Effect of cyclosporine on bile secretion in rats

Short-term effects of cyclosporine were studied in the isolated perfused rat liver model. Bile flow was inhibited by cyclosporine in 2 mg/kg and 20 mg/kg doses but not by a 0.2 mg/kg dose. Cholestasis was accompanied by a decrease in bile acid secretion, indicating an inhibitory effect on the bile acid-dependent fraction of bile flow. Perfusate bilirubin levels increased threefold in rat livers given 20 mg/kg of cyclosporine, but did not change in control animals. Alkaline phosphatase and transaminase levels did not differ from those of control animals. The isolated perfused rat liver was able to excrete cyclosporine, as demonstrated by a continual decrease in perfusate cyclosporine levels. No light microscopic evidence of cholestasis or hepatocellular damage was demonstrated on histologic staining. Our model appears to be a good one for the study of altered hepatic physiologic characteristics caused by administration of cyclosporine.     

Effect of cyclosporine administration on renal hemodynamics in conscious rats

The effect of acute and chronic administration of cyclosporine on systemic and renal hemodynamics was studied in conscious rats. Infusion of cyclosporine in a dose of 20 mg/kg (Cy 20) resulted in a significant fall in renal blood flow (RBF) (3.4 vs. 6.5 ml/min/g, P less than 0.05) and a rise in renal vascular resistance (RVR) (36.9 vs. 20.6 mm Hg/ml/min/g, P less than 0.05). Infusion of cyclosporine at a dose of 10 mg/kg (Cy 10) did not result in a significant change in RBF or RVR. Both doses of cyclosporine resulted in stimulation of plasma renin activity (PRA) from control values of 5.6 +/- 0.8 ng/ml/hr to 11.6 +/- 2.0 with 10 mg/kg and 26.7 +/- 5.6 with 20 mg/kg. Urinary 6-keto-PGF1 alpha excretion increased from control values of 14.0 +/- 2.0 ng/6 hr to 22.7 +/- 2.2 with 10 mg/kg and 25.0 +/- 2.0 with 20 mg/kg. Similar effects on RBF, RVR, PRA, and 6-keto-PGF1 alpha excretion were seen after chronic administration of cyclosporine (20 mg/kg i.p. for 7 days). Pretreatment of animals with captopril did not prevent the fall in RBF after cyclosporine, suggesting that the vasoconstriction was not mediated by angiotensin II. Animals treated with meclofenamate demonstrated reduction in RBF with 10 mg/kg cyclosporine (4.3 vs. 7.0 ml/min/g, P less than 0.05), suggesting that prostaglandins protect against the vasoconstrictor effect of cyclosporine. Administration of phenoxybenzamine after cyclosporine improved RBF (5.0 vs. 3.4 ml/min/g) and restored RVR to normal. Similarly, renal denervation dramatically reduced the fall in RBF after cyclosporine (innervated right kidney 3.6 vs. denervated left kidney 6.0 ml/min/g, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of FTY720 on chronic cyclosporine nephropathy in rats

BACKGROUND: Long-term treatment with cyclosporine A (CsA) causes tubulointerstitial inflammation and fibrosis in the kidney. To define the role of lymphocytes in this process, the novel lymphocyte-specific inhibitor FTY720 was administered to rats with experimental model of chronic CsA nephropathy. METHODS: Sprague-Dawley rats were treated daily for 4 weeks with CsA (7.5 mg/kg), or both CsA and FTY720 (0.125 mg/kg). The effects of FTY720 on CsA-induced renal injury were evaluated using renal function tests and histopathology, and the expression of mediators of CsA-induced renal injury (osteopontin, transforming growth factor-beta1 [TGF-beta1], betaig-h3, and angiotensin II). RESULTS: FTY720 treatment significantly decreased T-lymphocyte accumulation in kidneys compared with CsA treatment alone. FTY720 treatment improved not only CsA-induced renal dysfunction but also renal histopathology, demonstrated by decreased macrophage infiltration and interstitial fibrosis. Increased osteopontin, TGF-beta1, betaig-h3, and angiotensin II expression in CsA-treated rat kidneys were decreased with FTY720 treatment. CONCLUSIONS: FTY720 treatment prevents CsA-induced renal injury.     

Effects of cyclosporine on metalloproteinase in endothelial cells

Objectives

Cyclosporine (CsA) is a potent agent widely used after organ transplantations and in treatment of various autoimmune disorders. Some patients suffer severe complications including renal and vascular toxicity that are influenced by the degree of endothelial damage. Dysregulation of metalloproteinase (MMP) activity is known to contribute to renal and vascular diseases. To investigate the possible mechanisms of posttransplantation complications in the kidney and vessels by CsA, we examined its effects on metalloproteinases in endothelial cells using human umbilical vein endothelial cells (HUVECs).

