CsA-treated Rats Research Articles

Expression of Erythropoietin and Its Receptor in Kidneys From Normal and Cyclosporine-Treated Rats

Long-term treatment with cyclosporine A (CsA) is associated with various types of complications; however, CsA-induced anemia has not been reported. The present study examined the impact of CsA on hematopoietic parameters and intrarenal expression of erythropoietin (EPO) and the EPO receptor (EPOR) in a rat model of chronic CsA nephrotoxicity. Sprague-Dawley rats were fed a low-salt diet (0.05% sodium) and were treated daily for 4 weeks with vehicle (olive oil 1 mL/kg subcutaneously) or CsA (15 mg/kg subcutaneously). The expression of EPO and EPOR was evaluated by immunohistochemistry and immunoblotting, and hematopoietic parameters were assessed by measuring blood hemoglobin and hematocrit levels, and these variables were compared between treatment groups. Renal function, oxidative stress, histopathology (tubulointerstitial fibrosis), apoptotic cell death, and expression of transforming growth factor β–inducible gene-h3 (βig-h3) were also compared between treatment groups. In kidneys from vehicle-treated rats, endogenous EPO and EPOR protein were expressed constitutively in the outer stripe of the outer medulla and the cortex. EPO protein expression decreased significantly in kidneys from CsA-treated rats. By contrast, EPOR expression was higher in kidneys from CsA-treated rats than in vehicle-treated rats. These changes were accompanied by decreases in serum hemoglobin and hematocrit levels and correlated with the number of cells positive for terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (r = −0.769, P = .003) and βig-h3 protein expression (r = −0.910, P < .001). Long-term treatment with CsA suppresses renal endogenous EPO expression, resulting in anemia. Increases in apoptotic cell death and βig-h3 expression are closely associated with inhibition of EPO expression in chronic CsA nephrotoxicity.

Evaluation of P-glycoprotein (abcb1a/b) modulation of [18F]fallypride in MicroPET imaging studies

[18F]Fallypride ([18F]FP) is an important and routinely used D2/D3 antagonist for quantitative imaging of dopaminergic neurotransmission in vivo. Recently it was shown that the brain uptake of the structurally related [11C]raclopride is modulated by P-glycoprotein (P-gp), an important efflux transporter at the blood–brain barrier. The purpose of this study was to determine whether the brain uptake of [18F]FP is influenced by P-gp. For examination of this possible modulation microPET studies were performed in a rat and a mouse model. Hence, [18F]FP was applied to Sprague Dawley rats, half of them being treated with the P-gp inhibitor cyclosporine A (CsA). In a second experimental series the tracer was applied to three different groups of FVB/N mice: wild type, P-gp double knockout (abcb1a/1b (−/−)) and CsA-treated mice. In CsA-treated Sprague Dawley rats [18F]FP showed an elevated standard uptake value in the striatum compared to the control animals. In FVB/N mice a similar effect was observed, showing an increasing uptake from wild type to CsA-treated and double knockout mice. Since genetically or pharmacologically induced reduction of P-gp activity increased the uptake of [18F]FP markedly, we conclude that [18F]FP is indeed a substrate of P-gp and that the efflux pump modulates its brain uptake. This effect – if true for humans – may have particular impact on clinical studies using [18F]FP for assessment of D2/3 receptor occupancy by antipsychotic drugs.This article is part of the Special Issue Section entitled ‘Neuroimaging in Neuropharmacology’.

Bilirubin attenuates the renal tubular injury by inhibition of oxidative stress and apoptosis

BackgroundBilirubin (BIL) has been recognized as an endogenous antioxidant that shows a protective effect for cardiorenal diseases. We investigated whether administration of BIL had a protective effect on cyclosporine (CsA)-induced nephropathy (CIN), and examined the effects of BIL on the oxidative stress and apoptosis.MethodsBIL was pretreated intraperitoneally three times for a week (60 mg/kg), and CsA was injected for 4 weeks (15 mg/kg/day, subcutaneous). Proximal tubular epithelial (HK2) cells were pretreated with 0.1mg/ml of BIL for 24 hours, and then treated with 20 μM of CsA for another 24 hours.ResultsCsA induced marked increases in urine kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL) concentrations (P < 0.05). BIL reduced urine Kim-1 in CIN (P < 0.05), while urine NGAL exhibited a decreasing tendency. In CsA-treated rat kidneys, the protein expression of NOX4 and p22phox was reduced by BIL (P < 0.05). BIL ameliorated CsA-induced arteriolopathy, tubulointerstitial fibrosis, tubular injury, and the apoptosis examined by TUNEL assay (P < 0.01). In HK2 cells, BIL reduced intracellular reactive oxygen species in CsA-treated cells. CsA increased the protein expression of bax, cleaved caspase-9, caspase-3 and the activity of caspase-3; however, the anti-apoptotic bcl-2 protein was reduced. These changes were recovered by BIL (P < 0.05).ConclusionsThe direct administration of BIL protected against CsA-induced tubular injury via inhibition of oxidative stress and apoptosis.

