Renal expression of aquaporins in liver cirrhosis induced by chronic common bile duct ligation in rats.

Semiquantitative immunoblotting was used to investigate the expression levels of the four major renal aquaporins, the Na-K-2Cl cotransporter of the thick ascending limb, the type 3 Na-H exchanger, and the Na-K-ATPase in kidneys from rats with cirrhosis secondary to common bile duct ligation (CBDL). These rats had significant water retention and hyponatremia. In contrast to models of cirrhosis induced by carbon tetrachloride, aquaporin-2 expression in CBDL-induced cirrhosis was decreased. Thus, these results show that in the setting of extracellular fluid volume expansion, excessive water retention with hyponatremia can occur in the absence of increases in aquaporin-2 abundance. In addition, the expression levels of the two basolateral collecting duct aquaporins (aquaporin-3 and -4) were decreased in CBDL rats relative to sham-operated control rats. Similarly, the Na-K-2Cl cotransporter of the thick ascending limb and the type 3 Na-H exchanger showed decreases in expression. In contrast, the expression levels of aquaporin-1 and the all subunit of the Na-K-ATPase were not decreased. Thus, dysregulation of multiple water channels and ion transporters may play a role in water balance abnormalities associated with CBDL-induced cirrhosis in rats.     

Vitamin E prevents renal dysfunction induced by experimental chronic bile duct ligation

BACKGROUND: The mechanisms by which prolonged cholestasis alters renal hemodynamics and excretory function are unknown but may be related to increased oxidative stress, with subsequent formation of lipid peroxidation-derived products (e.g., F2-isoprostanes) and endothelin (ET). We investigated whether antioxidant therapy prevents chronic bile duct ligation (CBDL)-induced alterations in systemic and renal hemodynamics, and reduces F2-isoprostane and ET levels. METHODS: Sprague-Dawley rats were placed on either a normal or a high vitamin E diet for 7 days and then underwent either CBDL or sham surgery. They were then maintained on their respective diets for 21 more days, at which time the physiologic studies were performed. RESULTS: Thirty-three percent of the CBDL rats died by day 21. The remaining rats had a lower mean arterial pressure (MAP), renal blood flow (RBF), glomerular filtration rate (GFR), and sodium and water excretion than control rats. CBDL rats had higher portal pressure, renal venous pressure, and renal vascular resistance (RVR). These changes were associated with increased levels of systemic and renal venous F2-isoprostanes and ET. Vitamin E normalized MAP, RBF, GFR, RVR, and sodium and water excretion, and improved the 21-day survival without altering portal or renal venous pressures. Surprisingly, vitamin E did not alter the systemic levels of F2-isoprostanes but markedly reduced their levels in the renal venous circulation. CONCLUSION: Vitamin E improves MAP and renal function in CBDL rats, and selectively decreases renal levels of oxidative stress and ET, suggesting that local redox balance is implicated in CBDL-induced renal dysfunction.     

Increased cGMP phosphodiesterase activity mediates renal resistance to ANP in rats with bile duct ligation

