Effects of nitric oxide synthesis inhibition on FK506-induced nephrotoxicity in rats

This study was designed to evaluate the role of nitric oxide (NO) in FK506-induced nephrotoxicity by administering an inhibitor of NO synthesis, N omega-nitro-L-arginine methyl ester (L-NAME) to rats treated with FK506. After one week of treatment with FK506 (3.2 mg/kg/day, intramuscularly) and/or L-NAME (5 mg/100 mL of L-NAME in the drinking water), the arterial pressure, urinary NOx, and parameters for renal function were measured, and histological analysis of the kidney was made. In the L-NAME without FK506 group, L-NAME administration effectively inhibited urinary NOx excretion and increased mean arterial pressure (MAP) without any change in renal function. In the FK506 without L-NAME group, FK506 treatment showed increase in urinary NOx excretion and mild renal dysfunction. In the FK506 with L-NAME group, urinary NOx excretion was decreased by L-NAME administration and renal function was significantly worsened than FK506 without L-NAME group. The plasma creatinine, BUN and urinary N-acetyl-beta-D-glucosaminidase increased 2-, 3-, and 3-fold, respectively and the creatinine clearance was reduced by 50% as compared with that in the FK506 without L-NAME group. Histological analysis revealed severe interstitial fibrosis and tubular atrophy in the FK506 + L-NAME treatment group. Thus, results suggest that NO synthesis is enhanced in the kidney during FK506-induced nephrotoxicity and that NO synthesis inhibition aggravates FK506-induced nephrotoxicity. NO may play a protective role attributable to the balance of vasoactive substances in FK506-induced nephrotoxicity.     

EFFECTS OF NITRIC OXIDE SYNTHESIS INHIBITION ON FK506-INDUCED NEPHROTOXICITY IN RATS

This study was designed to evaluate the role of nitric oxide (NO) in FK506-induced nephrotoxicity by administering an inhibitor of NO synthesis, Nω-nitro-L-arginine methyl ester (L-NAME) to rats treated with FK506. After one week of treatment with FK506 (3.2 mg/kg/day, intramuscularly) and/or L-NAME (5 mg/100 mL of L-NAME in the drinking water), the arterial pressure, urinary NOx, and parameters for renal function were measured, and histological analysis of the kidney was made. In the L-NAME without FK506 group, L-NAME administration effectively inhibited urinary NOx excretion and increased mean arterial pressure (MAP) without any change in renal function. In the FK506 without L-NAME group, FK506 treatment showed increase in urinary NOx excretion and mild renal dysfunction. In the FK506 with L-NAME group, urinary NOx excretion was decreased by L-NAME administration and renal function was significantly worsened than FK506 without L-NAME group. The plasma creatinine, BUN and urinary N-acetyl-β-D-glucosaminidase increased 2-, 3-, and 3-fold, respectively and the creatinine clearance was reduced by 50% as compared with that in the FK506 without L-NAME group. Histological analysis revealed severe interstitial fibrosis and tubular atrophy in the FK506 + L-NAME treatment group. Thus, results suggest that NO synthesis is enhanced in the kidney during FK506-induced nephrotoxicity and that NO synthesis inhibition aggravates FK506-induced nephrotoxicity. NO may play a protective role attributable to the balance of vasoactive substances in FK506-induced nephrotoxicity.     