Serum and Renal Tissue Markers of Nephropathy in Rats Under Immunosuppressive Therapy: Cyclosporine Versus Sirolimus

Cyclosporin (CsA) has been progressively replaced by other drugs with putatively fever side effects, including nephrotoxicity and hypertension. Sirolimus (SRL) is one of the main options for management of kidney transplant patients in the post-CsA era. It shows identical efficacy with apparently less cardiorenal side effects than CsA. However, doubts remain concerning the mechanisms of putative renoprotection by SRL as well as the best serum and/or tissue markers for nephropathy, as assessed in this study employing CsA- and SRL-treated rats. Three groups (n = 6) were treated orally during a 6-week protocol: control (vehicle); CsA (5 mg/kg body weight per day Sandimmun Neoral); SRL (1 mg/kg body weight per day Rapamune). Blood pressure and heart rate were assessed with a “tail cuff”. Renal dysfunction and morphology were characterized using serum creatinine and blood urea nitrogen (BUN) levels as well as hematoxylin and eosin and periodic acid Schiff staining, respectively. We examined serum concentrations of interleukin (IL)-2, IL-1β, high-sensitivity C-reactive protein, tumor necrosis factor TNF-α, and vascular endothelial growth factor and kidney mRNA expression of interleukin-1β (IL-1β), tumor protein 53 (TP53), mammalian target of rapamycin (mTOR) and proliferating cell nuclear antigen (PCNA), as well as markers of lipid peroxidation in the kidney and serum. Both CsA and SRL induced significant increases in systolic and diastolic blood pressure, but only CsA caused tachycardia. CsA-treated rats also displayed increased serum creatinine and BUN levels, accompanied by mild renal lesions, which were almost absent among SRL-treated rats, which presented hyperlipidemic and hyperglycemic profiles. CsA-induced nephrotoxicity was accompanied by kidney overexpression of inflammatory and proliferative mRNA markers (IL-1β, mTOR and PCNA), which were absent among SRL group. In conclusion, the antiproliferative and antifibrotic character of SRL may explain its less nephrotoxic profile. Renal over expression of mTOR in the CsA-treated group, associated with renal dysfunction and structural damage, reinforces the potential beneft of SRL as a strategy to reduce CsA-evoked nephrotoxicity.

Sildenafil protects against nitric oxide deficiency-related nephrotoxicity in cyclosporine A treated rats

Cyclosporine A (CsA) is the most widely used immunosuppressant in organ transplant surgery and in treatment of autoimmune disease. Nevertheless, animal and clinical studies have demonstrated that nephrotoxicity is the major adverse effect limiting the prolonged CsA therapeutic use. The present study aimed to investigate possible protective effect of sildenafil, a phoshodiestrase-5 inhibitor, on CsA-induced nephrotoxicity and various mechanism(s) underlies this effect. Male Wistar rats were administered CsA (20mg/kg/day, s.c.) for 21 days alone or in combination with sildenafil (5mg/kg/day, p.o.). Sildenafil exhibited nephroprotective effects as evidenced by significant decrease in serum creatinine and urea levels, spot urine albumin–creatinine ratio, as well as levels of renal malondialdehyde and nitric oxide, with a concurrent increase in renal level of reduced glutathione and catalase activity compared to CsA-treated rats. Additionally, immunohistochemical analysis demonstrated that sildenafil treatment markedly reduced inducible nitric oxide synthase, tumor necrosis factor-alpha, and caspase-3 expressions, while expression of endothelial nitric oxide synthase was prominently enhanced. The protective effects of sildenafil were confirmed by renal histopathological examination. Pretreatment with l-nitro-arginine methyl ester (10mg/kg/day, i.p.), a non-selective nitric oxide synthase inhibitor, reversed the protection afforded by sildenafil. Taken together, the current study highlighted the renoprotective effects of sildenafil against CsA-induced nephrotoxicity in rats, which might be mediated, in part, through nitric oxide pathway as well as antioxidant, anti-inflammatory, and anti-apoptotic activities.