BACKGROUND: Liver disease resulting from common bile duct ligation (CBDL) causes abnormal sodium metabolism that is manifested by resistance to the natriuretic action of atrial natriuretic peptide (ANP). This resistance is corrected both in vitro and in vivo by zaprinast, a selective inhibitor of a guanosine cyclic-3'-5'-monophosphate (cGMP)-specific phosphodiesterase (PDE5). Several other PDEs with affinity for cGMP are expressed in kidney and could also be involved in this response. METHODS: We measured cGMP hydrolysis in inner medullary collecting duct (IMCD) cell homogenates from kidneys of sham-operated and CBDL rats and quantitated the amount of PDE5 protein by Western blotting and immunoprecipitation studies. We also characterized ANP responsiveness in vivo of kidneys of anesthetized sham and CBDL rats by measuring sodium excretion before and after volume expansion (VE). RESULTS: Kinetic analysis of PDE5 activity in homogenates of IMCD cells isolated from kidneys of sham-operated rats indicated a Vmax of 85.3 +/- 1.7 versus 157 +/- 2.9 pmol/mg/min from CBDL rats (P < 0.01), without a difference in Km. Enzyme activity was inhibited competitively by 1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazol[3,4d]-pyrimidin-4-(5H)-one (DMPPO), a potent and specific inhibitor of PDE5, with an apparent Ki of 4.5 +/- 0.7 and 4.9 +/- 0.7 nmol/L and an IC50 of 6.1 +/- 0.8 and 8.7 +/- 0.7 nmol/L in sham and CBDL rats, respectively (P = NS). DMPPO exhibited very poor inhibitory activity against the calcium-calmodulin-dependent PDE1 in IMCD homogenates from sham rats (Ki 1.3 +/- 0.1 micromol/L and IC50 1.9 +/- 0.2 micromol/L). Western analysis using an antiserum made against bovine lung PDE5 revealed a twofold increase in PDE5 protein in cytosolic extracts from IMCD of CBDL rat kidneys compared with sham-operated controls, and immunoprecipitation studies indicated that the increase in PDE5 protein accounted for the observed increase in cGMP hydrolysis. DMPPO (10 nmol/L) normalized the blunted ANP-dependent cGMP accumulation by IMCD cells from CBDL rats in vitro. Intrarenal infusion of DMPPO (0.5 nmol/min) in CBDL rats corrected both the impaired natriuretic response to VE and the blunted VE-related increase in urinary cGMP excretion from the infused, but not the contralateral kidney. CONCLUSION: These results demonstrate that renal resistance to ANP in CBDL rats is accompanied by heightened activity of PDE5, which is due largely to an increase in PDE5 protein. Other PDEs could contribute only a minor part to the enhanced cGMP hydrolysis observed in kidneys of CBDL rats. This PDE5-dependent ANP resistance may represent an important contributor to the sodium retention of liver disease.     

Microcomputed tomography of kidneys following chronic bile duct ligation

BACKGROUND: In hepatic cirrhosis, renal sodium and water retention can occur prior to decreases in renal blood flow (RBF). This may be explained in part by redistribution of the intrarenal microcirculation toward the juxtamedullary nephrons. To appreciate this three-dimensional spatial redistribution better, we examined the intrarenal microcirculatory changes using microcomputed tomography (micro-CT) in rats subjected to chronic bile duct ligation (CBDL). METHODS: Six kidneys from control rats and eight kidneys from rats that had undergone CBDL for 21 days were perfusion fixed in situ at physiological pressure, perfused with silicon-based Microfil containing lead chromate, embedded in plastic, and scanned by micro-CT. The microvasculature in the reconstructed three-dimensional renal images was studied using computerized image-analysis techniques. To determine the physiological condition of the rats, parallel experiments were conducted on six control and six CBDL rats to measure mean arterial pressure (MAP), RBF, glomerular filtration rate (GFR), urine flow (UF) rate, and sodium excretion by conventional methods. RESULTS: The percentage of vasculature in the renal cortex from CBDL rats was significantly decreased (10.8 +/- 0.4% vs. 16.8 +/- 2.7% control values). However, the vascular volume fractions of the medullary tissues were not significantly altered. There were no significant differences in the number of glomeruli between groups (36,430 +/- 1908 CBDLs, 36,609 +/- 3167 controls). The CBDL rats had a similar GFR than the controls but a reduced MAP, RBF, UF, and sodium excretion. CONCLUSIONS: The results indicate that after CBDL, there is a selective decrease in cortical vascular filling, which may contribute to the salt and water retention that accompanies cirrhosis.     

