CsA-treated Rats Research Articles

Omega-3 fatty acids upregulate Nrf2 expression and attenuate apoptosis, inflammation, and fibrosis in a rat model of cyclosporine-induced nephropathy

Background: Cyclosporine A (CsA)-induced kidney injury is characterized by renal impairment with inflammatory cell infiltrations, apoptosis, fibrosis, and hypoxic injury. It is not clear whether omega-3 fatty acids (O-3 FAs), which have anti-inflammatory and antioxidant roles, affect nuclear factor erythroid 2-related factor 2 (Nrf2) expression. The aim of this study was to investigate whether O-3 FAs affect Nrf2 expression and exert anti-inflammatory, anti-apoptotic, and anti-fibrotic effects in CsA-induced nephropathy.Methods: Male Sprague-Dawley rats were divided into control, CsA-treated, and CsA-treated with O-3 FA groups. Nrf2 expression was measured by Western blots and immunohistochemical staining.Results: Kidney function was impaired in the CsA-treated rats compared to the controls. Caspase-3 and caspase-7 were activated in the CsA-treated group, and the Bax/Bcl2 ratio was higher. O-3 FAs attenuated these apoptosis-related changes. ED-1 and inhibition of kappa B (IĸB) protein expression were significantly upregulated in the CsA-treated group. Compared to the control group, O-3 FA supplementation attenuated the increased expression of ED-1 and IĸB related to inflammation. Smad2/3, Smad4, and transforming growth factor-β1 were activated in the CsA group, and O-3 FA treatment prevented these changes related to renal fibrosis. The expression of Nrf2 was reduced in CsA-treated rats, but Nrf-2 was increased by O-3 FA treatment.Conclusions: We suggest that Nrf2 is a potential mediator induced by O-3 FA supplementation and that it attenuates pro-inflammatory pathways, fibrotic processes, and apoptosis. Further studies are needed to elucidate the crosstalk between Nrf2 expression and signals related to O-3 FA treatment.

Cyclosporin-induced hypertension is associated with the up-regulation of Na+-K+-2Cl- cotransporter (NKCC2).

The use of cyclosporin A (CsA) is hampered by the development of nephrotoxicity including hypertension, which is partially dependent on renal sodium retention. To address this issue, we have investigated in vivo sodium reabsorption in different nephron segments of CsA-treated rats through micropuncture study coupled to expression analyses of sodium transporters. To translate the findings in rats to human, kidney-transplanted patients having CsA treatment were enrolled in the study. Adult male Sprague-Dawley rats were treated with CsA (15 mg/kg/day) for 21days, followed by micropuncture study and expression analyses of sodium transporters. CsA-treated kidney-transplanted patients with resistant hypertension were challenged with 50mg furosemide. CsA-treated rats developed hypertension associated with reduced glomerular filtration rate. In vivo microperfusion study demonstrated a significant decrease in rate of absolute fluid reabsorption in the proximal tubule but enhanced sodium reabsorption in the thick ascending limb of Henle's loop (TAL). Expression analyses of sodium transporters at the same nephron segments further revealed a reduction in Na+-H+ exchanger isoform 3 (NHE3) in the renal cortex, while TAL-specific, furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and NHE3 were significantly upregulated in the inner stripe of outer medulla. CsA-treated patients had a larger excretion of urinary NKCC2 protein at basal condition, and higher diuretic response to furosemide, showing increased FeNa+, FeCl- and FeCa2+ compared with both healthy controls and FK506-treated transplanted patients. Altogether, these findings suggest that up-regulation of NKCC2 along the TAL facilitates sodium retention and contributes to the development of CsA-induced hypertension.

Long-term cyclosporine A treatment promotes anxiety-like behavior: Possible relation with glutamate signaling in rat hippocampus

• Long-term CsA treatment induces anxiety-like behavior; • Hippocampus of CsA-treated rats presents increased GluN2 levels, NOS and CREB activities; • Chronic CsA administration affects hippocampal adaptive responses that can induce anxiogenic behavior. Chronic immunosuppressive treatment with cyclosporine A (CsA) leads to multiple side effects, including some that occur in the central nervous system (CNS). This study aims to investigate the impact of long-term CsA treatment on anxiety behavior and verify the alterations related to glutamate signaling in the rat hippocampus. Adult male Wistar rats were intraperitoneally injected with 15 mg/kg/day CsA for eight weeks, subjected to anxiety tests, and had their brains collected for biochemistry and Western blot analysis. Herein, we reported that eight weeks of CsA treatment promoted an anxiety-like effect and increased N-methyl-D-aspartate receptor (NMDAR) GluN2 subunit levels, nitric oxide synthase, and cAMP-response element binding protein (CREB) activities. Future work should consider the impact of long-term CsA treatment on NMDAR function, as well as on other neurotransmitter signaling pathways. Peripheral side effects should also be considered a factor that could lead to CsA-induced neuropsychiatric effects. In conclusion, this work reports modifications to CNS adaptive responses following chronic CsA treatment, which can influence anxiogenic behavior and other associated neurotoxic events. Understanding the mechanisms underlying CsA-induced neuropsychiatric side effects is an important step toward better symptom management in patients submitted to immunosuppression in the clinical setting.

