Effect of cyclosporine and sirolimus on the expression of connective tissue growth factor in rat experimental chronic nephrotoxicity

BACKGROUND/AIMS: Connective tissue growth factor (CTGF) is a pro-fibrotic growth factor that acts downstream of transforming growth factor (TGF)-beta. However, CTGF regulation remains unknown. We tried to determine the effect of two commonly used immunosuppressants, cyclosporine (CsA) and sirolimus (SRL), on CTGF expression in a model of chronic nephrotoxicity. METHODS: Adult Sprague-Dawley rats kept on a low-salt diet were treated daily for 4 weeks with vehicle (VH), SRL (0.3 mg/kg), CsA5 (5 mg/kg), CsA10 (10 mg/kg) or both CsA5 and SRL. CTGF and TGF-beta1 expressions were evaluated by Northern blot. Functional and histologic parameters in addition to number of apoptotic cells were determined. RESULTS: At 28 days, both CsA doses were capable of inhibiting CTGF mRNA expression to levels similar to control. On the other hand, SRL increased CTGF expression by 3.5-fold. However, addition of CsA to SRL completely reversed that trend and returned levels to control. The results were different for TGF-beta1, which was increased by both CsA and SRL and to a greater extent by the drug combination. CONCLUSION: Unlike TGF-beta, CTGF does not seem to play an important role in CsA-induced chronic nephrotoxicity. In addition, calcineurin-dependent pathways are likely involved in CTGF regulation.     

Effect of etomidate on hepatic drug metabolism in humans

The authors studied the effect of etomidate on drug metabolism in vivo in humans and in vitro using human liver microsomes. When these liver microsomes were incubated with different concentrations of etomidate, dose-dependent inhibition of ketamine N-demethylation, a cytochrome P-450-dependent enzymatic process, was produced. Cytochrome P-450 binding spectra displayed type II binding with a UV light absorption maximum (lambda max) at a wavelength of 424 nm in the presence of etomidate. In vivo studies were conducted using ketamine and antipyrine as substrates. Evaluation of antipyrine's pharmacokinetic variables after an intravenous infusion of etomidate (0.34 +/- 0.17 mg/kg) revealed an 18% increase in its elimination half-life (P = 0.04). In addition, there were 16% and 11% decreases in the area under the curve (P = 0.05) and in the clearance rate (P = 0.07) for antipyrine, respectively. In patients administered a bolus dose of ketamine during brief outpatient operations, etomidate produced no significant changes in ketamine's pharmacokinetics compared to thiopental. The authors conclude that the etomidate-induced inhibition of hepatic drug metabolism can prolong the elimination of drugs with low hepatic clearance rates (e.g., antipyrine). However, etomidate would not be expected to alter the rate of elimination of high clearance anesthetics and analgesic drugs (e.g., ketamine, fentanyl).     

Influence of hepatic dysfunction on cyclosporine metabolism in the pig

Cyclosporine (CyA) is eliminated from the body via biliary excretion at a rate directly proportional to bile production and the functional status of the liver. Previous reports demonstrated that disturbances in the hepatic excretory function with a rise in the plasma bilirubin level are positively correlated with high blood concentrations of CyA and CyA plus metabolites (CyA + M). Less information is available about the blood concentrations of the CyA parental substance or CyA metabolites in the case of liver dysfunction when there was no elevation of serum bilirubin content. To answer this question, we compared the pharmacokinetic profile of CyA in a cholestatic and in a ischemic model in pigs. Our results show that in pigs receiving a single dose of CyA after liver ischemia, the blood concentrations of CyA and CyA + M are significantly increased independently of the serum bilirubin concentration, probably through a slow down of CyA metabolism by impairment of cytochrome P450 III A.     

Bone metabolism in renal transplant patients treated with cyclosporine or sirolimus

Sirolimus is a new immunosuppressive agent used as treatment to prevent acute renal allograft rejection. One of the complications of renal transplantation and subsequent long-term immunosuppression is bone loss associated with osteoporosis and consequent fracture. Two open-label, randomized, phase 2 studies comparing sirolimus versus cyclosporine (CsA) included indices of bone metabolism as secondary end-points. Markers of bone turnover, serum osteocalcin and urinary N-telopeptides, were measured over a 1-year period in 115 patients receiving either CsA or sirolimus as a primary therapy in combination with azathioprine and glucocorticoids (study A) or mycophenolate mofetil (MMF) and glucocorticoids (study B). Urinary excretion of N-telopeptides and the concentrations of serum osteocalcin were consistently higher in the CsA-treated patients and significantly different at week 24 for N-telopeptides and at weeks 12, 24, and 52 for osteocalcin. In conclusion, future trials are warranted to test whether a sirolimus-based regimen conserves bone mineral density compared with a CsA-based regimen.     