Methods

HUVECs isolated from umbilical cords by collagenase digestion were seeded in 6-well plates at a density of 1 × 10⁵ cells/well before treatment with 2-250 μmol/L CsA and a 24-hour incubation. Thereafter we performed gelatin zymography of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMp-13 to evaluate band density using a luminescent image analyzer system with controls calculated as 100%.

Results

MMP-1, MMP-3, MMP-8, MMP-9, and MMP-13 activities were increased after CsA treatment; MMP-1 = 121; MMP-3 = 164; MMP-8 = 133; MMP-9 = 124; and MMP-13 = 121. In contrast, MMP-2 activity was decreased after CsA treatment; MMP-2 = 79.

Conclusions

This study showed CsA to activate most MMPs (except MMP-2) in endothelial cells. This result suggests that CsA may dysregulate MMPs in endothelial cells.     

Effect of acute intestinal obstruction on the leakage of albumin from blood into the small intestine.

The effect of a simple, low intestinal obstruction has been investigated in dogs on the leakage of 131I-serum albumin from the circulation into the intestine. An increase leakage has been demonstrated. In the distended segment of the intestine above that ligation a significant increase in protein-bound radioactivity, from the normal value of 0.082 plus or minus 0.012 ml/10 cm intestine/hour to 0.276 plus or minus 0.068 ml/10 cm intestine/hour, was obseved which means a more than 3fold increase. The values for albumin leakage did not change in the more proximal segments of the intestine less involved in the distension namely in the duodenum and the jejunum, furthermore in the ileal segment below the ligation. The increase in albumin liadage observed during intestinal obstruction resulted in 33% rise of total catabolism.     

Acute modulation of albumin microvascular leakage by advanced glycation end products in microcirculation of diabetic rats in vivo.

Advanced glycation end products (AGEs) are nonenzymatic glycosylated adducts of proteins that accumulate in vascular tissue during diabetes and aging. The aim of this work was to study the role of AGEs and of the oxidative mechanisms in diabetes-induced changes in vascular permeability. Intravital videomicroscopy was used to study albumin microvascular leakage in cremaster muscle. The extravasation of a fluorescent macromolecular tracer (fluorescein isothiocyanate-albumin) was measured for 1 h and, after computer-aided image analysis, was expressed as variations of normalized gray levels (arbitrary units). Extravasation of the macromolecular tracer was much higher in diabetic rats than in control rats (slope of extravasation versus time increased by >100%, P < 10(-4)). This increase was significantly inhibited when we blocked AGEs binding to their endothelial receptor by intravenous bolus of soluble recombinant receptor to AGEs (rR-RAGE) (slope of extravasation versus time decreased by 19, 30, and 40%, for 0.5, 2.5, and 5.15 mg/kg rR-RAGE, respectively) or by a 6 mg/kg intravenous bolus of antibody against RAGE (slope decreased by 53%). Systemic injection of probucol (an antioxidant) also significantly inhibited the increase in the extravasation of the macromolecular tracer occurring in experimental diabetes (slope decreased by 51%, P < 10(-4)). These results strongly suggest that in experimental diabetes the interaction of circulating AGEs and endothelial RAGE mediates albumin micro-vascular leakage, possibly via AGE-RAGE-dependent enhanced oxidant stress.     

环孢素对大鼠睾丸缺血-再灌注损伤后生精细胞凋亡的影响

目的探讨环孢素对大鼠单侧睾丸缺血-再灌注损伤后生精细胞凋亡的影响,研究其保护性作用。方法雄性SD大鼠24只,随机分成假手术组、扭转组和环孢素组,每组8只,建立720°2h单侧睾丸扭转动物模型。扭转组和环孢素组于复位前15min分别腹腔注射生理盐水和环孢素,术后24h留取手术侧睾丸。应用流式细胞术检测各组患侧睾丸组织生精细胞凋亡;Quantitative Real-time PCR技术对Fas、FasL和Bax mRNA进行定量分析;Western-blot技术检测细胞色素C含量。结果与假手术组相比,扭转组患侧睾丸组织早期凋亡生精细胞百分比明显增多,Fas、FasL和Bax mRNA表达上调,同时胞质中细胞色素C含量显著升高,其差异均有显著性(P均<0.05)。环孢素干预能显著减轻上述变化,患侧睾丸组织正常细胞群百分比升高,Fas、FasL和Bax mRNA表达下调,同时胞质中细胞色素C含量显著降低,其差异均有显著性(P均<0.05)。结论环孢素可能参与调控生精细胞凋亡的分子途径,对睾丸扭转术后的生精功能具有明显的保护性效果。     

Effect of cyclosporine on carcinogenesis induced in rats by N-Methyl-N′-Nitro-N-Nitrosoguanidine