High Potassium Intake Enhances the Inhibitory Effect of 11,12-EET on ENaC

High dietary potassium stimulates the renal expression of cytochrome P450 (CYP) epoxygenase 2C23, which metabolizes arachidonic acid (AA). Because the AA metabolite 11,12-epoxyeicosatrienoic acid (11,12-EET) can inhibit the epithelial sodium channel (ENaC) in the cortical collecting duct, we tested whether dietary potassium modulates ENaC function. High dietary potassium increased 11,12-EET in the isolated cortical collecting duct, an effect mimicked by inhibiting the angiotensin II type I receptor with valsartan. In patch-clamp experiments, a high potassium intake or treatment with valsartan enhanced AA-induced inhibition of ENaC, an effect mediated by a CYP-epoxygenase–dependent pathway. Moreover, high dietary potassium and valsartan each augmented the inhibitory effect of 11,12-EET on ENaC. Liquid chromatography/mass spectrometry showed that the rate of EET conversion to dihydroxyeicosatrienoic acids (DHET) was lower in renal tissue obtained from rats on a high-potassium diet than from those on a control diet, but this was not a result of altered expression of soluble epoxide hydrolase (sEH). Instead, suppression of sEH activity seemed to be responsible for the 11,12-EET–mediated enhanced inhibition of ENaC in animals on a high-potassium diet. Patch-clamp experiments demonstrated that 11,12-DHET was a weak inhibitor of ENaC compared with 11,12-EET, whereas 8,9- and 14,15-DHET were not. Furthermore, inhibition of sEH enhanced the 11,12-EET–induced inhibition of ENaC similar to high dietary potassium. In conclusion, high dietary potassium enhances the inhibitory effect of AA and 11,12-EET on ENaC by increasing CYP epoxygenase activity and decreasing sEH activity, respectively.We previously demonstrated that cytochrome P450 (CYP) epoxygenase-dependent arachidonic acid (AA) metabolism inhibited epithelial sodium channel (ENaC) in the cortical collecting duct (CCD) and that 11,12-epoxyeicosatrienoic acid (11,12-EET) was responsible for mediating the effect of AA on ENaC.¹ Furthermore, the observation that AA failed to inhibit ENaC in the CCD of the mice with a low expression of CYP2C44 suggests that CYP2C44 and its orthologs may be responsible for mediating the inhibitory effect of AA.² The expression of CYP2C44 or its orthologs has been shown to be regulated by dietary Na intake: A high Na intake stimulates² whereas a low, Na intake suppresses the expression of CYP2C44 homologue.³ A large body of evidence has suggested that EET plays a role in the regulation of renal Na transport and salt-sensitive hypertension.^1,2,4,5 Inhibition of CYP epoxygenase-dependent AA metabolism results in the development of salt-sensitive hypertension^5,6; however, the BP returned to normal after the removal of the epoxygenase inhibitor, even when the animals were still kept on a high-Na diet. The high Na intake–induced increase in EET formation was defective in CYP4A10(−/−) mice.² Although CYP4A10 is not the enzyme responsible for generating EET, deleting the CYP4A10 gene impairs the expression of CYP epoxygenases, CYP2C44 in particular. Consequently, CYP4A10(−/−) mice had developed a salt-sensitive hypertension that was prevented by amiloride, suggesting that defective regulation of ENaC by CYP epoxygenase-dependent AA metabolism was responsible for the salt-sensitive hypertension in CYP4A(−/−) mice.² The expression of CYP2C44 or its ortholog is stimulated not only by high Na but also by high potassium (HK) intake.⁷ Because CYP2C44 is highly expressed in the connecting tubule and the CCD,¹ it is conceivable that a high expression of CYP2C44 should enhance the AA-induced inhibition of ENaC; therefore, the aim of this study was to examine whether HK intake enhances the CYP-epoxygenase–dependent inhibitory effect of AA on ENaC.     

Rosiglitazone treatment attenuates renal tissue inflammation generated by urinary tract obstruction

Aim: Peroxisome proliferator‐activated receptor (PPAR)‐γ activation by rosiglitazone decreases manifestation of intrarenal inflammatory hallmarks. Inflammation significantly aggravates renal injury following urinary tract obstruction. The effect of rosiglitazone on renal inflammation following unilateral ureteral obstruction was investigated. Methods: Ninety‐six Srague–Dawley rats were subjected to unilateral ureteral ligation, or to sham operation. Half of each group received rosiglitazone, 5 mg/kg bodyweight per day. The animals were killed and their kidneys allocated following 1 h, 24 h or 2 weeks, for pathological examination or for intrarenal transforming growth factor (TGF)‐β, interleukin (IL)‐4, IL‐6, IL‐10 and nitric oxide (NO) assessment by specific enzyme‐linked immunosorbent assays. Apoptosis rates, extracellular matrix deposition, PPAR‐γ, α‐smooth muscle actin (α‐SMA) expression and macrophage infiltration were assessed by specific immunohistological stainings. Results: PPAR‐γ receptor expression was downregulated, and infiltration of macrophages decreased, in all rosiglitazone‐treated kidneys. Rosiglitazone significantly decreased apoptosis, TGF‐β, IL‐6, α‐SMA expression and NO availability in obstructed kidneys. Synthesis of IL‐10 was unaltered, while IL‐4 augmented by Rosiglitazone. Rosiglitazone also affected NO and IL‐4 production in sham‐operated controls. Conclusion: (i) Rosiglitazone attenuates profibrotic and pro‐inflammatory responses in a rat model of ureteral obstruction‐induced renal inflammation; (ii) rosiglitazone stimulates counteractive anti‐inflammatory responses in the damaged kidneys; (iii) in part, rosiglitazone exerts comparable anti‐inflammatory effects on obstructed kidneys and unobstructed healthy controls. Taken together, this ascertains the importance of the anti‐inflammatory role of rosiglitazone treatment in amelioration of ureteral obstruction‐induced renal damage.     