Changes in nitric oxide production in the rat kidney due to CaOx nephrolithiasis

AIMS: The aim of the study was to test the hypothesis that the renal nitric oxide (NO) system is involved in the animal model of nephrolithiasis by evaluating the relationship between nitric oxide synthase (NOS) and oxidative stress. METHODS: Deposition of renal calculi was induced by adding 0.75% ethylene glycol (EG) to the drinking water of male Wistar rats. After 42 days of treatment, urinary biochemistry and urinary levels of oxalate, NO metabolites (nitrate and nitrite), cGMP, and lipid peroxides, and markers for renal damage and oxidative stress in the kidney were examined. In the second part of the experiment, two diuretic stimuli (intrarenal infusion of l-arginine or saline loading) were applied to test the renal NO system response. Finally, levels of three isoforms of NOS in renal tissues were evaluated by immunostaining. RESULTS: In the EG-treated rats, increased urinary excretion of enzymes and lipid peroxides and increased nitrotyrosine levels and oxidative injury markers in the kidneys indicated that peroxynitrite formation occurred during oxidative stress, while the 24-hr urinary excretion of NO metabolites and cGMP remained unchanged. In contrast to control rats, urinary excretion and NO metabolites and cGMP excretion were unresponsive to intrarenal l-arginine infusion; in response to saline loading, an increase in these factors was seen, but the increase was only 50% of that seen in the identically treated control group. A significant decrease in eNOS expression and increase in iNOS expression were observed in the renal medulla of the EG-treated group, whereas expression of nNOS was not affected. CONCLUSIONS: Although basal renal NO production remained unchanged, excessive peroxynitrite formation in the kidney was noted in this model. A decreased response of the NOS system was noted when diuretic stimuli were applied. How the imbalance between eNOS and iNOS expression influences CaOx stone formation requires detailed evaluation.     

Oral sorbent AST-120 increases renal NO synthesis in uremic rats.

OBJECTIVE: The urine level of nitric oxide (NO) metabolites, i.e., nitrates/nitrites (NOx), in chronic renal failure (CRF) is decreased because of reduced renal synthesis of NO. We determined whether the administration of an oral sorbent, AST-120, increases the urine level of NOx and the renal expression of nitric oxide synthase (NOS) isoforms in CRF rats. METHODS: Chronic renal failure rats were produced by 4/5 nephrectomy. Rats were randomized into two groups: CRF control rats, and AST-120-treated CRF rats. The AST-120 was administered to the rats at a dose of 4 g/kg with powder chow for 16 weeks, whereas powder chow alone was administered to control rats. The urine levels of NOx were measured by using a NOx colorimetric assay kit. The expression of endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) in the kidney was determined by immunohistochemistry. Serum and urine levels of indoxyl sulfate were determined by high-performance liquid chromatography. RESULTS: Urine levels of NOx and the expression of glomerular eNOS and tubulointerstitial nNOS were significantly decreased in CRF rats compared with normal rats. The administration of AST-120 to CRF rats significantly increased urine levels of NOx and the expression of glomerular eNOS and tubulointerstitial nNOS. The administration of AST-120 to CRF rats significantly decreased urine and serum levels of indoxyl sulfate. CONCLUSIONS: The oral sorbent AST-120 increases NO synthesis in the kidneys of uremic rats by increasing the renal expression of eNOS and nNOS, through alleviation of indoxyl sulfate overload on the kidney.     

Nitric oxide production and nitric oxide synthase expression in acute human renal allograft rejection

BACKGROUND: Nitric oxide (NO) is produced by nitric oxide synthases (NOS), which are either constitutively expressed in the kidney or inducible, in resident and infiltrating cells during inflammation and allograft rejection. NO is rapidly degraded to the stable end products nitrite and nitrate, which can be measured in serum and urine, and may serve as noninvasive markers of kidney allograft rejection. METHODS: Total nitrite and nitrate levels (NOx) were measured in serum and urine thrice weekly after an overnight fast in 18 consecutive patients following renal cadaveric transplantation. Inducible NOS (iNOS) and endothelial NOS (eNOS) expression was immunochemically determined in renal biopsy specimens with or without acute rejection (AR). RESULTS: Serum NOx levels increased days before AR and were significantly higher at the moment of AR (27+/-12.4 micromol/L) compared with recipients with an uncomplicated course (13+/-7.6 micromol/L), but not compared with recipients with cyclosporine (CsA) toxicity (20+/-13.0 micromol/L). Urinary NOx levels were significantly lower during AR (20+/-13.6 micromol/mmol creatinine) compared with an uncomplicated course (64+/-25.2 micromol/mmol creatinine) or CsA toxicity (53.8+/-28.3 micromol/mmol creatinine). Interstitial and glomerular iNOS expression was significantly increased in biopsy specimens showing AR. Unexpectedly, glomerular eNOS expression was significantly decreased in patients with AR. CONCLUSIONS: This study reports differences in NOx levels in serum and urine, which may help discriminate AR episodes from an uncomplicated course or CsA toxicity. As expected, renal iNOS expression is increased in acute allograft rejection. The decrease in glomerular eNOS expression suggests an intriguing link between acute and chronic rejection.     