Cyclosporine administration has been associated with the development of lymphomas in human transplant patients as well as animals. Its effect on the genesis of common epithelial carcinomas is unknown. To investigate this N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was administered in drinking water to Wistar rats. Seventy-five young healthy male animals were divided into six groups and received cyclosporine alone, cyclosporine followed by MNNG, MNNG alone, cyclosporine during MNNG administration, MNNG followed by cyclosporine, and no treatment. Cyclosporine seemed to have minimal overall health effects and no cancers were encountered in the group receiving this agent alone. Animals in all carcinogen-treated groups developed gastric and upper intestinal carcinomas by Week 39. No statistically significant differences among carcinogen-treated groups were evident with respect to tumor incidence, histology, or distribution. There appeared to be trends (not statistically significant) toward a greater incidence of small bowel carcinomas in animals receiving cyclosporine plus MNNG as compared to those receiving MNNG alone; greater multiplicity of small intestinal carcinomas in animals receiving cyclosporine after MNNG as compared to all other groups; and greater incidence of small bowel tumors > 1 cm³ in animals receiving cyclosporine after MNNG as compared to all other groups. The median total tumor volume in the animals receiving cyclosporine following carcinogen was significantly greater than in any other group. This study does not support a policy of aggressive surveillance for gastrointestinal carcinoma in the human population receiving cyclosporine.     

Apoptosis in endothelial cells by cyclosporine

Objectives

The immunosuppressive drug cyclosporine (CsA) is a potent agent widely used after organ transplantations and to treat various autoimmune disorders. After using CsA, some patients suffer severe complications including renal and vascular toxicity, which are influenced by the degree of the endothelial damage. Several studies have demonstrated CsA treatment to directly induce apoptosis in several cell types. Thus, CsA may induce endothelial damage via activation of proapoptotic proteins. The present study was undertaken to investigate the effects of CsA on apoptosis of endothelial cells using human umbilical vein endothelial cells.

Methods

Proliferation was measured by using the Cell Counting Assay Kit after cells were exposed to CsA (0 L, 10 L, 30 L, 50 L or 100 μg/mL). Apoptotic cells were identified by fluorescence microscopy of 4′, 6-diamidino-2-phenylidole-stained nuclei. Western blot analysis was done for poly(ADP-ribose) polymerase (PARP), p27, p53 and caspase.

Results

Cell viability decreased dependent on the CsA concentration. CsA treatment group showed chromatin condensation and nuclear fragmentation. CsA produced a dose-dependent induction of p27 and reduction of procasapase-3. CsA treatment induced the degradation of 116-kDa PARP into an 89-kDa fragment.

Conclusions

CsA induced apoptosis of endothelial cells.     

Beneficial effects of cyclosporine on postischemic liver injury in rats.

The discovery of cyclosporine has had a significant impact on preventing the rejection of transplanted organs in humans. In this study, we present another positive aspect of cyclosporine. Rats were pretreated with cyclosporine (10 mg/kg, i.v.), or untreated. After 2-hr ischemia or 1 hr of reperfusion following 2-hr ischemia, livers were isolated and liver adenine nucleotide concentrations were determined. Liver mitochondria were prepared and their function was estimated polarographically. Leakage of AST, ALT, LDH, and adenine nucleotides into the hepatic vein just after reperfusion was also measured. Cyclosporine treatment did not affect ischemia-induced mitochondrial dysfunction, nor did it prevent the associated decrease in adenosine triphosphate concentration. However, treatment with cyclosporine accelerated the recovery of mitochondrial function and of tissue adenosine triphosphate concentrations. Cyclosporine treatment also mitigated leakage of AST, ALT, LDH, and adenine nucleotides after reperfusion. These results indicate that cyclosporine shows a potent protective effect on ischemia-reperfusion-related liver injury.     

Effect of timing of cyclosporine administration on recovery from renal ischemia in rats

The effect of timing of cyclosporine administration on functional recovery from renal ischemia was studied in Sprague-Dawley rats. Animals were given cyclosporine and subjected to renal ischemia by temporarily occluding both the renal artery and vein. Our data demonstrate no significant difference in serum creatinine among rats subjected to renal ischemia, cyclosporine, or cyclosporine-vehicle cremophor EL administration, or the control group. On the other hand, renal ischemia in combination with cyclosporine resulted in rapid and marked deterioration in renal function with serum creatinine peaking on Day 2. The most significant rise was in rats that received cyclosporine 4 hr prior to induction of renal ischemia (4.7 +/- 0.5 mg/dl), followed by those that received cyclosporine 4 and 24 hr postischemia (2.8 +/- 0.5 and 3.2 +/- 0.7 mg/dl, respectively). Cyclosporine administration 24 hr prior to renal ischemia resulted in the least elevation of the serum creatinine (2.1 +/- 0.5 mg/dl) and the earliest return to the baseline value. Our data suggest that the timing of cyclosporine administration in rats subjected to renal ischemia influences the extent of renal injury and the subsequent recovery of renal function.     