The mineralocorticoid receptor antagonist eplerenone reduces renal interstitial fibrosis after long-term cyclosporine treatment in rat: antagonizing cyclosporine nephrotoxicity

BackgroundChronic cyclosporine-(CsA)-mediated loss of kidney function is a major clinical problem in organ transplantation. We hypothesized that the mineralocorticoid receptor antagonist eplerenone (EPL) prevents chronic CsA-induced renal interstitial volume increase, tubule loss, and functional impairment in a rat model.MethodsSprague–Dawley rats received CsA alone (15 mg/kg/d p.o.), CsA and EPL (approximately 100 mg/kg/day p.o.) or vehicle (control) for 12 weeks. At 11 weeks, chronic indwelling arterial and venous catheters were implanted for continuous measurements of arterial blood pressure (BP) and GFR (inulin clearance) in conscious, freely moving animals. Plasma was sampled for analysis and kidney tissue was fixed for quantitative stereological analyses.ResultsCompared to controls, CsA-treatment reduced relative tubular volume (0.73±0.03 vs. 0.85±0.01, p<0.05) and increased relative interstitial volume (0.080±0.004 vs. 0.045±0.003, p<0.05); EPL attenuated these changes (0.82±0.02, p<0.05, and 0.060±0.006, p<0.05, respectively). CsA-treated rats had more sclerotic glomeruli and a higher degree of vascular depositions in arterioles; both were significantly reduced in CsA+EPL-treated animals. CsA increased BP and reduced body weight gain and GFR. In CsA+EPL rats, weight gain, GFR and BP at rest (daytime) were normalized; however, BP during activity (night) remained elevated. Plasma sodium and potassium concentrations, kidney-to-body weight ratios and CsA whole blood concentration were similar in CsA and CsA+EPL rats.ConclusionsIt is concluded that in the chronic cyclosporine rat nephropathy model, EPL reduces renal tissue injury, hypofiltration, hypertension, and growth impairment. MR antagonists should be tested for their renoprotective potential in patients treated with calcineurin inhibitors.

Heteropterys tomentosa (A. Juss.) infusion counteracts Cyclosporin a side effects on the ventral prostate

BackgroundCyclosporin A (CsA) is an immunosuppressive drug widely used in treatment of auto-immune diseases or after organ transplants. However, several side effects are commonly associated with CsA long term intake, some regarding to loss of reproductive organ function due to oxidative damage. Considering that phytotherapy is an important tool often used against oxidative stress, we would like to describe the beneficial effects of Heteropterys tomentosa intake to minimize the damage caused by CsA to the ventral prostate tissue of Wistar rats under laboratorial conditions.MethodsThirty adult Wistar rats (Rattus norvegicus albinus) were divided into: control group (water); CsA group (Cyclosporin A); Ht group (H. tomentosa infusion) and CsA + Ht group (CsA and H. tomentosa infusion). Plasmic levels of hepatotoxicity markers, triglycerides, cholesterol and glucose were quantified. The ventral prostate tissue was analyzed under light microscopy, using stereological, morphometrical and immunohistochemical techniques.ResultsH. tomentosa did not cause any alterations either of the plasmic parameters or of the ventral prostate structure. CsA caused alterations of GOT, total and indirect bilirubin, cholesterol, triglycerides and glucose levels in the plasma; CsA-treated rats showed alterations of the ventral prostate tissue. There were no alterations regarding the plasma levels of GOT, triglycerides and glucose of CsA + Ht animals. The same group also showed normalization of most of the parameters analyzed on the ventral prostate tissue when compared to the CsA group. The treatments did not alter the pattern of AR expression or the apoptotic index of the ventral prostate epithelium.ConclusionsThe results suggest a protective action of the H. tomentosa infusion against the side effects of CsA on the ventral prostate tissue, which could also be observed with plasmic biochemical parameters.