转化生长因子-β1在他克莫司肾毒性中的作用

目的 观察转化生长因子(TGF)-β1在他克莫司大鼠肾毒性中的作用.方法 将SD大鼠24只随机分成对照组、CsA组、FK506组和FK506+Dil组,用药4周后建立起各组大鼠模型.观察各组大鼠的肾功能,应用免疫组织化学技术检测各组大鼠TGF-β1的表达.结果 FK506组大鼠的血肌酐值为(34.17±4.54)μmol/L,肌酐清除率为(0.58±0.39)ml·min-1·100g-1,与对照组比较差异有统计学意义(P<0.05).FK506组TGF-β1的阳性表达率均为100%(6/6),对照组TGF-β1的阳性表达率为16%(1/6),两者差异有统计学意义(P<0.05). 结论 TGF-β1可能介导了FK506引起的肾毒性.     

Increased nitric oxide synthase-3 expression in kidneys of deoxycorticosterone acetate-salt hypertensive rats.

In addition to its hemodynamic effects, nitric oxide (NO) may play a role in the renal tubular handling of sodium. Experiments were conducted to determine possible changes in renal nitric oxide synthase-3 (NOS3) expression in rats treated with deoxycorticosterone acetate (DOCA) and high salt. All rats were uninephrectomized, and either a placebo or DOCA pellet was implanted subcutaneously. Placebo-treated rats were then given tap water to drink ad libitum, and DOCA-treated rats received a 0.9% NaCl solution to drink. Once a week, rats were placed in metabolic cages so that a 24-h urine sample could be collected. After 3 wk, the animals were sacrificed and the kidneys removed and prepared for subsequent immunohistochemical or Western blot analysis. Urinary excretion of nitrate and nitrite (NOx) was measured to provide an indication of the intrarenal production of NO. DOCA-salt hypertensive rats exhibited increased urinary NOx excretion (2.43 +/- 0.48 micromol NOx/mg creatinine) compared with the placebo control animals (1.17 +/- 0.06 micromol NOx/mg creatinine). Western blot analysis revealed that NOS3 protein levels in both the cortex and medulla were greater in DOCA-salt rats compared with placebo-treated animals. Immunohistochemical analysis of kidneys revealed that NOS3 expression in placebo rats was localized in vascular endothelial cells with slight, but detectable, immunoreactivity in medullary collecting ducts. In DOCA-salt rats, a very large increase in the intensity of immunostaining was detected in tubular epithelia of the proximal tubule, thick ascending limb of Henle's loop, and cortical and medullary collecting duct; immunoreactivity in endothelial cells appeared unchanged. These data suggest that increased tubular expression of NOS3 is responsible, at least in part, for the increased renal production of NO in DOCA-salt hypertension, and are consistent with a role for NO in the renal tubular response to salt loading.     

骨质疏松发生一氧化氮机制及雌激素调控作用研究

目的　研究骨质疏松发生的一氧化氮机制及雌激素防治骨质疏松作用的一氧化氮通路 ,深入理解骨质疏松症病理过程中一氧化氮作用环节。方法　 45只SD雌性大鼠分为①假手术组(Sham组 ) ;②卵巢切除组 (OVX组 ) ;③OVX +倍美力治疗 (Premarin组 )。进行iNOS原位杂交及iNOS、eNOS免疫组化研究 ,测定其平均灰度值及平均积分光密度 (ODI)作统计指标。结果　iNOS原位杂交实验结果显示 :正常大鼠去卵巢后 ,骨髓腔内及骨小梁表面iNOSmRNA有强阳性表达 ,差异有非常显著性 (P <0 0 1) ,iNOS免疫组化实验结果也显示OVX组iNOS阳性表达明显较Sham组增加 ;而Pre marin组iNOS表达强度较OVX组明显降低 (P <0 0 5 )。OVX组与Sham组eNOS表达差异无显著性 ,Premarin组较OVX组eNOS表达强度则明显增加 (P <0 0 5 )。结论　NO是调节OB、OC功能活动的一种重要效应因子 ,NO导致细胞因子诱导性骨吸收增强与绝经后雌激素缺乏OP密切相关。雌激素可下调iNOSmRNA表达 ,抑制iNOS蛋白合成 ,从而减轻功能亢进的OC性骨吸收 ,而对骨组织正常生理活动中的eNOS合成 ,有适度促进作用 ,有利于骨组织的重建及骨量的恢复 ,从而重建骨形成 吸收新平衡偶联     