Effect of albumin solutions on endothelial oxidant injury: A microfluidic study

BackgroundStudies have suggested a beneficial effect of early plasma-based resuscitation in patients following trauma-hemorrhagic shock. The underlying mechanism(s) are unknown but may be owing to protective effects of plasma components on the endothelium and its glycocalyx layer. Albumin, the major protein in plasma, influences vascular integrity and has antioxidant properties in vivo. Sphingosine 1-phosphate is a bioactive sphingolipid with diverse signaling functions, which include endothelial barrier protection in part owing to preservation of the glycocalyx. Sphingosine 1-phosphate is bound mainly to albumin and high-density lipids in the plasma. Debate continues about the beneficial effect of albumin solutions in shock resuscitation. Pharmacologic preparations may modify constituents of albumin solutions for clinical use. We examined the relative effects of sphingosine 1-phosphate concentrations in albumin solutions on the endothelial-glycocalyx barrier in an in vitro microfluidic platform.MethodsEndothelial cell monolayers were established in microfluidic perfusion devices and exposed to control or biomimetic shock conditions followed by 5% plasma or different albumin solutions ± exogenous sphingosine 1-phosphate perfusion. Biomarkers of endothelial and glycocalyx activation, damage, and oxidant injury were then determined.ResultsEndothelial cell and glycocalyx barriers were damaged after biomimetic shock conditions. Plasma and sphingosine 1-phosphate loaded albumin solutions protected against barrier injury. Modest protective effects were noted with albumin alone; the efficacy varied with sphingosine 1-phosphate content of the albumin solution.ConclusionThe protective effect of albumin on the endothelia-glycocalyx barrier against oxidant injury was dependent on its sphingosine 1-phosphate concentration. Our data may help explain the discrepancies regarding the effectiveness of albumin solutions in shock resuscitation.     

Glomerular endothelial changes in cyclosporine A-treated rats: scanning and transmission electron microscopic studies

This study focused on the glomerular structural changes observed after cyclosporine A (CsA)-induced nephrotoxicity. Renal structural changes were examined in rats treated with oral CsA, given as a daily dose of 50 mg/kg for periods of up to 49 days. By means of scanning electron microscopy and morphometry, we first demonstrated that the most conspicuous and reproducible ultrastructural changes could be detected in the endothelial cells of the glomerular capillaries. These changes included a reduction in the fenestral pore size and partial disappearance of endothelial fenestration, the appearance of microvilli-like projections on the endothelial surface, and flattening and widening of the cytoplasmic folds. We believe that the ultrastructural changes observed in this study are partially responsible for the alterations in renal function seen in the cyclosporine A-treated model, and that these alterations are caused by CsA-induced vasospasm.     

Glomerular endothelial changes in cyclosporine A-treated rats: Scanning and transmission electron microscopic studies

This study focused on the glomerular structural changes observed after cyclosporine A (CsA)-induced nephrotoxicity. Renal structural changes were examined in rats treated with oral CsA, given as a daily dose of 50 mg/kg for periods of up to 49 days. By means of scanning electron microscopy and morphometry, we first demonstrated that the most conspicuous and reproducible ultrastructural changes could be detected in the endothelial cells of the glomerular capillaries. These changes included a reduction in the fenestral pore size and partial disappearance of endothelial fenestration, the appearance of microvilli-like projections on the endothelial surface, and flattening and widening of the cytoplasmic folds. We believe that the ultrastructural changes observed in this study are partially responsible for the alterations in renal function seen in the cyclosporine A-treated model, and that these alterations are caused by CsA-induced vasospasm.     

Reduction of nutrient absorption in normal rats by cyclosporine.

The indications for using cyclosporine are expanding rapidly beyond immune suppression for transplantation. We have previously described reduced active glucose uptake by small bowel following CsA treatment in rats. This study examined the effect of varying the dose and route of administration of CsA on bowel function. Male Lewis rats were given CsA via subcutaneous injection at doses of 5 mg/kg or 30 mg/kg on alternate days, or orally via gavage at 0 (control solvent oil), 7.5 mg/kg, or 30 mg/kg daily. Weight gain and feed intake were followed for 1 month when a balance study was performed to quantify in vivo nutrient absorption from the feed. In vitro studies of glucose and fatty acid uptake studies were then performed. Weight gain was reduced by high-dose CsA whether given orally or by subcutaneous injection. Oral CsA reduced in vivo fat and energy absorption from the diet, and all doses and routes of administration of CsA caused a reduction in both active glucose uptake and passive fatty acid absorption by the bowel in vitro. Thus, CsA has significant effects on bowel function in the normal rat. We suggest that further studies are indicated to determine the effects of CsA in man, especially in conditions with already-impaired bowel function.