Sodium-glucose cotransporter 2 inhibition prevents renal fibrosis in cyclosporine nephropathy

AimsSodium-glucose cotransporter 2 (SGLT2) inhibitors, a new class of antidiabetic drugs, are nephroprotective in case of diabetes, but whether a similar beneficial effect may be detectable also in case of chronic non-diabetic kidney diseases remains still unknown. The aim of this study was to evaluate the effects of empagliflozin, a SGLT-2 inhibitor, on the progression of cyclosporine nephropathy, in the absence of diabetes.MethodsSprague Dawley rats (n = 27) have been fed with low-salt diet starting 10 days before the beginning and finished at the end of the experimental period. Cyclosporine-A (CsA, 15 mg/kg/day, intraperitoneal injection, n = 8) and CsA plus empagliflozin (Empa, 10 mg/kg/day, per os, n = 7) were administered for 4 weeks. The control groups were treated with placebo (Control, n = 7) or empagliflozin (Control + Empa, n = 5). Blood pressure (plethysmographic method) was measured at the beginning and at the end of the experimental period. At the end of the experimental protocol, the kidneys were excised for histomorphometric analysis of renal fibrosis and for immunohistochemical evaluation of inflammatory infiltrates (monocytes/macrophages), type I and type IV collagen expression, and tyrosine hydroxylase expression, used as marker of sympathetic nerve activity.ResultsCsA-treated rats showed a significant increase (p < 0.01) in blood pressure, which was reduced by administration of empagliflozin (p < 0.05). CsA administration caused an increase in glomerular and tubulo-interstitial fibrosis (p < 0.05), renal inflammatory infiltrates (p < 0.05), type I and type IV collagen expression (p < 0.01), and tyrosine hydroxylase expression (p < 0.01) as compared to the control rats and control + Empa-treated rats. Treatment with empagliflozin in CsA-treated rats reduced glomerular (p < 0.01) and tubulo-interstitial fibrosis (p < 0.05), type I and type IV collagen expression (p < 0.01), inflammatory cell infiltration (p < 0.01) and tyrosine hydroxylase expression (p < 0.05), as compared to rats treated with CsA.ConclusionEmpagliflozin administration caused a reduction in blood pressure in CsA-treated rats and showed a protective effect on CsA nephropathy by decreasing renal fibrosis, type I and type IV collagen expression, macrophage infiltration and tyrosine hydroxylase expression. These data suggest that empagliflozin promotes nephroprotection also in non-diabetic kidney disease.

P0984SGLT2 INHIBITION PREVENTS RENAL FIBROSIS IN CYCLOSPORINE NEPHROPATHY

Abstract Background and Aims Sodium glucose cotransporter 2 (SGLT2) inhibitors, a new class of antidiabetic drugs, showed nephroprotection in type 2 diabetic patients. The mechanisms underlying nephroprotection are not completely known and it is unclear whether the nephroprotective effects are present also in non-diabetic nephropathy. The aim of this study was to evaluate the effects of empagliflozin, a SGLT-2 inhibitor, in cyclosporine nephropathy in the absence of diabetes. Method Ten days before the beginning and then during the entire experimental periods, low-salt diet (Teklad 7034) was administered to Sprague Dawley rats. Cyclosporine-A (CsA, 15 mg/kg/day, intraperitoneal injection; n=6) and CsA plus empagliflozin (Empa, 10 mg/kg /day, per os; n=6) were administered for 4 weeks. Control group was treated with placebo (n=6). Blood pressure was measured by plethysmographic method at the beginning and at the end of the experimental period. At the end of the protocol, the kidneys were excised for histomorphometric analysis of renal fibrosis and for immunohistochemical evaluation of inflammatory infiltrates and tyrosine hydroxylase expression, used as marker of symphatetic nerve activity. Results The rats treated with CsA showed a significant increase (p &amp;lt;0.01) in blood pressure, which was slightly reduced by administration of empagliflozin. CsA administration caused an increase in glomerular and tubulo-interstitial fibrosis (p &amp;lt;0.05), renal inflammatory infiltrates (p &amp;lt;0.05) and tyrosine hydroxylase expression (p &amp;lt;0.01) as compared to the control rats. Treatment with empagliflozin reduced glomerular and tubulo-interstitial fibrosis (p &amp;lt;0.05), inflammatory cell infiltration (p &amp;lt;0.01) and tyrosine hydroxylase expression (p &amp;lt;0.01), as compared to CsA-treated rats. Conclusion Empagliflozin administration showed protective effects on cyclosporine nephropathy, decreasing renal fibrosis, macrophage infiltration and tyrosine hydroxylase expression. These data suggest that the nephroprotective role of empagliflozin could not be restricted only to diabetic nephropathy.