Effect of severe aortic banding above the renal arteries on nitric oxide synthase isotype expression

BACKGROUND: Severe aortic stenosis above the renal arteries leads to a reduction in renal perfusion, increased renin secretion, and elevation of arterial blood pressure above the stenotic site. Nitric oxide (NO) plays an important role in regulation of renal and systemic vascular resistance, renal blood flow, and Na(+) handling. Abdominal aortic banding provides an excellent model for simultaneous testing of the effects of increased and decreased pressure, flow, and shear stress in the same animal. METHODS: We studied protein expressions of endothelial NO synthase (eNOS), inducible NOS (iNOS), and neuroneal NOS (nNOS) isotypes in the renal cortex, renal medulla, heart, brain, and aorta segments above and below the stenosis site three weeks after abdominal aortic banding above the renal arteries. The results were compared with those obtained in the sham-operated controls. NOS isotype proteins were measured by Western blot. RESULTS: Compared with the control group, the banded group showed significant up-regulations of eNOS, iNOS, and nNOS in renal cortex and medulla. Likewise, heart eNOS, brain nNOS, and thoracic aorta eNOS proteins were significantly increased in the banded group. However, eNOS and iNOS expressions were unchanged in the aorta segment below the stenotic site. Likewise, iNOS expression in the heart and thoracic aorta remained unchanged in the banded animals. No significant difference was found in creatinine clearance or urinary protein excretion between the two groups. CONCLUSIONS: These findings clearly demonstrate the up-regulatory action of increased pressure on eNOS expression in the thoracic aorta and heart and of nNOS expression in the brain. These data further show up-regulation of all NOS isotypes in the kidney, which must have helped to mitigate the associated hypoperfusion.     

Functional and morphological changes in rat kidney induced by FK506 and its reversal by various vasodilators]

FK506, a newly developed immunosuppressive agent, has recently been used for human liver and kidney transplantation. The present study was carried out to assess the functional and morphological changes by acute or chronic administration of FK506 in heminephrectomized rats. FK506 was given intravenously at the dose of 0.384 mg/kg/hr for 90 min in acute experiment. FK506 was administered by gastric gavage at doses of 1, 2.5, 5 mg/kg for 21 days. Blood and urinary biochemistry were monitored every week. Inulin and PAH clearance studies were conducted during the infusion of FK506 for acute study, and at day 21 for chronic study. Some of the rats were treated with diltiazem (Dilt), captopril or prazosine for 21 days to prevent FK506 nephrotoxicity. Acute infusion of FK506 did not change renal and systemic hemodynamics. Creatinine clearance showed a dose dependent decrease by 10-20% in the rats with chronic administration of FK506. Inulin/PAH clearance indicated a decrease in glomerular filtration rate and renal plasma flow with an increase in renal vascular resistance. The renal histology showed vacuolization in proximal tubuli and media of smooth muscle cells of arterioles. The administration of Dilt functionally and morphologically improved renal impairment induced by FK506. In conclusion, FK506 induces a dose dependent decrease in renal function with significant histological changes in tubuli and arterioles in rat kidney. Intracellular calcium deregulation seems to be involved in FK506-induced nephrotoxicity.     