Diabetes-induced microvascular dysfunction in the hydronephrotic kidney: role of nitric oxide.

BACKGROUND: Renal hemodynamics in early diabetes are characterized by preglomerular and postglomerular vasodilation and increased glomerular capillary pressure, leading to hyperfiltration. Despite intensive research, the etiology of the renal vasodilation in diabetes remains a matter of debate. The present study investigated the controversial role of nitric oxide (NO) in the renal vasodilation in streptozotocin-induced diabetic rats. METHODS: In the renal microcirculation, basal tone and response to NO synthase blockade were studied using the in vivo hydronephrotic kidney technique. L-arginine analog N-nitro-L-arginine methyl ester (L-NAME) was administered locally to avoid confounding by systemic blood pressure effects. The expression of endothelial NO synthase (eNOS) was investigated in total kidney by immunocytochemistry and in isolated renal vascular trees by Western blotting. Urinary excretion of nitrites/nitrates was measured. RESULTS: Diabetic rats demonstrated a significant basal vasodilation of all preglomerular and postglomerular vessels versus control rats. Vasoconstriction to L-NAME was significantly increased in diabetic vessels. After high-dose L-NAME, there was no difference in diameter between diabetic and control vessels, suggesting that the basal vasodilation is mediated by NO. Immunocytochemically, the expression of eNOS was mainly localized in the endothelium of preglomerular and postglomerular vessels and glomerular capillaries, and was increased in the diabetic kidneys. Immunoblots on isolated renal vascular trees revealed an up-regulation of eNOS protein expression in diabetic animals. The urinary excretion of nitrites/nitrates was elevated in diabetic rats. CONCLUSION: The present study suggests that an up-regulation of eNOS in the renal microvasculature, resulting in an increased basal generation of NO, is responsible for the intrarenal vasodilation characteristic of early diabetes.     

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.     

Renal damage in rats induced by myocardial ischemia/reperfusion: Role of nitric oxide

BACKGROUND: It has been demonstrated that myocardial ischemia/reperfusion (MI/R) causes renal damage. However, the mechanism underlying this damage in kidneys during revascularization of myocardium is unclear. Direct renal ischemia/reperfusion has been implicated in the induction of inducible nitric oxide synthase (iNOS) that leads to increase production of nitric oxide (NO). Recently, excessive production of NO has been found to be involved in causing renal injury by formatting peroxinitrite (ONOO(-)). The aim of this study was to investigate whether NO has a role in this damage, using aminoguanidine (AMG), a known iNOS inhibitor and an antioxidant, in rats undergoing MI/R. METHODS: Male Wistar rats were used for the experiments (n = 7 each group). In the MI/R group, the left coronary artery was occluded for 30 min and then reperfused for 120 min; the same procedure was used for the AMG group, with the additional step of AMG (200 mg/kg) administered 10 min prior to ischemia. A control group underwent sham operation. At the end of the reperfusion period, all rats were killed and their kidneys removed for biochemical determination and histopathological analysis. RESULTS: Myocardial ischemia/reperfusion in the rat kidney was accompanied by a significant increase in malondialdehyde and NO production, and a decrease in glutathione content. Administration of AMG reduced malondialdehyde and NO production and prevented depletion of glutathione content. These beneficial changes in the biochemical parameters were also associated with parallel changes in histopathological appearance. CONCLUSION: These findings suggest that MI/R plays a causal role in kidney injury and AMG exerts renal-protective effects, probably by inhibiting NO production and antioxidant activities.     