Urotensin receptor antagonist palosuran attenuates cyclosporine-a-induced nephrotoxicity in rats.

Cyclosporine-A (CsA) is widely used for immunosuppressive therapy in renal transplantation. Nephrotoxicity is the main dose-limiting undesirable consequence of CsA. Urotensin II (U-II), a novel peptide with a powerful influence on vascular biology, has been added to the list of potential renal vascular regulators. Upregulation of the urotensin receptors and elevation of plasma U-II levels are thought to possibly play a role in the etiology of renal failure. The present study examines this hypothesis by evaluating renal function and histology with regard to the potential role of U-II and its antagonist, palosuran, in the pathogenesis of CsA-induced nephrotoxicity in rats. Male Sprague-Dawley rats were treated with CsA (15 mg/kg, for 21 days, intraperitoneally) or CsA + palosuran (300 mg/kg, for 21 days). Renal function was measured and histopathology, U-II immunostaining and protein detection with western blotting of the kidneys were performed. Cyclosporine-A administration caused a marked decline in creatinine clearance (Ccr). Fractional sodium excretion (FENa) tended to increase in the CsA-treated rats. Plasma U-II levels decreased in the CsA-treated rats. Cyclosporine-A treatment resulted in a marked deterioration in renal histology and an increase in the expression of U-II protein in the kidneys. Palosuran's improvement of renal function manifested as a significant decrease in serum creatinine levels and a significant increase in urine creatinine levels, resulting in a marked increase in Ccr. Palosuran produced a significant normalization of kidney histology and prevented an increase in U-II expression. Cyclosporine-A-induced renal impairment was accompanied by an increase in U-II expression in kidneys and a contrary decrease in systemic U-II levels. Palosuran improved the condition of rats suffering from renal dysfunction by preventing the decrease in renal U-II expression without affecting the systemic levels of U-II. The protective effect of palosuran in CsA nephrotoxicity is possibly independent of its U-II receptor antagonism.

The protective effect of resveratrol against cyclosporine A-induced oxidative stress and hepatotoxicity

The immunosuppressive agent cyclosporine A (CsA) has hepatotoxic potential. Increased reactive oxygen species (ROS) formation is among the causes leading to hepatotoxicity. This study aimed to investigate the effect of resveratrol (RES) on CsA-induced oxidative stress and hepatotoxicity in rats. Rats were treated with RES (10 mg/kg/day; i.p.) for 14 days. CsA (25 mg/kg/day; s.c.) was given during the last seven days together with RES. Serum alanine aminotransferase and aspartate aminotransferase activities together with hepatic histopathological examinations were performed. ROS, thiobarbituric acid reactive substances (TBARS), advanced oxidation protein products (AOPPs), ferric reducing antioxidant power, and glutathione levels as well as superoxide dismutase, and glutathione peroxidase activities were measured in the liver tissue. RES ameliorated histopathological changes and decreased hepatic ROS, TBARS, and AOPP levels significantly. However, antioxidant parameters did not change in CsA-treated rats. Our results indicate that RES treatment may be effective in decreasing CsA-induced oxidative stress and hepatotoxicity.