Kidney function and morphology after short-term combination therapy with cyclosporine A, tacrolimus and sirolimus in the rat.

BACKGROUND: Sirolimus (SRL) may supplement calcineurin inhibitors in clinical organ transplantation. These are nephrotoxic, but SRL seems to act differently displaying only minor nephrotoxic effects, although this question is still open. In a number of treatment protocols where SRL was combined with a calcineurin inhibitor indications of a synergistic nephrotoxic effect were described. The aim of this study was to examine further the renal function, including morphological analysis of the kidneys of male Sprague-Dawley rats treated with either cyclosporine A (CsA), tacrolimus (FK506) or SRL as monotherapies or in different combinations. METHODS: For a period of 2 weeks, CsA 15 mg/kg/day (given orally), FK506 3.0 mg/kg/day (given orally) or SRL 0.4 mg/kg/day (given intraperitoneally) was administered once a day as these doses have earlier been found to achieve a significant immunosuppressive effect in Sprague-Dawley rats. In the 'conscious catheterized rat' model, the glomerular filtration rate (GFR) was measured as the clearance of Cr(EDTA). The morphological analysis of the kidneys included a semi-quantitative scoring system analysing the degree of striped fibrosis, subcapsular fibrosis and the number of basophilic tubules, plus an additional stereological analysis of the total grade of fibrosis in the cortex stained with Sirius Red. RESULTS: CsA, FK506 and SRL all significantly decreased the GFR. A further deterioration was seen when CsA was combined with either FK506 or SRL, whereas the GFR remained unchanged in the group treated with FK506 plus SRL when compared with treatment with any of the single substances. The morphological changes presented a similar pattern. The semi-quantitative scoring was significantly worst in the group treated with CsA plus SRL (P<0.001 compared with controls) and the analysis of the total grade of fibrosis also showed the highest proportion in the same group and was significantly different from controls (P<0.02). The FK506 plus SRL combination showed only a marginally higher degree of fibrosis as compared with controls (P=0.05). CONCLUSION: This rat study demonstrated a synergistic nephrotoxic effect of CsA plus SRL, whereas FK506 plus SRL was better tolerated.     

Thoracic duct drainage pretreatment and low dose cyclosporine and low dose steroid immunosuppressive treatment in living related kidney transplantation

The results of 20 HLA 1-haploidentical donor kidney transplant patients treated with preoperative lymphocyte deletion through thoracic duct drainage and low dose of cyclosporine and steroid immunosuppressive therapy, were presented. The number of removed lymphocytes was 114 +/- 36 X 10(9) (mean +/- SD) and the duration of thoracic duct drainage was 35 +/- 7 days. Graft survival was 100% at 3-9 months and 89% at 1-2 years after transplantation. Patient survival was 100% at 3-9 months and 89% at 1-2 years. A patient died from lung cancer (adenocarcinoma) in the 9th posttransplant month. Acute rejection was not seen in 20 patients during the first 3 months. Life-threatening infectious disease was never seen either. Diabetes mellitus was observed in 1 patient. No other complications were observed. These results indicated that thoracic duct drainage and low dose cyclosporine and steroid postoperative immunosuppressive treatment might yield complete success in HLA 1-haploidentical kidney transplant patients.     

Effect of starvation on renal metabolism in the rat.

The effects of starvation on the acid-base status of the rat and on the glucoeogenic and ammoniagenic capacity of rat renal-cortical slices were examined. Starvation for 48 or 72 hr did not affect acid-base status, and urinary ammonia production did not change. Kidney cortical slices from starved as compared to fed rats showed increased gluconeogenic capacity when incubated with the substrated pyruvate, succinate, fumarate, malate, 2-oxyoglutarate, glutamine and glutamate. Renal cortical tissue from starved rats also had increased activity of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase. Renal cortical slices from starved rats did not differ from those from fed rats in the ability to produce ammonia from glutamine or glutamate, nor was there any difference inhe activity of glutaminase between these groups. These results show that renal gluconeogenic capacity is increased in starved rats in the absence of systemic acidosis, and starvation does not lead to an increase in urinary ammonia excretion or renal ammoniagenic capacity.     