Impaired Kupffer cell function and effect of immunotherapy in obstructive jaundice

BACKGROUND: Obstructive jaundice is frequently associated with septic complications. This study examined the influence of biliary obstruction on bacterial clearance and translocation. The study focused on the phagocytic and killing activities of Kupffer cells and the preventive effect on bacterial translocation of OK-432, which is a hemolytic streptococcal preparation developed as a biological response modifier. METHODS: To study the mechanism of sepsis in obstructive jaundice, two groups of Wistar rats were examined: rats subjected to common bile duct ligation (CBDL) and rats subjected to a sham operation. Bacterial clearance, organ distribution, hepatic blood flow, and phagocytic function of Kupffer cells were examined. To evaluate the effect of OK-432 on bacterial translocation, rats were divided into three groups: sham operation + phosphate-buffered saline (PBS), CBDL + PBS, and CBDL + OK-432. RESULTS: In this study, clearance of Escherichia coli. from the peripheral blood in CBDL rats was decreased significantly compared with that in sham-operated rats. Significant decreases in E.coli trapped in the liver and in hepatic blood flow were observed in CBDL rats compared with sham-operated rats. Phagocytic activity and superoxide production of Kupffer cells isolated from CBDL rats were significantly lower than in sham-operated rats. The incidence of bacterial translocation in CBDL rats was increased significantly, and oral administration of OK-432 prevented it. CONCLUSION: The results suggest that susceptibility to infection in obstructive jaundice is due to impaired phagocytic function of Kupffer cells. Furthermore, obstructive jaundice promotes bacterial translocation, and OK-432 may be useful in preventing this translocation.     

Downregulation of hepatic cytochrome P450 in chronic renal failure

Chronic renal failure (CRF) is associated with a decrease in drug metabolism. The mechanism remains poorly understood. The present study investigated the repercussions of CRF on liver cytochrome P450 (CYP450). Three groups of rats were defined: control, control paired-fed, and CRF. Total CYP450 activity, protein expression of several CYP450 isoforms as well as their mRNA, and the in vitro N-demethylation of erythromycin were assessed in liver microsomes. The regulation of liver CYP450 by dexamethasone and phenobarbital was assessed in CRF rats. Compared with control and control paired-fed rats, creatinine clearance was reduced by 60% (P: < 0.01) in CRF rats. Weight was reduced by 30% (P: < 0.01) in control paired-fed and CRF rats, compared with control animals. There was no difference in the CYP450 parameters between control and control paired-fed. Compared with control paired-fed rats, total CYP450 was reduced by 47% (P: < 0.001) in CRF rats. Protein expression of CYP2C11, CYP3A1, and CYP3A2 were considerably reduced (>40%, P: < 0.001) in rats with CRF. The levels of CYP1A2, CYP2C6, CYP2D, and CYP2E1 were the same in the three groups. Northern blot analysis revealed a marked downregulation in gene expression of CYP2C11, 3A1, and 3A2 in CRF rats. Although liver CYP450 was reduced in CRF, its induction by dexamethasone and phenobarbital was present. N-demethylation of erythromycin was decreased by 50% in CRF rats compared with control (P: < 0.001). In conclusion, CRF in rats is associated with a decrease in liver cytochrome P450 activity (mainly in CYP2C11, CYP3A1, and 3A2), secondary to reduced gene expression.     

Soluble epoxide hydrolase inhibition protects the kidney from hypertension-induced damage