钙离子通道阻滞剂在预防他克莫司肾毒性中的作用

目的 观察他克莫司(FK506)肾毒性模型中钙离子代谢的变化情况,探讨钙离子通道阻滞剂地尔硫草(Dil)对FK506肾毒性的预防作用.方法 按公式将肾移植术后FK506、环孢素(CsA)和Dil的首剂治疗剂量换算成大鼠的治疗剂量.雄性SD大鼠24只随机分成对照组、CsA组(25 mg·kg-1·d-1)、FK506组(0.8 mg·kg-1·d-1)和FK506加Dil组(0.8 mg·kg-1·d-1及8 mg·kg-1·d-1),每组6只,用药4周后建立各组大鼠肾毒性模型.观察各组大鼠SCr、血电解质、肾组织的病理改变(HE染色)、电子显微镜F肾脏细胞内超微结构的改变.结果 CsA组和FK506组大鼠SCr值分别为(36.00±2.61)和(34.17±4.54)μmol/L,均高于FK506加Dil组和对照组[(28.50±2.07)和(29.17±3.43)μmol/L,P＜0.05].CsA组和FK506组大鼠血钙浓度分别为(2.00±0.04)和(2.05_4-0.04)mmol/L,均低于FK506加Dil组和对照组(P＜0.05).CsA组和FK506组均可观察到肾小管细胞轻微肿胀及空泡变性、线粒体肿胀及空泡化等病理改变.与FK506组和CsA组相比,FK506加Dil组上述各项指标的变化明显减轻或接近正常.结论 钙离子代谢紊乱可能介导了FK506引起的肾毒性,Dil可用于预防FK506的肾毒性.     

Inhibition of nuclear factor-kappaB activation by pyrrolidine dithiocarbamate prevents chronic FK506 nephropathy

BACKGROUND: Chronic tacrolimus (FK506) nephrotoxicity is characterized by renal fibrosis with interstitial inflammation. Since nuclear factor-kappaB (NF-kappaB) plays a key role in chronic inflammatory diseases including renal disease, the present study was conducted to elucidate the role of NF-kappaB in the pathogenesis of chronic FK506-induced nephropathy. METHODS: FK506 (1 mg/kg/day, SC) was administered daily to rats maintained on low sodium diet for 42 days. Some rats were treated with a putative NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC; 100, 200 mg/kg/day, by gavage). The renal function, renal histology, renal NF-kappaB-DNA binding activity and gene expression profile were examined. RESULTS: FK506 caused a decline in glomerular filtration and induced characteristic renal morphologic changes including arteriolopathy, tubular atrophy and interstitial fibrosis. FK506 markedly activated renal cortical NF-kappaB-DNA binding. PDTC administration inhibited NF-kappaB-DNA binding activity in a dose dependent manner. With higher dose, NF-kappaB-DNA binding activity was decreased to a control level. PDTC had little effect on FK506-induced renal dysfunction. Renal cortical monocyte/macrophage infiltration observed in FK506-treated rats was dramatically suppressed by PDTC. FK506 up-regulated renal cortical gene expression of chemoattractant proteins, monocyte chemoattractant protein-1 (MCP-1) and osteopontin. PDTC significantly blocked MCP-1 gene expression but had no effect on osteopontin gene expression. Tubular atrophy and tubulointerstitial fibrosis, but not arteriolopathy, were significantly attenuated by PDTC. FK506 increased renal mRNA expression of fibrogenic molecules and extracellular matrices that also were attenuated by PDTC treatment. CONCLUSIONS: NF-kappaB plays an important role in mediating cortical monocyte/macrophage infiltration and in the pathogenesis of tubular injury and interstitial fibrosis in experimental FK506-induced chronic nephropathy.     

Human recombinant erythropoietin augments serum asymmetric dimethylarginine concentrations but does not compromise nitric oxide generation in mice.

