using Roche/Hitachi Cobas – C system. Experimental design Thirty-six mice were divided into three groups as follows: Group 1: Control group (subcutaneously received 2 ml/kg olive oil daily for 28 days); Group 2: CsA group (subcutaneously received 20 mg/kg of CsA daily for 28 days); Group 3: Aliskiren group (orally received 10 mg/kg of aliskiren twice daily for 28 days). Assessment of blood pressure
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Histopathology The second kidney from each group was stored into 10% paraformaldehyde solution and embedded in wax. After dewaxing, 4 mm sections were processed and stained with Hematoxylin and Eosin (H&E).
Table 1 Functional alterations in dTG C57 CsA challenged mice. BUN, serum creatinine, serum urea and serum uric acid were used as indicators of renal function. Results are expressed as mean SEM. N = 6 for each treatment group.
BUN (mg/dl) Serum creatinine (mg/dl) Serum urea (mg/dl) Serum uric acid (mg/dl)
Statistical analysis All data were expressed as mean standard error mean (SEM). All data were compared by one-way analysis of variance (ANOVA). Each test was followed by the Bonferroni’s multiple comparison test. p values less than 0.05 (p < 0.05) were considered statistically significant. Result Effect on blood pressure Systolic blood pressure (SBP) in mice was measured and observed that mice challenged with CsA (20 mg/kg, sc) showed significant elevation of blood pressure as compared to Control group. Aliskiren (10 mg/kg, po, bid) treated CsA challenged animals showed significant prevention in blood pressure as compared to CsA group. These results suggested that aliskiren could antagonize the hypertensive effect of CsA in dTG C57 mice (Fig. 1). Effect on renal function In order to evaluate renal functional alterations in mice, kidney function tests were performed in the serum at study termination. These estimations indicated deterioration of the renal function indicating kidney disorder wherein CsA significantly increased serum biomarker concentration (BUN, serum creatinine, serum urea and serum uric acid). These results suggested significant decline in renal function of CsA challenged mice. Chronic treatment with aliskiren significantly prevented the anomalous rise in concentrations of serum biomarkers as compared to CsA group. These, results suggested significant improvement in the renal function with aliskiren treatment as compared to CsA group and directed toward it renoprotective action (Table 1).
****
Tail Cuff Systolic BP (mmHg)
120
110
####
100
90
*
Control
CsA
Aliskiren
20.60 0.75 0.22 0.02 44.20 1.9 1.98 0.19
43.00 5.18*** 0.30 0.01** 92.00 1.2*** 2.95 0.17**
22.33 0.56## 0.22 0.02## 44.67 2.39### 1.80 0.15###
and # indicates statistically significant difference within groups. p < 0.001 as compared to Control and ##p < 0.01, ###p < 0.001.
**
p < 0.01,
***
Effect on cytokine levels The changes in the cytokine levels (TNF-a, IL-6 and TGFb-1) were estimated in the experimental groups with the respective ELISA methods. CsA group significantly raised the TNF-a level both in serum as well as the kidney tissue homogenate. Moreover, CsA increased the IL-6 concentration in the serum and kidney tissue and tissue TGF b-1 levels were also raised. Chronic treatment with aliskiren significantly prevented the increase in TNF-a (Fig. 2A and B) and IL-6 (Fig. 2C and D) levels both in the serum as well as the kidney homogenates as compared to CsA group. Secondly, aliskiren could also significantly prevent the increase in levels of TGF b-1 in the kidney homogenates (Fig. 2E). Taken together these results became indicative of the anti-inflammatory and property of aliskiren that could substantiate its role in the prevention of hypertension induced renal damage. Effect on newer kidney injury biomarker Estimation of the newer kidney injury biomarker i.e. Kidney Injury Molecule-1 (KIM-1) was performed with the help of competitive ELISA method. At 28 day after CsA administration, there was significant increase in the urinary and tissue KIM-1 concentration. However, aliskiren prevented the rise in KIM-1 concentrations in mouse urine as well as kidney tissue homogenates and thereby indicated renoprotective action (Fig. 3). Histopathological alterations Sections of mouse kidney stained with Hematoxylin and Eosin (H&E) were examined by light microscopy. Nephropathic changes in kidney architecture in different experimental groups were observed. Glomerulosclerosis, tubular dilatation and atrophy, interstitial cell infilteration and interstitial fibrosis, hyalinization of the glomerular foci, proteinaceous material in the tubules, vacuolar degeneration of tubular epithelium, hyperplasic tubules, Necrosis, etc. were observed to indicate nephropathic changes. Animals challenged with CsA showed significant nephropathic alterations as described compared to that of Control group (Fig. 4A and B). Aliskiren treated animals did not elicit such nephropathic changes and were found to be similar to normal (Fig. 4C). Hence, aliskiren could prevent the kidney from undergoing nephropathic changes as those seen in CsA group. Discussion
80 Control
CsA
Aliskiren
Fig. 1. Effect of drug treatment on systolic blood pressure (SBP) after 25 days. All values are expressed as mean SEM (N = 10). **** indicates p < 0.0001 as compared to Control group, #### indicates p < 0.0001 as compared to CsA group. Data was analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test.