Epoxyeicosatrienoic acids (EET) have antihypertensive and anti-inflammatory properties and play a role in the maintenance of renal vascular function. A novel approach to increase EET levels is to inhibit epoxide hydrolase enzymes that are responsible for conversion of biologically active EET to dihydroxyeicosatrienoic acids (DHET). We hypothesized that soluble epoxide hydrolase (SEH) inhibition would improve renal vascular function and ameliorate hypertension induced renal damage. Chronic administration of the specific SEH inhibitor 1-cyclohexyl-3-dodecylurea (CDU, 3 mg/d) for 10 d lowered BP in angiotensin hypertensive rats. The contribution of renal vascular SEH to afferent arteriolar function in angiotensin hypertension was also assessed. SEH protein expression was increased in renal microvessels from hypertensive rats. Although CDU did not change afferent arteriolar responsiveness to angiotensin in normotensive animals, CDU treatment significantly attenuated afferent arteriolar diameter responses to angiotensin in hypertensive kidneys from 51% +/- 8% to 28% +/- 7%. Protection of the renal vasculature and glomerulus during chronic CDU administration was demonstrated by histology. Urinary albumin excretion, an index of renal damage, was also lower in CDU-treated hypertensive rats. These data demonstrate that SEH inhibition has antihypertensive and renal vascular protective effects in angiotensin hypertension and suggests that SEH inhibitors may be a useful therapeutic intervention for cardiovascular diseases.     

Downregulation of intestinal cytochrome p450 in chronic renal failure

Chronic renal failure (CRF) is associated with a decrease in intestinal drug metabolism. The mechanisms remain poorly understood, but one hypothesis involves a reduction in cytochrome P450 levels. This study aimed to investigate the effects of CRF on intestinal cytochrome P450. Two groups of rats were defined, i.e., rats with CRF (induced by 5/6 nephrectomy) and control pair-fed rats. Total cytochrome P450 levels and protein and mRNA expression of cytochrome P450 isoforms, as well as in vitro N-demethylation of erythromycin (a probe for CYP3A activity) and 7-ethoxyresorufin o-deethylase activity (a probe for CYP1A), were assessed in intestinal microsomes. Body weights were similar in the two groups. Creatinine clearance was reduced by 77% (P < 0.001) in CRF rats, compared with control pair-fed animals. Total intestinal cytochrome P450 activity was reduced by 32% (P < 0.001) in CRF rats. CYP1A1 and CYP3A2 protein expression was considerably reduced (>40%, P < 0.001) in rats with CRF. CYP2B1, CYP2C6, and CYP2C11 levels were the same in the two groups. RT-PCR assays revealed marked downregulation of CYP1A1 and CYP3A2 gene expression in CRF rats (P < 0.001). Although intestinal cytochrome P450 levels were reduced in CRF, induction by dexamethasone was present. N-Demethylation of erythromycin and 7-ethoxyresorufin o-deethylase activity were decreased by 25% (P < 0.05) in CRF rats, compared with control rats. In conclusion, CRF in rats is associated with decreases in intestinal cytochrome P450 activity (mainly CYP1A1 and CYP3A2) secondary to reduced gene expression.     

细胞外信号调节激酶及胞质磷脂酶A2α在血管紧张素Ⅱ调节肾近曲小管Na+-HCO3-转运中的作用

目的 探讨血管紧张素Ⅱ（AngⅡ）在肾近曲小管Na+-HCO3-转运中的作用及细胞外信号调节激酶（ERK）、胞质磷脂酶A2α(cPLA2α)通路对其调节的机制。 方法 从野生小鼠和血管紧张素1a型受体 (AT1aR)基因缺陷小鼠分离新鲜单根肾近曲小管，在不同浓度AngⅡ（10－10、10－8、10－6 mol/L）及AT1、AT2受体阻滞剂或促分裂原活化蛋白激酶（MAPK）、cPLA2、P450抑制剂存在下对比Na+-HCO3-离子转运活动度变化。Western印迹方法测定ERK磷酸化（p-ERK）。RT-PCR测定AT1bR在两种小鼠肾小管中的表达。 结果 （1）在野生小鼠，低浓度AngⅡ（10－10 mol/L）刺激Na+-HCO3-转运并被AT1受体阻滞剂及MAPK阻滞剂PD98059阻滞；而高浓度AngⅡ（10－6 mol/L）抑制Na+-HCO3-的转运，被AT1受体阻滞剂阻滞，但PD98059对其无阻滞作用。显示AngⅡ在肾脏近曲小管双向性调节Na+-HCO3-转运，ERK通路仅参与低浓度AngⅡ的刺激作用。（2）在AT1aR基因缺陷小鼠，只有高浓度AngⅡ（10-6 mol/L）能刺激Na+-HCO3-转运，并被AT1受体阻滞剂及PD98059阻滞。显示在AT1aR缺乏时，AT1bR起到部分代偿作用，RT-PCR也证实AT1bR在肾小管的存在。（3）在野生小鼠，cPLA2 阻滞剂或P450阻滞剂存在下，所有浓度AngⅡ均显示刺激作用，并被AT1受体阻滞剂及PD98059阻滞。Western印迹检测也证实上述结论。这显示经由AT1受体，低浓度AngⅡ通过ERK通路仅参与刺激肾近曲小管Na+-HCO3-离子转运作用，而高浓度AngⅡ经cPLA2α-P450通路抑制Na+-HCO3-离子转运，cPLA2α-P450通路同时也参与了抑制ERK的激活作用。 结论 不同浓度AngⅡ经由AT1受体介导了ERK和cPLA2α通路的平衡，从而决定AngⅡ调节在肾近曲小管水和钠的重吸收。     