BACKGROUND: Patients with advanced chronic renal disease (CRD) suffer from excessive morbidity and mortality due to complications of accelerated atherosclerosis. Recombinant human erythropoietin (EPO), which is routinely used to treat the anaemia present in approximately 90% of dialysis-dependent patients with end-stage renal disease, may induce vascular dysfunction by reducing nitric oxide (NO) availability. Pathophysiologic concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), are found in patients with CRD and correlate with vascular disease and cardiovascular mortality. The aim of the current study was to investigate the effect of EPO on ADMA concentrations and NO generation in vitro and in vivo. Furthermore, we wanted to study the effect of EPO on the expression of the enzymes that regulate ADMA metabolism and NO generation. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to therapeutic concentrations of EPO. The expression and metabolic activity of dimethylarginine dimethylaminohydrolase II (DDAH II), the enzyme that degrades ADMA, was evaluated. Following subcutaneous administration of EPO to Balb/c mice for 10 weeks, serum ADMA concentrations were determined. Systolic blood pressure was measured noninvasively. Urinary nitrite and nitrate (NOx) concentrations were assessed by Griess assay. Protein expression of DDAH and NOS in livers and kidneys was measured by western blotting. RESULTS: EPO suppressed ADMA elaboration by HUVECs. Systolic blood pressure and serum concentrations of ADMA were significantly elevated in EPO-treated mice. The protein expression of DDAH I in the kidney and liver was upregulated while hepatic expression of DDAH II was decreased and renal DDAH II expression remained unchanged by EPO administration. However, EPO augmented urinary NOx concentrations as well as the expression of NOS 1 and NOS 2 in the kidney. CONCLUSION: In spite of elevating serum ADMA concentrations, EPO does not appear to compromise overall NO generation in Balb/c mice.     

Comparison of expression of TGF-beta1, its receptors TGFbeta1R-I and TGFbeta1R-II in rat kidneys during chronic nephropathy induced by cyclosporine and tacrolimus

OBJECTIVE: Chronic rejection is a major cause of graft dysfunction after kidney transplantation. Long-term treatment with cyclosporine (CsA) or tacrolimus (FK506) results in chronic nephrotoxicity. Transforming growth factor-beta1 (TGFbeta1) and its receptors type I (TR-I) and type II (TR-II) have been known to contribute to this side effect. The expression of TGF-beta1, TR-I, and TR-II in rat kidneys has not been compared during chronic nephropathy induced by CsA or FK506. METHODS: Rat models of chronic CsA- or FK506-induced nephropathy were established using Sandimun Neoral or Prograf administration. The kidneys were dissected and TGF-beta1, TR-I, and TR-II proteins and TR-I and TR-II mRNAs measured by immunohistochemistry and in situ hybridization, respectively, to compare the results of the two groups. RESULTS: The functional and morphologic studies showed that in the rats the nephrotoxic effects of FK506 were not as significant as those of CsA. The results of immunohistochemistry and in situ hybridization showed that the expression of renal TGFbeta1, TR I, TR-II proteins and TR and TR II mRNA in the FK506 group were lower than those in the CsA groups. CONCLUSION: These results showed that both FK506 and CSA display nephrotoxicity, but that the nephrotoxicity of FK506 was less than that of CsA in chronic nephropathy.     

Expression of nitric oxide and inducible nitric oxide synthase in acute renal allograft rejection in the rat

BACKGROUND: Recent studies have shown that nitric oxide (NO) synthases, particularly inducible nitric oxide synthase (i-NOS), are induced in acute rejection episodes following heart, liver, pancreas and kidney allotransplantation. Furthermore, tissue and cellular injury has been demonstrated to be mediated by peroxynitrite (ONOO-), a metabolite of NO as well as a potent oxidant. However, a detailed relationship between NO, i-NOS and graft injury in transplantation remains elusive. METHODS: The present study used the following models of renal transplantation in rats: allografts (n = 5, Brown-Norway to Lewis [LEW] rats), isografts (n = 5, LEW to LEW) and allografts treated with aminoguanidine (AG), an i-NOS inhibitor (n = 5). Blood urea nitrogen (BUN), serum creatinine (SCr) and urinary and serum nitrosocompounds (NOx) were measured on days 2, 4 and 7 post-transplant. Western blot analysis of i-NOS protein expression and measurement of i-NOS activity were carried out in grafts harvested on Day 7, along with immunohistochemical and histopathological examinations. RESULTS: In the allograft group, both BUN and SCr levels increased markedly on Day 7, in parallel with a sharp increase in NOx. A band stained by anti-i-NOS antibody was detected at approximately 130 kDa, along with high levels of i-NOS activity and diffusely distributed i-NOS-positive cells (macrophages). Histologically, an acute rejection episode was confirmed (Grade 3 according to Banff classifications). In the AG group, reduced renal function and graft injury were significantly less severe than in the allograft group. CONCLUSIONS: In rat renal allograft acute rejection, markedly increased levels of serum NOx were observed, along with enhanced tissue i-NOS activity, together resulting in graft injury. AG administration suppressed the increase of serum NOx levels, with concomitant mitigation of tissue injury and renal function impairment.     