Role of CXCR1 (CKR-1) in Inflammation of Experimental Mesangioproliferative Glomerulonephritis

CXCR1 (CKR-1), a receptor of IL-8, is expressed in various cells including neutrophils and monocytes, both of which play a major role in proliferating glomerular diseases. We investigated time-dependent expression of CXCR1 and the effect of single-dose cyclosporine A (CsA) treatment on this expression in experimental mesangioproliferative glomerulonephritis induced by anti-thymocyte serum (ATS). Wistar rats were divided into three groups. Group 1 (control, n = 24) received non-immune serum. Group 2 (nephritis, n = 24) received ATS. Group 3 (nephritis + CsA, n = 24) received ATS and CsA concomitantly. Kidneys from six rats in each group were removed at sixth hour, 3 days, 5 days, and 7 days. ATS induced proteinuria compared to controls (p < 0.001) and CsA precluded the development of proteinuria. Glomerular inflammation and mesangial proliferation were significantly higher in ATS group than control and CsA-treated rats (p < 0.001). ATS injection caused marked interstitial inflammation that was precluded by CsA (p < 0.001). CXCR1 was not expressed in control kidneys. However, ATS induced expression of CXCR1 in both glomeruli and tubulointerstitium. CsA treatment precluded CXCR1 expression in both glomeruli and tubulointerstitium only in the first 6 h. CXCR1 may contribute to inflammation in experimental mesangioproliferative glomerulonephritis. CsA may be beneficial by inhibiting CXCR1 expression and corresponding inflammation.

The Role of Coenzyme Q10 against Cyclosporine-A Induced Cardiotoxicity in Adult Male Albino Rats (Histological and Toxicological Study)

Cyclosporine-A (CsA) has a potent immunosuppressive activity and is commonly used as anti-rejection drug after organ transplantation and for treatment of some autoimmune diseases. Several toxic effects on the cardiovascular system have been reported with CsA therapy. The toxicity induced by CsA is attributed to the formation of free oxygen radicals. Coenzyme Q10 (CoQ10) is a fat soluble, vitamin-like benzoquinone compound that functions primarily as an antioxidant, and a cofactor in the oxidative phosphorylation processes. The level of CoQ10 is reduced under the conditions of illness for example after renal transplantation. The present study was done aiming to detect the possible morphological and structural changes that may occur in the cardiac muscle of the adult male albino rats duo to CsA therapy and to test the protective effect of CoQ10 against CsA-induced cardiotoxicity. Thirty adult male albino rats were used in this study. They were classified into 3 groups and orally given the following materials for 28 days: Group (1) (Control group): that was further subdivided into 2 equal subgroups, each of 5 animals: subgroup 1a: received olive oil 1 ml/kg. Subgroup 1b: (CoQ10 group): received 5 mg/kg/day of CoQ10. Group (2) (CsA-treated group): The rats received 25 mg/kg/day of CsA. Group (3)(CoQ10 & CsA treated group): The rats were treated with the previously mentioned doses of both CoQ10 and CsA. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) were recorded three times per week. At the time of sacrifice, the hearts were dissected out and processed for light and electron microscopic examination. Oral administration of CsA, produced a significant (p<0.05) elevation in SBP, DBP, and HR in comparison to the control group. Treatment with CoQ10 significantly (p<0.05) reduced the increments in arterial blood pressure and heart rate. However, the recorded values were still significantly (p<0.05) higher than that of the control group. The light microscopic study of sections of cardiac muscle of CsA-treated adult male albino rats showed increase in the number of infiltrated cells and disorganization of myocardial fibers with increase in the amount of connective tissue and interstitial fibrosis with perinuclear cytoplasmic vacuolation and decrease in the amount of myofilaments. Electron microscopy showed distension of the Z lines with disintegration of some of them, loss of some microfilaments and disorganized intercalated discs. CoQ10 partially prevented most of the pathological changes revealed by the light and electron microscope. From the above mentioned results we could conclude that treatment with CoQ10 partially prevented the CsA-induced cardiotoxicity in adult male albino rats. We could recommend using CoQ10 during CsA therapy to prevent its cardiotoxicity.

Rosuvastatin Attenuates Inflammation, Apoptosis and Fibrosis in a Rat Model of Cyclosporine-Induced Nephropathy