Nitric oxide synthesis and oxidative stress in the renal cortex of rats with diabetes mellitus.

Experiments were performed to test the hypothesis that diabetes mellitus disrupts the balance between synthesis and degradation of nitric oxide (NO) in the renal cortex. Diabetes was induced by injection of streptozotocin, and sufficient insulin was provided to maintain moderate hyperglycemia for the ensuing 2 wk. Despite an 80% increase in total NO synthase activity measured by L-citrulline assay, nicotinamide adenine dinucleotide phosphate-diaphorase staining was unaltered, and no changes in NO synthase isoform protein levels or their distribution were evident in renal cortex from diabetic rats. Superoxide anion production was accelerated twofold in renal cortical slices from diabetic rats, with an associated 50% increase in superoxide dismutase activity. Western blots prepared by use of a monoclonal antinitrotyrosine antibody revealed an approximately 70-kD protein in renal cortex from sham rats, the nitrotyrosine content of which was threefold greater in cortical samples from diabetic rats. These observations indicate that the early stage of diabetes mellitus provokes accelerated renal cortical superoxide anion production in a setting of normal or increased NO production. This situation can be expected to promote peroxynitrite formation, resulting in the tyrosine nitration of a single protein of unknown identity, as well as a decline in the bioavailability of NO. These events are consistent with the postulate that oxidative stress promotes NO degradation in the renal cortex during the early stage of diabetes mellitus.     

Inhibition of nitric oxide synthase does not influence urinary nitrite plus nitrate excretion after renal ischemic injury

BACKGROUND AND AIMS: Whether renal nitric oxide production caused by ischemia/reperfusion (I/R) influences the urinary excretion of nitric oxide (NO) metabolites (nitrite and nitrate) is far from being elucidated. In the present study, we evaluated the role of NO synthase inhibition using N(G)-nitro- L-arginine methyl ester ( L-NAME) in a model of experimental renal I/R injury. METHODS: Male Wistar rats were used in our experiments, and renal I/R injury was achieved after a 30-min occlusion of the bilateral renal artery followed by a 60-min period of reperfusion. Renal function including nitrite plus nitrate excretion and hemodynamics in reperfused kidneys were measured in the presence and absence of L-NAME. RESULTS: Intravenous application of L-NAME (5 mg/kg body weight) resulted in a marked reduction of urine flow, renal plasma flow (0.7 +/- 0.3 ml/min), and the glomerular filtration rate (0.1 +/- 0.01 ml/min), but a significant increase in NO excretion (FENOx, 67.9 +/- 10.5%). In addition we found after L-NAME injection a significant increase of the fractional excretion of sodium (FENa, 49.3 +/- 7.7%) and lithium (FELi, 70.2 +/- 1.6%), as well as the renal vascular resistance compared with animals with renal I/R but non-treated with L-NAME ( P<0.001). Furthermore, we observed a high correlation between FENOx and FELi (r(2)=0.80, P<0.01). CONCLUSION: Our results suggest that renal excretion of NO derivatives is not influenced by NO production during renal I/R injury, although NO contributes to the tubular transport capacity in the ischemia/reperfused kidney.     