依普拉芬对去卵巢大鼠骨质疏松骨保护效果及NO调控机制研究

目的 研究人工合成的植物雌激素-依普拉芬对去卵巢大鼠骨密度及生物力学的影响;同时与雌激素对照,探讨其骨保护效果及NO调控作用机制.方法 60只6月龄SD雌性大鼠,随机分成6组:假手术组和手术切除大鼠双侧卵巢组,后者分为阴性对照组、依普拉芬低、中、高剂量组和雌激素对照组, 分别给予基础饲料和不同剂量受试物,12周后进行骨密度、血清NO及NOS浓度测定;免疫组化方法检测股骨NOS表达.结果 与假手术组相比,去卵巢大鼠阴性对照组股骨密度、生物力学指标和血清NO、eNOS浓度以及股骨eNOS表达明显降低,血清iNOS浓度以及股骨iNOS表达增加,依普拉芬组骨密度、生物力学指标血清NO、eNOS浓度以及股骨eNOS表达均高于阴性对照组,与假手术组差异无显著意义.同时低于雌激素组.血清iNOS浓度以及股骨iNOS表达均低于阴性对照组,与假手术组以及雌激素组差异无显著意义.结论 一定浓度的依普拉芬可以通过提高去卵巢大鼠eNOS的表达来提高NO的浓度,促进成骨,增加骨密度,达到防治骨质疏松症的作用.     

Chronic diabetic nephropathy: role of inducible nitric oxide synthase

Nitric oxide (NO) is a multifunctional mediator that has been implicated in the short-term hemodynamic alterations that occur in acute streptozocin (STZ)-induced diabetes. We investigated the role of NO produced by inducible nitric oxide synthase (iNOS) in chronic STZ diabetic nephropathy. Diabetes was induced in C57BL/6 and iNOS knockout (KO) mice with two intraperitoneal injections of STZ, 100 mg/kg. Animals were maintained without insulin treatment for 40 weeks. There were no significant differences between the strains in blood urea nitrogen (BUN), serum creatinine or glucose concentration, or urinary protein excretion during the entire observation period. Urinary nitrite + nitrate excretion was significantly lower in iNOS KO mice compared to control animals at all time points; in C57 mice, urinary nitrite declined progressively with more prolonged duration of diabetes. Renal hypertrophy (kidney weight/body weight) was noted in both strains of mice. However, histopathological assessment of renal tissue specimens at 16 and 40 weeks demonstrated increased mesangial hypercellularity and expansion as well as more prominent tubulointerstitial fibrosis in iNOS KO versus C57 mice. These changes were accompanied by increased interstitial deposition of type I collagen at 16 and 40 weeks in iNOS KO mice. Glomerular basement membrane staining for type IV collagen was also increased at 40 weeks in diabetic iNOS KO mice. While iNOS protein was undetectable in any of the kidney specimens obtained from either strain, eNOS was present throughout the course of chronic STZ diabetes. Moreover, eNOS expression was significantly increased by approximately 40% at 16 and 40 weeks of observation in iNOS KO versus C57 mice. There was no difference in renal cortical malondialdehyde content between the strains early or late in the disease course. In time control animals, there was no evidence of renal histopathological damage in iNOS KO or C57 mice after 40 weeks. We conclude that iNOS-derived NO modulates glomerulosclerosis and tubulointerstitial fibrosis in chronic STZ nephropathy. This action is probably a result of the direct actions of NO on the synthesis and degradation of extracellular matrix proteins. Received: 28 February 2001 / Revised: 10 August 2001 / Accepted: 13 August 2001     

Paradoxical increase in nitric oxide synthase activity in hypercholesterolaemic rats with impaired renal function and decreased activity of nitric oxide.