Inhibition of PPARα target genes during cyclosporine A-induced nephrotoxicity and hepatotoxicity

Cyclosporine A (CsA) is an immunosuppressant used to improve the rate of acute rejection and graft survival in transplant patients. However, CsA has serious adverse effects including high blood pressure as well as liver and kidney dysfunction. Although several toxicological mechanisms of CsA have been suggested, the investigations have been limited to evaluate the initiating events of CsA-induced toxicity in liver and kidney. To understand the events during initial hepatotoxicity and nephrotoxicity induced by CsA, we analyzed the comparative gene expression profiles of CsA-treated rats. We performed the microarray experiment using Affymetrix GeneChip Rat 230 2.0 array from the liver and kidney of CsA-treated rats (10 and 25 mg/kg of body weight, once daily for 4 weeks). Differentially expressed genes (DEGs) were selected using the Gene- Spring program (analysis of variance test, fold change ≥1.3), and functional analysis of the DEGs was conducted using the Ingenuity Pathways Analysis program. The most significant biofunctions of the DEGs were fatty acid oxidation, immune response, and molecular transport. In particular, fatty acid β-oxidation and lipopolysaccharide/interleukin-mediated inhibition of retinoid X receptor function were selected as significantly changed gene sets for each DEG in the liver and kidney. Fatty acid β-oxidation is closely correlated with peroxisome proliferator activated receptor α (PPARα), which regulates the expression of genes involved in lipid and glucose metabolism. In this study, Pparα was identified as a common upstream regulator of the DEGs, and the expression of genes related to lipid metabolism, as well as the damage of renal tubules, was significantly influenced by CsA treatment. We confirmed that fatty acid β-oxidation-related genes are downregulated by CsA treatment in the liver and kidney and using quantitative real-time RT-PCR. Our findings suggest that PPARα agonists may be candidates for reducing the toxicity of CsA treatment and encourage translational research.

Differential Glycosylation Expression in Injured Rat Spinal Cord Treated with Immunosuppressive Drug Cyclosporin-A.

Glycosylation is ubiquitous throughout the central nervous system and altered following spinal cord injury (SCI). The glial scar that forms following SCI is composed of several chondroitin sulfate proteoglycans, which inhibit axonal regrowth. Cyclosporin-A (CsA), an immunosuppressive therapeutic, has been proposed as a potential treatment after SCI. We investigated CsA treatment in the spinal cord of healthy, contusion injured, and injured CsA-treated rats. Lectin histochemistry using fluorescently labeled lectins, SBA, MAA, SNA-I, and WFA, was performed to identify the terminal carbohydrate residues of glycoconjugates within the spinal cord. SBA staining decreased in gray and white matter following spinal cord injury, whereas staining was increased at the lesion site in CsA-treated animals, indicating an increase in galactose and N-acetylgalactosamine terminal structures. No significant changes in MAA were observed. WFA staining was abundant in gray matter and observed to increase at the lesion site, in agreement with increased expression of chondroitin sulfate proteoglycans. SNA-I-stained blood vessels in all spinal cord regions and dual staining identified a subpopulation of astrocytes in the lesion site, which expressed α-(2,6)-sialic acid. Glycosylation were altered in injured spinal cord treated with CsA, indicating that glycosylation and alteration of particular carbohydrate structures are important factors to consider in the examination of the environment of the spinal cord after injury.

Expression of brain-derived neurotrophic factor in kidneys from normal and cyclosporine-treated rats

BackgroundAccumulating evidence suggests that a decrease in brain-derived neurotrophic factor (BDNF) level induces a variety of psychiatric and neurological disorders. However, the expression and role of BDNF in the kidney have not been explored. The present study examined the expression of BDNF and tropomyosin-related kinase (Trk) receptors in an experimental model of chronic cyclosporine A (CsA) nephropathy.MethodsSprague-Dawley rats on a salt-deplete diet were treated daily for four weeks with vehicle or CsA. Urine profiles, apoptotic cell death, oxidative stress (8-hydroxy-2′-deoxyguanosine, 8-OHdG), and expression of BDNF and Trk receptors (TrkB and TrkC) were compared between groups. The impact of vasopressin infusion on the urine-concentrating ability was examined by measuring the expression of aquaporin-2 (AQP-2) and BDNF and urine profiles in normal and CsA-treated rats.ResultsCompared with the vehicle-treated rats, rats given CsA had enhanced urine volume and declined urine osmolality. Immunohistochemistry and immunoblotting showed that BDNF and Trk receptors were constitutively expressed in kidneys from vehicle-treated rats. This was confirmed by double immunofluorescent staining for Na-K-ATPase-α1, AQP-1, and AQP-2. By contrast, the expression of these factors decreased in kidneys from CsA-treated rats (BDNF: 51.1 ± 19.5% vs. 102.0 ± 30.3%, p < 0.01). Downregulation of BDNF was accompanied by impairment of urine osmolality, and this was reversed by exogenous infusion of vasopressin. Notably, the number of TUNEL-positive cells correlated negatively with BDNF expression and positively with urinary 8-OHdG excretion.ConclusionsBDNF is expressed in the collecting duct of the kidney and may be associated with urine-concentrating ability in an experimental model of chronic CsA-induced nephropathy. Our study provides a new avenue for further investigation of chronic CsA nephropathy.