Hemodynamic function in dogs with chronic obstructive jaundice: effects of radiocontrast medium

Chronic obstruction of the biliary tract alters circulatory function. The effects of radiocontrast medium (iothalamate meglumine) on systemic and renal hemodynamics were studied in normal dogs and dogs with chronic common bile duct ligation (CBDL). After intravenous injection of radiocontrast, cardiac output and stroke volume were not altered in normal and CBDL dogs; arterial pressure was stable in normal dogs but decreased significantly in CBDL dogs and was accompanied by reduced systemic vascular resistance. In normal and CBDL dogs the renal hemodynamic responses to radiocontrast medium were characterized by initial vasoconstriction (independent of renal renin release) and later vasodilation. This vasodilatation in CBDL dogs was particularly striking as it occurred despite reduced renal perfusion pressure and the augmented renin-angiotensin system. Inhibition of prostaglandin synthesis with indomethacin in CBDL dogs abolished both the hypotensive and the renal vasodilator responses to radiocontrast medium. We conclude that enhanced prostaglandin activity contributes to the labile hemodynamic function noted in obstructive jaundice.     

Effect of Bile Acid Replacement on Endotoxin-induced Tumor Necrosis Factor-a Production in Obstructive Jaundice

There is a high incidence of perioperative morbidity and mortality in patients with obstructive jaundice due to sepsis. Tumor necrosis factor-a (TNF-a) is considered a crucial mediator in inducing and processing the inflammatory cascade. We hypothesize that obstructive jaundice leads to an increased endotoxin-induced TNF-a production and that intestinal bile acid replacement can prevent this phenomenon. Sprague-Dawley rats were randomized to three groups of 12 animals each. Group 1 underwent common bile duct ligation (CBDL) with oral intestinal bile acid (deoxycholic acid 5 mg/100 g body weight/3 times daily) replacement (CBDL + bile acid); group 2 underwent common bile duct ligation with the same amount of normal saline replacement orally (CBDL + saline); and group 3 underwent a sham operation (sham control). After 2 days, endotoxin was given to the animals, and after 90 minutes, tissues (liver and lung) and blood were collected for checking the TNF-a levels and biochemical analyses. Comparisons among these three groups were performed and recorded. While serum and tissue (liver and lung) TNF-a levels of group 2 (CBDL + saline) were significantly increased after endotoxin challenge, these elevations were reduced to control levels (sham control) following oral replacement of intestinal bile acid (CBDL + bile acid). Obstructive jaundice leads to an increased endotoxin-induced TNF-a production and intestinal bile acid replacement can inhibit this phenomenon.     

Nitrite-derived nitric oxide protects the rat kidney against ischemia/reperfusion injury in vivo: role for xanthine oxidoreductase

In normal conditions, nitric oxide (NO) is oxidized to the anion nitrite, but in hypoxia, this nitrite may be reduced back to NO by the nitrite reductase action of deoxygenated hemoglobin, acidic disproportionation, or xanthine oxidoreductase (XOR). Herein, is investigated the effects of topical sodium nitrite administration in a rat model of renal ischemia/reperfusion (I/R) injury. Rats were subjected to 60 min of bilateral renal ischemia and 6 h of reperfusion in the absence or presence of sodium nitrite (30 nmol) administered topically 1 min before reperfusion. Serum creatinine, serum aspartate aminotransferase, creatinine clearance, fractional excretion of Na(+), and plasma nitrite/nitrate concentrations were measured. The nitrite-derived NO-generating capacity of renal tissue was determined under acidic and hypoxic conditions by ozone chemiluminescence in homogenates of kidneys that were subjected to sham, ischemia-only, and I/R conditions. Nitrite significantly attenuated renal dysfunction and injury, an effect that was abolished by previous treatment of rats with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (2.5 mumol intravenously 5 min before ischemia and 50 nmol topically 6 min before reperfusion). Renal tissue homogenates produced significant amounts of NO from nitrite, an effect that was attenuated significantly by the xanthine oxidoreductase inhibitor allopurinol. Taken together, these findings demonstrate that topically administered sodium nitrite protects the rat kidney against I/R injury and dysfunction in vivo via the generation, in part, of xanthine oxidoreductase-catalyzed NO production. These observations suggest that nitrite therapy might prove beneficial in protecting kidney function and integrity during periods of I/R such as those encountered in renal transplantation.