BACKGROUND: We have shown that acute exposure of oxidized low-density lipoprotein (OX-LDL) induces vasoconstriction in renal vessels and reduces glomerular filtration rate (GFR) in an isolated perfused rat kidney model by decreasing the activity of nitric oxide (NO). L-arginine has a protective role against OX-LDl-induced vasoconstriction. Micropuncture studies have demonstrated that short-term diet-induced hypercholesterolaemia is associated with decreased GFR and renal blood flow and increased glomerular capillary pressure. This may be mediated by decreased activity of NO. METHODS: Rats were made hypercholesterolaemic by supplementing the standard chow with 4% cholesterol and 1% sodium cholate. A group of rats on hypercholesterolaemic diet also received L-arginine in the drinking water. After 4 and 6 weeks, blood samples and 24-h urine samples were collected for the measurement of biochemical parameters. After 6 weeks, all rats were subjected to isolated perfusion of kidneys at a constant pressure of 100 mmHg. During isolated perfusion, the unused contralateral kidney was taken for morphological studies and for assessing the activity of nitric oxide synthase enzyme by beta-NADPH diaphorase histochemistry. RESULTS: Rats fed a high-cholesterol diet had LDL levels 3-6 times greater than the rats fed standard chow. Rats that received L-arginine in the drinking water had serum L-arginine levels 5-6 times greater than control rats. At 6 weeks, creatinine clearance was significantly lower in the rats on the high-cholesterol diet compared to the rats on standard chow and rats on high-cholesterol diet plus L-arginine. Twenty-four-hour urinary total nitrate and nitrite excretion in the hypercholesterolaemic rats was 1.5-2 times greater than that of control rats. Twenty-four-hour urinary cGMP excretion was significantly lower in the rats on a high-cholesterol diet, but in the rats on high-cholesterol diet and L-arginine, 24-h urinary cGMP excretion was not significantly different from that of control rats. During isolated perfusion of kidneys, renal perfusate flow was found to be significantly reduced in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation in the drinking water almost completely reversed the effect of a high-fat diet. Inulin clearance was also significantly reduced in kidneys on a high-fat diet in contrast to controls but not in kidneys on high fat-diet and L-arginine. Basal cGMP excretion in urine was significantly lower in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation restored the basal cGMP excretion in these kidneys. NO synthase (NOS) enzyme activity as assessed by NADPH diaphorase activity showed that kidney sections taken from the rats on a high-fat diet showed more intense staining, indicating increased activity compared to the kidney sections taken from the rats on a normal diet. CONCLUSION: Though activity of NO is diminished in hypercholesterolaemic rats with impaired renal function, there is a paradoxical increase in NO production and NOS activity. L-arginine reverses the effects of a high-fat diet.     

FK506对糖尿病大鼠早期肾脏肥大的抑制作用及机制

目的 探讨FK506对糖尿病大鼠早期肾脏肥大的抑制作用及机制。 方法 应用链脲菌素(65 mg/kg)腹腔注射建立大鼠糖尿病模型。每日分别给予FK506 0.5、1.0 mg/kg 灌胃，共4周。观察大鼠肾质量/体质量、尿白蛋白排泄率（AER）、Ccr及肾组织病理形态学变化。应用Western印迹和免疫组化方法检测肾组织钙神经蛋白(calcineurin，CaN)、1αⅣ型胶原、α-平滑肌肌动蛋白(α-SMA)及转化生长因子β1(TGF-β1)蛋白表达。 结果 FK506 1.0组大鼠相对肾质量明显低于模型组(P < 0.05)。FK506 0.5与1.0组大鼠AER水平与肾小球平均体积明显低于模型组(P < 0.05、0.01)。FK506 1.0组肾小管间质?鄄损伤指数也明显低于模型组(P < 0.01)。Western印迹显示，模型组肾组织CaN蛋白表达较对照组增加2.4倍；FK506 0.5与1.0 组肾组织CaN蛋白表达比模型组下降38.0%与73.2%。模型组肾组织1αⅣ型胶原、α-SMA及TGF?鄄β1蛋白表达明显高于对照组；FK506组肾组织上述蛋白表达明显低于模型组(P < 0.05、0.01)。 结论 FK506对糖尿病大鼠早期肾脏肥大有明显抑制作用，其机制与下调肾组织增高的CaN表达有关。