Evaluating the Effect of Resveratrol on the Healing of Extraction Sockets in Cyclosporine A–Treated Rats

The objective of this study was to investigate the effects of resveratrol on alveolar socket healing after tooth extraction in normal and cyclosporin A (CsA)-treated rats. Seventy-two female Sprague-Dawley rats were separated into 4 groups of 18. Group 1 was injected with a placebo solution intraperitoneally. Group 2 was injected with resveratrol (10μmol/kg) intraperitoneally. Groups 3 and 4 were injected with CsA (10mg/kg) subcutaneously for 8days once daily before tooth extraction. Next, the teeth were extracted and CsA injection continued until the animals were sacrificed. Eight days after commencing the CsA injections, group 4 was injected with resveratrol while continuing with CsA injections. Nine rats from each group were sacrificed on days 14 and 28, and sections were examined to assess the degree of inflammation, formation of connective tissue, and new bone formation. Immunohistochemical analysis was used to evaluate the alveolar socket healing process using osteocalcin and osteopontin markers. A P value less than .05 was considered significant. There was more new bone formation in group 2 than in the other 3 groups on day 14 after tooth extraction (P<.05), and there was more new bone formation in group 2 than in groups 3 and 4 on day 28 after extraction (P<.05). Based on the immunohistochemical assessment, the amount of osteocalcin and osteopontin labeling was greater in group 2 compared with the other 3 groups on day 14 (P<.05); however, on day 28 after extraction, it was greater in group 4 compared with group 3 (P<.05). Resveratrol improves alveolar socket healing in normal and CsA-treated rats. Resveratrol also increases levels of osteocalcin and osteopontin in normal and CsA-treated rats. These results suggest that this natural compound is useful for alveolar socket healing after tooth extraction.

Cyclosporine A exhibits gender-specific nephrotoxicity in rats: Effect on renal tissue inflammation

Cyclosporine A (CSA) is a widely used immunosuppressant drug known to commonly cause cardio and nephrotoxicity. A study looking at the sex specificity of the cardiotoxicity of CSA revealed that sexual dimorphism existed when looking at the electrocardiographs and left ventricles of CSA-treated rats. We hypothesized that cyclosporine A exhibited gender-specific nephrotoxicity by testing various parameters of kidney function in male and female rats treated for 21 days with 15 mg/kg CSA versus control male and female rats that received a vehicle consisting of 18% kolliphore and 2% ethanol in sterile saline. It was found that male rats treated with CSA had significantly higher levels of serum creatinine and lower creatinine clearance than control males. However, serum creatinine and creatinine clearance were not affected by CSA treatment in females. Histopathological examination of kidney cross-sections revealed a heavy aggregation of inflammatory cells and significant vascular congestion in males treated with CSA, which was less prominent in female rats receiving CSA. In addition CSA treated male rats had higher levels of serum cholesterol compared with control while, CSA did not affect serum cholesterol in female rats. Kidney tumor necrosis factor alpha (TNF-α) levels were found to drop in female rats following CSA treatment, whereas no change was observed in male rats before and after treatment. These results suggest that CSA exhibits gender-related nephrotoxicity in rats that might be mediated by differences in the inflammatory response between males and females.

Dysbiosis in chronic intestinal inflammation and tumorigenesis: Can we apply Koch's postulates?