Effect of cyclosporine on steroidogenesis in rat Leydig cells

Cyclosporine induces hypoandrogenism in adult male rats. In order to assess whether this effect of CsA may be due to a direct inhibitory effect on Leydig cell function, CsA (0, 50, 500, and 5000 ng/ml) was added to a collagenase-dispersed mixed Leydig cell preparation and incubated with and without hCG (0, 0.1, 0.3, 1.0, 3.0, and 10.0 ng/ml). Testosterone (T) production, mitochondrial cholesterol side chain cleavage (CSCC) and microsomal 17,20-desmolase enzyme activities in Leydig cells were determined after 3 hr of incubation. In the absence of CsA, stimulation of T production was maximal (about 16-fold) with 1.0 ng/ml hCG. With 50 and 500 ng/ml CsA there were no changes in either the hCG-stimulated T levels or the two enzymatic activities. However, 5000 ng/ml CsA significantly (P less than 0.05) reduced the hCG (1 ng/ml)-stimulated T levels, CSCC and 17,20-desmolase activities. The high dosage of CsA (5000 ng/ml) also caused a significant decrease in cell viability (P less than 0.05) during the incubation period. These effects of CsA were not due to cremophor EL, the CsA vehicle. This in vitro data indicate that high dosages of CsA (greater than or equal to 5000 ng/ml) appear to have a cytotoxic effect on rat Leydig cells that results in a decrease in T production. However, lower doses of CsA (less than 500 ng/ml) do not have any direct inhibitory effect on the rat Leydig cells, suggesting that the hypoandrogenic effect of in vivo CsA in rats is not due to any direct effect on the testis.     

The effect of chronic renal failure on hepatic drug metabolism and drug disposition

There is abundant evidence that chronic renal failure (CRF) and end-stage renal disease (ESRD) alter drug disposition by affecting protein and tissue binding and reducing systemic clearance of renally cleared drugs. What is not fully appreciated is that CRF can significantly reduce nonrenal clearance and alter the bioavailability of drugs predominantly metabolized by the liver. Animal studies in CRF have shown a major down-regulation (40-85%) of hepatic cytochrome P-450 metabolism involving specific isozymes. Phase II reactions such as acetylation and glucuronidation are also involved, with some isozymes showing induction and others inhibition. Hepatic enzymes exhibiting genetic polymorphisms such as N-acetyl-transferase-2 (NAT-2), which is responsible for the rapid and slow acetylator phenotypes, have been shown to be inhibited by ESRD and reversed by transplantation. There is some evidence pointing to the possibility of inhibitory factors circulating in the serum in ESRD patients which may be dialyzable. This review includes all significant animal and clinical studies using the search terms "chronic renal failure,"cytochrome P-450," and "liver metabolism" over the past 10 years obtained from the National Library of Medicine MEDLINE database, including relevant articles back to 1969.     

Influence of hemodilution on hepatic energy metabolism in rat

The effects and safety limits of acute hemodilution on hepatic energy status were investigated in relation to arterial blood ketone body ratio and hepatic energy charge in a hemodilution rat model. As long as the hematocrit value was maintained above 20%, ketone body ratio and energy charge level at 6 h after hemodilution remained at the same levels as those of the sham-diluted groups. However, when hematocrit value was less than 15%, the ketone body ratio markedly decreased from the control value of 0.686 +/- 0.044 to 0.278 +/- 0.048 (p less than 0.001), and energy charge decreased from the control value of 0.856 +/- 0.012 to 0.0806 +/- 0.011 (p less than 0.01). From these results, it was suggested that hemodilution exerts no influence on the energy status of the liver as long as hematocrit is maintained above 20%.     

Organ-specific response to inhibition of mitochondrial metabolism by cyclosporine in the rat

To evaluate organ-specific metabolic changes after in vivo cyclosporine (CyA) treatment, male Wistar rats were treated with 10 mg/kg per day CyA orally for 6 days. Blood, kidney, liver, and heart tissues were extracted and analyzed by magnetic resonance spectroscopy (MRS). CyA decreased the energy balance [adenosine triphosphate (ATP)/adenosine diphosphate (ADP)] in all organs (kidney [control]: 50%, liver: 64%, and heart: 62%, all P<0.01) due to decreased activity of the mitochondrial Krebs cycle and oxidative phosphorylation. As a compensatory effect, anaerobic glycolysis (lactate) was increased. This was reflected in the low glucose level in the kidney and heart, but not in the liver where a significant decrease in glycogen was seen. Only in the kidney was mitochondrial inhibition accompanied by decreased polyunsaturated fatty acid (PUFA) concentrations and elevated lipid peroxidation. The metabolic marker for nephrotoxicity, trimethylamine-N-oxide (TMAO), was elevated. While CyA decreased mitochondrial homeostasis in all organ systems, cellular adaptation was different and most efficient in the liver.The results of this study were presented in part at the 2002 Winter Symposium of the American Society of Transplant Surgeons, Miami, 25–27 January 2002     

Effect of high dose and low dose aspirin on survival of random pattern flaps in rats.