Background/Aim: Cyclosporine (CsA)-induced kidney injury is characterized by renal dysfunction with inflammatory cell infiltrations, apoptosis and fibrosis. Pleiotropic effects of statins may exert anti-inflammatory, antiapoptotic and antifibrotic actions beyond lipid control. The aim of this study is to investigate whether rosuvastatin (RUS) has anti-inflammatory, antiapoptotic and antifibrotic effects on chronic CsA-induced nephropathy in a rat model. Methods: Male Sprague-Dawley rats fed a low-sodium diet were divided into three treatment groups: control (0.9% saline injection), CsA (15 mg/kg/day by subcutaneous injection), CsA + RUS (10 mg/kg/day by gastric gavage). Renal function, CsA level and lipid levels were measured at the end of 4 weeks. The expression of ED-1, transforming growth factor-β₁ (TGF-β₁) and α-smooth muscle actin (α-SMA) for inflammation and fibrosis were examined by Western blot analysis. The expression levels of apoptosis-associated factors were examined by Western blot analysis. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated biotin nick end-labeling (TUNEL) method. Results: Kidney function was decreased in CsA-treated rats compared with controls, which was attenuated by RUS. RUS did not affect the lipid level or the blood CsA level. TUNEL staining showed that RUS inhibited CsA-induced tubular apoptosis. RUS decreased CsA-induced increased expression of Bax/Bcl-2 ratio. The expressions of ED-1, α-SMA, TGF-β₁, Smad2/3, Smad4 and p-JNK were increased in CsA-treated rats, which were attenuated by RUS. Tubular atrophy and interstitial fibrosis in CsA-treated rats were attenuated by RUS supplementation. Conclusion: RUS supplementation attenuates proinflammatory and apoptosis-related factors and inhibits the fibrotic pathways including the smad-dependent and smad-independent pathways in a rat model of CsA-induced nephropathy.

Aqueous extract of Terminalia arjuna prevents cyclosporine-induced renal disorders

Cyclosporine (CsA), a powerful immunosuppressant, had a significant impact on transplantation medicine, and exposure to this chemical is known to induce oxidative stress and causes renal injury by the formation of free radicals. Acute and chronic renal damage are very common pathophysiologic disturbances caused by CsA. The present study has been conducted to evaluate the protective role of the aqueous extract of the bark of Terminalia arjuna (TA), an important Indian medicinal plant widely used in the preparation of ayurvedic formulations, on CsA-induced oxidative stress and resultant dysfunction in the rat kidney. Animals were treated with the aqueous extract of TA (100 mg/kg body weight (b.wt)) and then treated with CsA (25 mg/kg b.wt) in olive oil for 14 days. The level of urea, uric acid, and creatinine was determined from serum sample. The enzymes, namely, alkaline phosphatase (ALP), acid phosphatase (ACP), aspartate transaminase (AST), and alanine transaminase (ALT), and enzymic indices of membrane integrity such as Na+K+ATPase, Ca2+ATPase, and Mg2+ATPases were estimated in the tissue of all study groups. Antioxidant status in kidney tissues was estimated by determining the activities of the antioxidative enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione transferase (GST) and the levels of reduced glutathione, vitamin E, and vitamin C. In addition, the kidney phospholipids, cholesterol, triglycerides, and free fatty acids were determined in the tissue. Results showed that CsA caused a marked increase in the levels of urea, uric acid, and creatinine in serum and decreases the levels of ALP, ACP, AST, ALT, Na+K+ATPase, Ca2+ATPase, Mg2+ATPases, SOD, CAT, glutathione reductase, GST, GPx, vitamin E, and vitamin C whereas cholesterol, triglycerides, free fatty acid, and phospholipid levels were found to be increased in the kidney tissue homogenates of CsA-treated rats. Aqueous extract of TA successfully prevented the alterations of these effects in the experimental animals. Data also showed that the extract possessed strong free radical scavenging activity comparable to that of vitamin C. Our study demonstrated that the aqueous extract of the bark of TA could protect the kidney tissues against CsA-induced oxidative stress probably by increasing antioxidative defense activities.

Redox imbalances incite the hypertensive, baroreflex, and autonomic effects of cyclosporine in rats

Previous studies including ours showed that cyclosporine (CSA) causes baroreflex dysfunction and hypertension. Here we tested the hypothesis that oxidative damage in central and peripheral tissues underlies the hypertensive, baroreflex and autonomic actions elicited by CSA in rats. We investigated the effects of individual and combined 7-day treatments with CSA (25mg/kg/day, n=7) and 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol, superoxide dismutase mimetic, 100mg/kg/day, n=7) on blood pressure, reflex heart rate responses to peripherally mediated pressor and depressor responses, and biomarkers of oxidative stress. CSA elevated blood pressure and reduced reflex bradycardic (phenylephrine) and tachycardic (sodium nitroptrusside) responses. The ability of muscarinic (atropine, 1mg/kg i.v.) or β-adrenoceptor blockade (propranolol, 1mg/kg i.v.) to reduce reflex heart rate responses was reduced in CSA-treated rats, suggesting the impairment by CSA of reflex cardiac autonomic control. Concurrent administration of tempol abolished CSA-induced hypertension and normalized the associated impairment in baroreflex gain and cardiac autonomic control. Tempol also reversed the CSA-induced increases in aortic and brainstem nitrite/nitrate and malondialdehyde (MDA) and decreases in aortic superoxide dismutase (SOD). These findings implicate oxidative stress in peripheral and central cardiovascular sites in the deleterious actions of CSA on blood pressure and baroreceptor control of heart rate.