The endothelium-derived hyperpolarizing factor (EDHF) serves as a back-up mechanism that compensates for reduced nitric oxide (NO)/prostanoids bioavailability. Here we investigated whether (i) under conditions of vascular endothelium dysfunction, the immunosuppressant drug cyclosporine (CSA) upregulates EDHF-dependent renal vasodilations through altering CYP4A/CYP2C signaling, and (ii) calcium channel blockers modulate the CSA/EDHF/CYP interaction. Rats were treated with CSA, verapamil, nifedipine, or their combinations for 7 days. Blood pressure (BP) was measured by tail-cuff plethysmography. Kidneys were then isolated, perfused with physiological solution containing L-NAME (NOS inhibitor) and diclofenac (cyclooxygenase inhibitor, DIC), and preconstricted with phenylephrine. CSA (25 mg kg− 1 day− 1 for 7 days) increased BP and augmented carbachol renal vasodilations. The co-treatment with verapamil (2 mg kg− 1 day− 1) or nifedipine (3 mg kg− 1 day− 1) abolished CSA hypertension and conversely affected carbachol vasodilations (increases vs. decreases). Infusion of MSPPOH (epoxyeicosatrienoic acids, EETs, inhibitor) reduced carbachol vasodilations in kidneys of all rat groups, suggesting the importance of EETs in these responses. By contrast, 20-Hydroxyeicosatetraenoic Acid (20-HETE) inhibition by HET0016 increased carbachol vasodilations in control rats, an effect that disappeared by CSA treatment, and reappeared in rats treated with CSA/verapamil or CSA/nifedipine. Renal protein expression of CYP2C and CYP4A as well as their vasoactive products (EETs/20-HETE) were increased in CSA-treated rats. Whereas the CYP2C/EETs effects of CSA were abolished by verapamil and intensified by nifedipine, the CYP4A/20-HETE effects were reduced by either CCB. Overall, nifedipine and verapamil blunts CSA hypertension but variably affected concomitantly enhanced EDHF-dependent renal vasodilations and alterations in CYP2C/CYP4A signaling.

CYP4A/CYP2C modulation of the interaction of calcium channel blockers with cyclosporine on EDHF-mediated renal vasodilations in rats

The endothelium-derived hyperpolarizing factor (EDHF) serves as a back-up mechanism that compensates for reduced nitric oxide (NO)/prostanoids bioavailability. Here we investigated whether (i) under conditions of vascular endothelium dysfunction, the immunosuppressant drug cyclosporine (CSA) upregulates EDHF-dependent renal vasodilations through altering CYP4A/CYP2C signaling, and (ii) calcium channel blockers modulate the CSA/EDHF/CYP interaction. Rats were treated with CSA, verapamil, nifedipine, or their combinations for 7days. Blood pressure (BP) was measured by tail-cuff plethysmography. Kidneys were then isolated, perfused with physiological solution containing L-NAME (NOS inhibitor) and diclofenac (cyclooxygenase inhibitor, DIC), and preconstricted with phenylephrine. CSA (25mgkg−1day−1 for 7days) increased BP and augmented carbachol renal vasodilations. The co-treatment with verapamil (2mgkg−1day−1) or nifedipine (3mgkg−1day−1) abolished CSA hypertension and conversely affected carbachol vasodilations (increases vs. decreases). Infusion of MSPPOH (epoxyeicosatrienoic acids, EETs, inhibitor) reduced carbachol vasodilations in kidneys of all rat groups, suggesting the importance of EETs in these responses. By contrast, 20-Hydroxyeicosatetraenoic Acid (20-HETE) inhibition by HET0016 increased carbachol vasodilations in control rats, an effect that disappeared by CSA treatment, and reappeared in rats treated with CSA/verapamil or CSA/nifedipine. Renal protein expression of CYP2C and CYP4A as well as their vasoactive products (EETs/20-HETE) were increased in CSA-treated rats. Whereas the CYP2C/EETs effects of CSA were abolished by verapamil and intensified by nifedipine, the CYP4A/20-HETE effects were reduced by either CCB. Overall, nifedipine and verapamil blunts CSA hypertension but variably affected concomitantly enhanced EDHF-dependent renal vasodilations and alterations in CYP2C/CYP4A signaling.

Epithelial to mesenchymal transition in Cyclosporine A-induced rat gingival overgrowth

Background and objectiveEpithelial-mesenchymal transition (EMT) has been proved to occur in drug-induced gingival overgrowth. However, the specific pathogenic mechanism remains uncertain. The aim of this study is to examine the expression of EMT markers in cyclosporine A (CsA)-induced gingival overgrowth in rat models. Material and methodsThirty-six rats were randomly divided into two groups. The experimental group received CsA therapy subcutaneously in a daily dose of 10mg/kg, and the other group was used as a control. Six rats per group were sacrificed at 20, 40 and 60days, and the gingivae were obtained. The expression of TGF-β1, E-Cadherin, ZEB1, ZEB2, and Snail1 were examined by quantitative real time PCR (qRT-PCR), western blotting, and immunohistochemistry. In addition, a group of microRNAs associated with EMT and fibrosis were also detected in gingival tissue by qRT-PCR. ResultsThe mRNA and protein levels of TGF-β1, ZEB1, and ZEB2 in gingivae were significantly upregulated after 40 and 60days of CsA administration. Conversely, the levels of E-cadherin were significantly downregulated in overgrowth sample at day 40 and 60. Intense immunohistochemmical staining for TGF-β1 were observed in the samples from CsA group at day 40 and 60. Concomitantly, the densities of E-cadherin were gradually decreased in the basal layers of epithelium with time. Three members of miR–200s (miR-200a, miR-200b and miR-200c) were significantly downregulated in CsA-treated rats at 40 and 60days, while miR-9, miR-23a and miR-155 were significantly upregulated when compared with those of the control group. ConclusionsThe process of EMT in CsA-induced rat gingival overgrowth is associated with increased expression of TGF-β1, ZEB1, and ZEB2, and decreased expression of E-cadherin.