This paper studies which of the physiological effects of aspirin is responsible for increasing the survival of random flaps in rats, found in an earlier experiment. We wished to confirm that the antiaggregating--antithrombotic effect was responsible for the increased survival of flaps without microvascular anastomosis. Three groups of rats with standardised random pattern flaps were used. The first two were given aspirin 200 mg/kg (high dose, n = 27) or 40 mg/kg (low dose, n = 21) and the third (n = 28) acted as controls. The beneficial effects of aspirin were restricted to the high dose group. Since the low dose group also showed antiaggregation of platelets, but without the anti-inflammatory or vasodilatory effects, the results indicate that the antiaggregating effect alone was not responsible for the increased survival of the flaps.     

Synergistic effects of sirolimus with cyclosporine and tacrolimus: analysis of immunosuppression on lymphocyte proliferation and activation in rat whole blood

BACKGROUND: Whole-blood analysis of lymphocyte function was used to investigate the pharmacodynamic (PD) interaction of sirolimus (SRL) with cyclosporine (CsA) or tacrolimus (TRL) in vitro and to determine the relation between PD and pharmacokinetics (PK) of SRL in an in vivo rat model. METHODS: In vitro, experiments involved incubation of increasing concentrations (0.25-25 [corrected] nM) of SRL with either CsA or TRL in rat whole blood. For the in vivo study, rats were orally treated with different doses of SRL alone (1, 3, 5, or 8 mg/kg) or with a combination of 3 mg/kg SRL plus 2.5 or 5 mg/kg CsA. Blood was obtained before and at different times after dosing. Inhibition of lymphocyte proliferation (proliferating cell nuclear antigen [PCNA]) and activation (CD25, CD71, CD11a, CD134) in mitogen-stimulated blood was determined using fluorescence-activated cell sorter analysis. SRL and CsA blood concentrations were determined at the same time points by light chromatography tandem mass spectrometry (LC-MS). RESULTS: In vitro, concentrations of SRL between 0.25-25 [corrected] nM acted synergistically in combination with CsA or TRL at concentrations between 0.25-1.0 [corrected] nM. Higher SRL concentrations did not further increase inhibition of lymphocyte function in these combinations. In vivo, good correlations (r=0.68-0.94) were observed between PD parameters of lymphocyte function and SRL-PK and dose. Increasing SRL doses produced higher blood concentrations, but SRL doses of 8 mg/kg did not further increase inhibition of lymphocyte function. PD effects on lymphocyte function were prolonged, but maximal inhibition was not increased when SRL was applied in combination with CsA as compared to SRL mono therapy. CONCLUSIONS: The results suggest that analysis of lymphocyte function in whole blood may be useful to optimize dosing of SRL in combination with CsA or TRL and that PD monitoring of immunosuppressive drugs will enhance the value of PK monitoring.     

PDZK1敲除对小鼠肝脏胆固醇代谢和胆囊结石形成影响的实验研究

目的探讨敲除PDZK1(Postsynaptic density-95,disks-large,ZO-1-domain K1,PDZK1)基因对小鼠肝脏胆固醇代谢调节和胆囊结石形成的影响。方法雄性成年PDZK1基因敲除小鼠(PDZK1 knockout,KO组)和野生型小鼠(wild type,WT组),每组各10只,以成石饲料分别喂养4周,观察胆囊成石情况,并收集肝脏和胆囊组织。采用蛋白印迹法测定肝脏PDZK1和清道夫受体B族1型(scavenger receptor B type 1,SRB1)表达。采用胆总管插管收集肝胆汁,测定胆汁分泌率和胆汁胆固醇含量。采用试剂盒酶法测定胆囊胆汁成分并计算胆汁胆固醇饱和指数(cholesterol saturation index,CSI)。以实时定量PCR检测肝脏脂质代谢相关基因的mRNA表达。结果成石饲料喂养4周后,WT组小鼠全部成石(10/10),KO组小鼠则为40%(4/10)成石。两组小鼠肝胆汁分泌率差异无统计学意义,但KO组小鼠肝胆汁胆固醇含量显著降低(P0.05),胆汁酸含量增加(P0.05),且CSI降低(P0.05)。KO组小鼠肝脏SRB1蛋白表达降低(P0.05),甾醇氧-酰基转移酶基因1/2mRNA表达降低(P0.05),而肝型脂肪酸结合蛋白1和胆汁酸转运相关蛋白(ATP结合盒b11)表达则显著增加(P0.05)。结论 PDZK1影响SRB1在小鼠肝脏中表达,降低对高密度脂蛋白胆固醇摄取,减少胆汁胆固醇分泌,继而降低胆囊结石形成。