Effect of cyclosporine A on hepatic carbohydrate metabolism and hepatic gene expression in rat

Objective: Cyclosporine A (CsA)-based therapy has been implicated in the development of diabetes. Hence, its effects on hepatic carbohydrate metabolism and gene expression will be investigated.Methods: Sprague-Dawley rats given 15 mg/kg body weight/day of CsA for 20 days, as well as healthy untreated animals, received a glucose load enriched with [U-13C]glucose and deuterated water to resolve load and endogenous contributions to plasma glucose. Blood glucose and plasma insulin levels were assayed and at 60-min post-load, plasma glucose 13C and 2H-enrichments were analyzed by nuclear magnetic resonance spectroscopy and liver tissue analyzed for hepatic gene expression.Results: CsA-treated rats were glucose intolerant relative to controls (AUCglucose = 21,297 ± 857 versus 14,183 ± 1094, p < 0.01). Contributions from endogenous glucose production (EGP) were significantly elevated in CsA-treated rats (179 ± 16 versus 123 ± 13 mg/dl, p < 0.05). The increased endogenous contributions were attributable to glycogenolysis or glucose-G6P cycling and not to gluconeogenesis. Significantly higher expressions of fatty acid synthase and acetyl-CoA carboxylase 1 and 2 genes were observed in CsA-treated rats.Conclusions: CsA-altered glucose metabolism and gene expression could reflect increased hepatic insulin resistance. In the liver of CsA-treated animals, EGP suppression is impaired whereas hepatic de novo lipogenesis is enhanced contributing to dysregulated glucose and lipid metabolism.

Protective Effect of Paricalcitol on Cyclosporine-Induced Renal Injury in Rats

We evaluated the protective effect of paricalcitol on cyclosporine (CsA)–induced renal injury using an experimental model of chronic CsA nephropathy. Paricalcitol (50 and 200 ng/kg/d) was concomitantly administered with CsA (15 mg/kg/d) for 28 days in rats. We assessed the effects of paricalcitol by measuring degree of the tubulointerstitial fibrosis (TIF) and inflammation, a profibrotic cytokine (βig-h3), a proapoptotic gene (caspase-3), apoptotic cell death, and oxidative stress. The CsA-treated rats showed increased TIF and inflammatory cell infiltration, but paricalcitol treatment (200 ng/kg) significantly decreased those compared with the CsA-alone group. The expression of βig-h3, a biologic marker of transforming growth factor β1, which was increased in the CsA group, also decreased with paricalcitol treatment. The increased rates of excretion of urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) and expression of tissue 8-OHdG produced by CsA treatment were significantly attenuated by paricalcitol treatment. The increased expression of caspase-3 and number of TUNEL-positive cells in the CsA group were decreased with concomitant paricalcitol treatment. The effect of paricalcitol was more evident high among the rather than low-dose cohort. In conclusion, paricalcitol showed antiinflammatory and antifibrotic effects. This finding may provide a rationale for use of paricalcitol in CsA-induced renal injury.

Cyclosporine A Impairs Norepinephrine-Induced Vascular Contractility

Usage of cyclosporine A (CsA) after kidney transplantation may be associated with development of nephrotoxicity and vasculopathy, but the mechanisms by which CsA causes vascular dysfunction are still under scrutiny. We established a transplantation model and investigated the effect of CsA on vascular contractility with the aid of a pressurized myograph in comparison with control and unilaterally nephrectomized rats. Results were correlated with mRNA expression studies of α- and β-adrenoreceptors, in mesenteric resistance arteries versus the thoracic aorta. Consequences of everolimus on functional properties as well as adrenoreceptor expression were also studied. CsA significantly downregulated expression of mesenteric adrenoreceptors, whereas no effect on aortic adrenoreceptors was seen. Administration of everolimus had no influence on mRNA adrenoreceptor expression in mesenteric resistance arteries. Furthermore, contractile responses of mesenteric resistance arteries to norepinephrine were markedly reduced after treatment with CsA, while there was no difference in contraction by endothelin. Everolimus did not alter the contractility response at all. In summary, norepinephrine-induced, but not endothelin-induced, contractile responses of mesenteric resistance arteries are blunted in CsA-treated rats. This finding was accompanied by a marked downregulation of adrenoreceptors in mesenteric resistance arteries and was limited to the usage of CsA.