Role of alpha-lipoic acid in ameliorating Cyclosporine A-induced pancreatic injury in albino rats: A structural, ultrastructural, and morphometric study

ABSTRACTThe aim of this study was to investigate the effectiveness of alpha-lipoic acid (α-LA) against cyclosporine A (CsA)-induced pancreatic toxicity in rats. Thirty-two male albino rats were divided into four equal groups. Group I treated orally (per os, p.o.) with vehicles and served as control; Group II received α-LA (100 mg/kg b.w. /day, p.o.) for 21 days; Group III received CsA (25 mg/kg b.w./day, p.o.) for 21 days; and Group IV received α-LA 1 hr before oral treatment by CsA for 21 days. Histological examination of the pancreas of CsA-treated rats showed marked changes represented by wide interlobular septae that contained congested blood vessels, cytoplasmic vacuolation of some acinar cells, and distortion of the other cells. Most of the islets of Langerhans showed vacuolation, degenerative changes, and loss of uniform cellular distribution. Some of the islets appeared shrunken with few cells. In the CsA group, the immunohistochemical and morphometric study demonstrated a decrease in the number of insulin-secreting β-cells and also a reduction in islet diameters, with statistically significant difference (p < 0.001 and p = 0.004), respectively, compared with the control group. Ultrastructure of the exocrine and endocrine pancreatic cells of the CsA-treated group confirmed the light microscopic observation and showed dilated rough endoplasmic reticulum, decreased zymogen and secretory granules, damaged mitochondria, and abnormal nuclei. However, α-LA administration simultaneously with CsA resulted in some sort of regression of the previously mentioned effects. Conclusion: α-LA attenuated CsA-induced structural and ultrastructural changes in pancreatic cells, which were nearly reverted to their normal structure.

The inflammatory state provokes sexual dimorphism in left ventricular and electrocardiographic effects of chronic cyclosporine in rats

Although cardiotoxicity has been recognized as an adverse effect of cyclosporine A (CSA), no information exists regarding sex specificity of CSA cardiotoxicity. We tested the hypothesis that left ventricular (LV) and electrocardiographic (ECG) effects of CSA and related inflammatory/histopathological derangements are sex related. CSA reduced the LV slope of end-systolic pressure volume relationship and increased isovolumic relaxation constant. These effects were more pronounced in male compared to female rats, suggesting LV systolic and diastolic dysfunction. ECG recordings showed elevated ST segments and increased QTc and T peak trend intervals in CSA-treated male rats, markers of LV ischemia and arrhythmogenesis. In female rats, CSA delayed AV conduction, as reflected by prolonged PR interval. Other sex-related effects for CSA included (i) increased blood cholesterol, and reduced rates of rise and fall in LV pressure and nuclear factor kappa B and angiotensin receptors type 1 expressions in male rats, and (ii) increased LV adiponectin in females. Histopatholgically, CSA caused vascular congestion, blood extravasation, and pyknotic or even absent nuclei in both sexes. In conclusion, rats exhibit sex-independent susceptibility to negative LV and histopathological influences of CSA. These effects become more intensified in male rats, perhaps on account of aggravated ischemic and inflammatory milieus.

Facilitation by the renin-angiotensin system of cyclosporine-evoked hypertension in rats: Role of arterial baroreflexes and vasoreactivity