Many chronic renal diseases evolve progressively with few outward clinical symptoms until the onset of renal dysfunction and, often, structural damage. An appropriate therapeutic agent is therefore one that effectively treats existing as well as developing renal injury. In order to address this critical issue, a mouse model that developed renal lesions similar to clinical nephropathic condition was employed in the present study. Documented reports have indicated that human renin (hREN) and human angiotensi-
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B
*
25
Serum TNF-apha (pg/ml)
TNF-apha in mouse kidney tissue (pg/mg of protein)
A
20 15 10
*
5 0 Control
CsA
Aliskiren
***
1200
#
1000 800 600 400 200 0 Control
CsA
Aliskiren
D
Serum IL-6 (pg/ml)
30
IL-6 in mouse Kidney tissue (pg/mg of protein)
C
1400
*
20
10
# 0 Control
CsA
1200
**
1000
Aliskiren
800 600
#
400 200 0
Control
CsA
Aliskiren
TGF Beta-1 in mouse Kidney Tissue (pg/mg of protein)
E 200
***
150 100
### 50 0 Control
CsA
Aliskiren
Fig. 2. Effect of drug treatment on cytokine levels. (A) Effect on TNF-a level in mouse serum, (B) effect on TNF-a level in mouse kidney tissue homogenate, (C) effect on IL-6 level in mouse serum, (D) effect on IL-6 level in mouse kidney tissue homogenate, (E) effect on TGF-b1 level in mouse kidney tissue homogenate. All values are expressed as mean SEM, N = 6. * and # indicates statistically significant difference within groups. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to Control and #p < 0.05, ##p < 0.01, ### p < 0.001.
hypertensive nephropathic response was verified. The following findings explain, at least partly, aliskiren counteraction of CSA induced hypertensive response wherein aliskiren significantly prevented the rise in systolic blood pressure induced due to chronic CsA administration. These findings established convincing evidence that aliskiren offsets the hypertensive effect of CSA via ameliorating significant RAS derangements caused by CSA. We reasoned that concurrent exposure to aliskiren would mitigate or at least minimize the hypertensive action of CSA and potentially related nephropathic manifestations. This assumption was ascertained in the current study because the hypertensive effect of CSA
nogen (hAGT) gene – harboring double transgenic (dTG) rats develop hypertension and severe hypertension-related end-organ damage due to local Ang II formation in the heart, kidney, and vasculature [10,19]. The current study is the first to report on the potential protective effect of the orally acting direct renin inhibitor drug aliskiren against the hypertensive and deleterious nephropathic effects of chronic CsA administration in dTG mice. Initially, we verified that CsA did in fact induce renal dysfunction in dTG C57 mice. We examined a range of well-established patho-physiological features associated with CsA nephropathy. Further, concurrent aliskiren administration virtually abolished the CsA induced
B
Urine KIM-1 (ng/ml)
6
* #
4
2
0 Control
CsA
Aliskiren
KIM-1 in mouse Kidney Tissue (ng/mg of protein)
A
2.0
***
1.5 1.0 0.5
### 0.0 Control
CsA
Aliskiren
Fig. 3. Effect of drug treatment on kidney injury molecule-1. (A) KIM-1 level in mouse serum and (B) KIM-1 level in mouse kidney tissue homogenate. All values are expressed as mean SEM, N = 6. * and # indicates statistically significant difference within groups. *p < 0.05, ***p < 0.001 as compared to Control and #p < 0.05, ###p < 0.001.
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Fig. 4. Histopathological alterations in kidney tissue. (A) Control group showed overall no abnormalities except few instances of mild tubular dilatation and presence of proteinaceous material in the tubule were observed, (B) CsA Group eilicited presence of proteinaceous material in the tubules, foci of lymphocytic infiltration (FLI), dilatation, basophilic tubules, hyalinization of the glomerular foci, vacuolation, and necrotic changes and (C) Aliskiren group elicited overall improvement as compared to CsA group.