Metoprolol Ameliorates Cyclosporine A–induced Hypertension and Nephrotoxicity in Rats

Despite their clinical relevance as important cardiovascular modulators, there are few studies regarding the potential protective effect of β-blockers against immunosuppressive-induced cardiovascular side effects. This study investigated the possible ameliorating effect of β1-blocker, metoprolol (MTP), against both hypertensive and nephrotoxic effects of cyclosporine A (CSA). Compared with vehicle (olive oil)-treated rats, chronic treatment with CSA (20 mg·kg·d subcutaneous, for 14 days) increased systolic blood pressure, elevated renal function indices and plasma renin activity, impaired renovascular responsiveness of isolated perfused rat kidneys to endothelium-dependent vasodilations induced by carbachol. These effects were abolished upon concurrent administration of MTP (5 mg·kg·d for 14 days, intraperitoneal). The possibility that alterations in the antioxidant and/or circulating cytokine levels contributed to the CSA-MTP interaction was also investigated. MTP abrogated the oxidative (superoxide dismutase, catalase), lipid peroxidation (malondialdyde), and elevated the cytokine (TNF-α and TGF-β) effects of CSA. Histologically, CSA caused tubular brush border loss and isometric vacuolization clustered in the proximal tubule; this effect disappeared in rats cotreated with MTP. The use of a nonhypotensive dose of MTP (1.25 mg/kg) countered in part endothelium dysfunction altered oxidative stress parameters and cytokine levels in CSA-treated rats. Collectively, MTP abrogates both the hypertensive and the nephrotoxic effects of CSA via ameliorating endothelium dysfunction, oxidative stress, and upregulated cytokine levels caused by CSA. The demonstration of nephroprotection by the low dose of MTP suggests that its renal effect may be partially unrelated to its hemodynamic activity.

Use of Uric Acid-Lowering Agents Limits Experimental Cyclosporine Nephropathy

Background: Hyperuricemia frequently complicates cyclosporine (CsA) therapy. Previous studies have shown that hyperuricemia exacerbates interstitial and vascular lesions in the cyclosporine model. We tested the hypothesis that normalization of uric acid could prevent the development of cyclosporine toxicity. Methods: CsA nephropathy was induced by administering CsA (15 mg/kg/day) for 7 weeks to rats on a low salt diet (CsA group). The effect of preventing hyperuricemia was determined by concomitant treatment with a xanthine oxidase inhibitor, allopurinol (CsAALP), or with a uricosuric, benzbromarone (CsABENZ), in drinking water. Control groups included vehicle-treated rats. Results: CsA-treated rats developed mild hyperuricemia with arteriolar hyalinosis, tubular atrophy, striped interstitial fibrosis, increased cell proliferation and decreased VEGF expression. Treatment with allopurinol or benzbromarone limited renal disease, with reduced interstitial fibrosis, cell proliferation, macrophage infiltration, osteopontin expression and arteriolar hyalinosis, in association with restoration of VEGF expression. Both drugs provided comparable protection. Conclusions: An increase in uric acid exacerbates CsA nephropathy in the rat. Concomitant treatment with allopurinol or benzbromarone reduced the severity of renal disease. The similar protection observed with both drugs suggests that the effect is associated more with lowering uric acid levels than the antioxidant effect of allopurinol.

Expression of renal distal tubule transporters TRPM6 and NCC in a rat model of cyclosporine nephrotoxicity and effect of EGF treatment

Renal magnesium (Mg(2+)) and sodium (Na(+)) loss are well-known side effects of cyclosporine (CsA) treatment in humans, but the underlying mechanisms still remain unclear. Recently, it was shown that epidermal growth factor (EGF) stimulates Mg(2+) reabsorption in the distal convoluted tubule (DCT) via TRPM6 (Thébault S, Alexander RT, Tiel Groenestege WM, Hoenderop JG, Bindels RJ. J Am Soc Nephrol 20: 78-85, 2009). In the DCT, the final adjustment of renal sodium excretion is regulated by the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which is activated by the renin-angiotensin-aldosterone system (RAAS). The aim of this study was to gain more insight into the molecular mechanisms of CsA-induced hypomagnesemia and hyponatremia. Therefore, the renal expression of TRPM6, TRPM7, EGF, EGF receptor, claudin-16, claudin-19, and the NCC, and the effect of the RAAS on NCC expression, were analyzed in vivo in a rat model of CsA nephrotoxicity. Also, the effect of EGF administration on these parameters was studied. CsA significantly decreased the renal expression of TRPM6, TRPM7, NCC, and EGF, but not that of claudin-16 and claudin-19. Serum aldosterone was significantly lower in CsA-treated rats. In control rats treated with EGF, an increased renal expression of TRPM6 together with a decreased fractional excretion of Mg(2+) (FE Mg(2+)) was demonstrated. EGF did not show this beneficial effect on TRPM6 and FE Mg(2+) in CsA-treated rats. These data suggest that CsA treatment affects Mg(2+) homeostasis via the downregulation of TRPM6 in the DCT. Furthermore, CsA downregulates the NCC in the DCT, associated with an inactivation of the RAAS, resulting in renal sodium loss.