AimsCyclosporine (CSA) elevates blood pressure (BP) and alters arterial baroreflex sensitivity (BRS) and vasoreactivity. In this study we determined whether the renin-angiotensin system (RAS) interplays with other vasopressor pathways in mediating the CSA actions. Materials and methodsWhole animal and isolated vascular preparations were employed to determine the effects of pharmacologic interruption of angiotensin II (Ang II), endothelin (ET), or thromboxane (TXA2) signaling on the adverse cardiovascular effects of CSA. Key findingsCSA (25mg/kg/day i.p. for 7days) caused significant increases in BP that were paralleled with (i) reduced BRS measured by phenylephrine (BRSPE) or sodium nitroprusside (BRSSNP), (ii) enhanced aortic contractile responses to Ang II and U-46619 (thromboxane analogue), and (iii) reduced aortic eNOS expression and acetylcholine, but not SNP, vasorelaxations. Except for the reduced BRSSNP, the CSA effects disappeared upon concurrent administration of losartan (angiotensin AT1 receptor antagonist), captopril (angiotensin converting enzyme inhibitor), or their combination. Moreover, CSA augmentation of Ang II contractions was abolished after cyclooxygenase inhibition (indomethacin) or endothelin ETA/ETB receptor blockade (atrasentan/BQ788). By contrast, the blockade of thromboxane receptors (terutroban) failed to alter the CSA-evoked facilitation of Ang II responsiveness. SignificanceThe facilitation of baroreflex control and inhibition of vascular responsiveness to Ang II and thromboxane contribute to the BP lowering effect of RAS inhibitors in CSA-treated rats. Further, endothelin receptors and vasoconstrictor prostanoids contribute to the CSA-evoked exaggeration of Ang II vascular responsiveness and hypertension.

The effects of propolis on sperm quality, reproductive organs and testicular antioxidant status of male rats treated with cyclosporine-A

This study was designed to determine the effects of propolis on the quality of sperm and reproductive organs in male rats treated with cyclosporine-A. In this study, 24 male Sprague-Dawley rats (280-300 g BW) at 8-10 weeks of age were used. The rats were randomly divided into control and 3 treatment groups. Each rat was placed into a cage. During 21 days (experimental period), Group 1 served as control group; Group 2 (CsA) was given 15 mg/kg BW/day of CsA by subcutaneously; Group 3 (P) was given 100 mg/kg BW/day of propolis by gavage; Group 4 (CsA+P) was given 15 mg/kg BW/day of CsA subcutaneously and 100 mg/kg BW/day of propolis by gavage. Administration of CsA to rats decreased sperm motility (P < 0.01) and sperm concentration (P < 0.01), and it increased total abnormal sperm rates (P < 0.05) as compared with the control group. Significant improvements were observed in the sperm motility (P < 0.01) and total abnormal sperm rates (P < 0.05) in CsA+P group. No significant differences were observed in the weights of right and left testes among all groups. The weight values of right and left epididymis were found similar to each other in CsA administrated groups. Seminal vesicle (P < 0.01) and prostat glands weights (P < 0.01) were significantly higher in CsA+P group than CsA group. As a result, it was determined that oral supplementation of 100 mg/kg of propolis had amendatory effect on the quality of sperm and reproductive organs treated with CsA administered rats. CsA treatment caused a significant increase in MDA level (P < 0.01) and significant decreases (P < 0.01) in GSH level and CAT activity when compared with the control group. Ingestion of propolis by CsA-treated rats significantly decreased the MDA level and significantly increased the GSH level when compared with the CsA alone group (P < 0.01).

Cyclosporine A Inhibits Apoptosis of Rat Gingival Epithelium

The use of cyclosporine A (CsA) induces hyperplasia of the gingival epithelium in a site-specific response manner, but the molecular mechanism via which the lesion occurs is unclear. The present research aims to investigate the site-specific effect of CsA on the apoptosis of gingival epithelium associated with gingival hyperplasia. Forty Wistar rats were divided into CsA-treated and non-treated groups. Paraffin-embedded sections of mandibular first molars were selected for hematoxylin and eosin staining, immunohistochemistry analyses of bcl-2 and caspase-3, and the staining of terminal deoxynucleotidyl transfer-mediated dUTP nick-end labeling (TUNEL). The area of the whole gingival epithelium and the length of rete pegs were measured, and the number of bcl-2- and caspase-3-positive cells in the longest rete peg were counted. The analysis of variance for factorial designs and Fisher least significant difference test for post hoc analysis were used to determine the significance levels. In CsA-treated rats, bcl-2 expression was significantly upregulated, whereas caspase-3 expression was downregulated, along with a reduced number of TUNEL-positive cells. The site-specific distribution of bcl-2 was consistent with the site-specific hyperplasia of the gingival epithelium in CsA-treated rats. CsA inhibited gingival epithelial apoptosis via the mitochondrial pathway and common pathway. The antiapoptotic protein bcl-2 might play a critical role in the pathogenesis of the site-specific hyperplasia of gingival epithelium induced by CsA. There were mechanistic differences in the regulation of apoptosis for cells in the attached gingival epithelium, free gingival epithelium, and junctional epithelium.