disappeared when mice were treated simultaneously with aliskiren. In order to evaluate renal functional alterations in mice, kidney function tests were performed. Dieterle et al. have demonstrated that serum creatinine in conjunction with other kidney function parameters are excellent indicators of renal dysfunction [7]. In the present study, significantly elevated levels of creatinine, urea and uric acid in mice serum and increased BUN levels were evident after 28 day administration of CsA which became indicative of its nephrotoxic response. However, concurrent treatment with aliskiren elicited significant prevention of the renal dysfunction caused due to chronic CsA administration. This substantiated the protective role of aliskiren in CsA induced renal dysfunction. Data in animal models suggest that the inflammatory response during kidney injury is dysregulated. The effect of kidney injury on the production and elimination of proinflammatory cytokines may be a key mechanism by which patients with kidney injury have increased distant organ dysfunction and increased mortality. Since cytokine production also increases in the kidney, renal cytokine production may contribute to renal injury and cause the increase in serum cytokines. Circulating cytokines may then contribute to extra renal organ injury. Hence, for the purposes of this study putative nephrotoxicity markers were designated as those identified in the literature as having a significant role in nephrotoxicity in a number of in vitro and in vivo models of renal disease. These included cytokines like TNF-a, IL-6 and TGF-b1. In patients with chronic renal failure not on dialysis, serum IL-6 and TNF-alpha are increased, suggesting that impaired kidney function results in increased cytokine levels. Additionally, increasing levels of serum IL-6 are significantly correlated with decreasing levels of glomerular filtration rate, further suggesting that impaired kidney function may affect cytokine clearance. Although filtration and excretion cytokines may occur, current evidence suggests that cytokines are not primarily cleared via filtration and excretion, but may be filtered, reabsorbed and metabolized by the proximal tubule [12]. ESRD patients have lower urinary IL-6 receptor excretion than controls [20]. Recently links between the IL-6 system and the residual renal function, showing an association between sIL-6R and the progression rate of renal function in the predialysis phase, as well as an association between changes in glomerular filtration rate and changes in IL-6 during peritoneal dialysis (PD) treatment were described [25]. From the present study it is inferred that mice challenged with CsA elicit significant rise in the levels of TNF-alpha and IL-6 both in the serum as well as in the kidney homogenates which demonstrates signs of inflammation and the deterioration of renal function that can be associated with a significant increase in serum cytokine levels [6]. The profibrotic cytokine TGF-b1 is a major contributor to
tubulointerstitial fibrosis and nephropathy [17]. Increased TGF-
b1 levels after CsA treatment suggests a role for TGF-b1 in the current model, and this is in agreement with previous studies [18,27,28]. Moreover, despite some initially promising results in experimental models of CsA nephropathy [8,17], TGF-b1 blockade has not yet translated into an effective therapeutic strategy in human patients. However, concomitant treatment with aliskiren significantly prevented the inflammatory responses of CsA that led to deleterious nephrotoxic reaction. The effect of aliskiren on the cytokine levels, as observed from the present study indicated its protective action as an antiinflammatory over and above its antihypertensive activity. This property of aliskiren can be attributed to its renin blocking action that in turn prevented the detrimental effects of angiotensin II which is reported to play a key role in the progression of CsA induced nephrotoxicity [4,24] Another evidence that suggested the protective role of aliskiren against CsA induced nephrotoxicity was the evaluation of its effect on newer kidney injury biomarker KIM-1. KIM-1 has recently been identified as the first non-myeloid phosphatidylderine receptor that confers a phagocytic phenotype on injured epithelial cells both in vivo and in vitro [14]. Urinary KIM-1 is a non-invasive, rapid, sensitive and reproducible biomarker for the early detection of kidney injury. In the present study, a sandwich KIM-1 ELISA test was used. Initially, we verified the role of KIM-1 in the pathophysiology of CsA induced nephropathy wherein it was evident that CsA challenge for 28 days illustrated significant increase in the urinary as well as kidney tissue KIM-1 concentration. Following this, it was observed that concurrent treatment with aliskiren significantly prevented the rise in the level of KIM-1 both in the urine and the mouse kidney homogenate. This further substantiated the renoprotective action of aliskiren against CsA induced nephrotoxicity. Nephropathic changes in kidney architecture in different experimental groups were observed where sections of mouse kidney stained with H&E were examined by light microscopy. Glomerulosclerosis, tubular dilatation and atrophy, interstitial cell infilteration and interstitial fibrosis, hyalinization of the glomerular foci, proteinaceous material in the tubules, vacuolar degeneration of tubular epithelium, hyperplasic tubules, necrosis, etc. were observed to indicate nephropathic changes. Groups challenged with CsA showed significant nephropathic alterations as described. However, aliskiren treated animals did not elicit nephropathic changes and showed improved architecture similar to those observed in the Control group. Hence, histomorphologically also it could be indicated that aliskiren indeed is a protective agent against CsA induced hypertensive nephropathy. This research has identified a number of potential markers of CsA hypertensive nephropathy. Further investigation will determine if these indicators translate to a clinical setting, and to what extent they may be useful as therapeutic targets.
M.S. Saraswat et al. / Pharmacological Reports 66 (2014) 62–67
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