Tranilast Prevents the Progression of Chronic Cyclosporine Nephrotoxicity Through Regulation of Transforming Growth Factor β/Smad Pathways

Purpose Our aim was to investigate the role of tranilast in transforming growth factor (TGF) β/Smad pathways using a rat model of chronic cyclosporine (CsA) nephrotoxicity. Methods Thirty Sprague-Dawley (SD) rats were equally randomized in to 5 groups for gavage treatments daily for 4 weeks: normal control (N), olive oil; CsA (25 mg/kg), (M) CsA plus low-dose tranilast group (T1; CsA 25 mg/kg and tranilast 100 mg/kg); CsA plus medium-dose tranilast group (T2; CsA 25 mg/kg and tranilast 200 mg/kg); and CsA plus high-dose tranilast group (T4; CsA 25 mg/kg and tranilast 400 mg/kg). Kidneys were harvested at the end of the fourth week. TGF-β1 as well as Smad3 and Smad7 were detected by reverse-transcription polymerase chain reaction and immunohistochemistry. Results The administration of tranilast decreased the expression of TGF-β1 and Smad3 by CsA-treated rats, whereas it increased both mRNA and protein levels of Smad7. Semiquantitative analysis of mRNA production revealed these treatments to markedly reduce the amount of TGF-β1: T1: 0.8452 ± 0.0825 vs 0.8529 ± 0.0606 ( P < .05); T2: 0.8414 ± 0.0696 vs 0.8529 ± 0.0606 ( P < .05); T4: 0.8336 ± 0.0592 vs 0.8529 ± 0.0606 ( P < .05). For Smad3: T1: 0.8581 ± 0.0328 vs 0.8613 ± 0.0542 ( P < .05); T2: 0.8528 ± 0.0599 vs 0.8613 ± 0.0542 ( P < .05); T4: 0.8436 ± 0.0185 vs 0.8613 ± 0.0542 ( P < .05). The significantly elevated dose-dependent amounts of Smad7 were: T1: 0.9026 ± 0.0522 vs 0.8678 ± 0.0246, ( P < .05); T2: 0.9087 ± 0.0506 vs 0.8678 ± 0.0246 ( P < .05); T4: 0.9151 ± 0.0793 vs 0.8678 ± 0.0246 ( P < .05). Conclusion Regulation of TGF-β/Smad pathways is one of the mechanisims by which tranilast mitigates the progression of chronic CsA nephrotoxicity in rats.

Cyclosporin Treatment Alters Prostanoid and Thromboxane Production by Rat Isolated Kidney Mitochondria

This study was designed to investigate the effects of chronic treatment with cyclosporin A (CSA) on the endogenous synthesis of prostanoids (PGs) and thromboxane (Tx) by renal isolated medullary and cortical mitochondria. The administration of CSA, dissolved in 10% ethanol in olive oil, to male Wistar rats (20 mg kg-1 day-1 i.p.) for 14 days resulted in alterations in mitochondrial biosynthesis of immunoreactive PGs. The endogenous synthesis of thromboxane by medullary and cortical mitochondria isolated from CSA-treated rats was significantly enhanced by 120 and 55%, respectively, whereas the synthesis of prostaglandin E2 by medullary mitochondria was reduced by 35%. The synthesis of prostaglandin F2 alpha and prostacyclin was not affected by CSA treatment. The conversion of exogenous arachidonic acid to PGs and Tx by cortical mitochondria isolated from CSA-treated rats was significantly increased. In addition, CSA treatment resulted in i) a reduced acylation of arachidonic acid into medullary phospholipids by 25% and into medullary and cortical triglycerides by 33 and 27%, respectively, and ii) an increase in cortical and medullary triglycerides. We suggest that the alterations in the endogenous mitochondrial production of PGs and Tx caused by CSA, may play a role in the impairment of membrane mediated functions.