Abstract-The present study characterized the biochemical pathways responsible for superoxide (O 2 Ϫ· ) production in different regions of the rat kidney and determined the role of O 2 Ϫ· in the control of renal medullary blood flow (MBF) and renal function. By use of dihydroethidium/DNA fluorescence spectrometry with microtiter plates, the production of O 2 Ϫ· was monitored when tissue homogenate from different kidney regions was incubated with substrates for the major O 2 Ϫ· -producing enzymes, such as NADH/NADPH oxidase, xanthine oxidase, and mitochondrial respiratory chain enzymes. The production of O 2 Ϫ· via NADH oxidase was greater (PϽ0.05) in the renal cortex and outer medulla (OM) than in the papilla. The mitochondrial enzyme activity for O 2 Ϫ· production was higher (PϽ0.05) in the OM than in the cortex and papilla. Compared with NADH oxidase and mitochondrial enzymes, xanthine oxidase and NADPH oxidase produced much less O 2 Ϫ· in the kidney under this condition. Overall, the renal OM exhibited the greatest enzyme activities for O 2 Ϫ· production. In anesthetized rats, renal medullary interstitial infusion of a superoxide dismutase inhibitor, diethyldithiocarbamate, markedly decreased renal MBF and sodium excretion. Diethyldithiocarbamate (5 mg/kg per minute by renal medullary interstitial infusion [RI]) reduced the renal medullary laser-Doppler flow signal from 0.6Ϯ0.04 to 0.4Ϯ0.03 V, a reduction of 33%, and both urine flow and sodium excretion decreased by 49%. In contrast, a membrane-permeable superoxide dismutase mimetic, 4-hydroxytetramethyl-piperidine-1-oxyl (TEMPOL, 30 mol/kg per minute RI) increased MBF and sodium excretion by 34% and 69%, respectively. These effects of TEMPOL on renal MBF and sodium excretion were not altered by pretreatment with N G -nitro-L-arginine methyl ester (10 g/kg per minute RI). We conclude that (1) Key Words: free radicals Ⅲ oxygen Ⅲ hemodynamics, renal Ⅲ kidney I n contrast to the conventional idea that reactive oxygen species (ROS) are of only pathological consequence, recent studies have indicated that under physiological conditions, low concentrations of ROS play an important role in the normal regulation of cell and organ function. 1-4 Redoxmediated signaling is emerging as a fundamental regulatory mechanism in cell biology and physiology. 2,4 In this regard, ROS have been reported to participate in the control of vascular tone, and the interaction of superoxide (O 2 Ϫ· ) and NO has been considered as one of the important mechanisms regulating cardiovascular function. 1,2,4,5 It has been demonstrated that O 2 Ϫ· inactivates the endothelium-dependent relaxing factor, thereby reducing the arteriolar dilation to acetylcholine or other endothelium-dependent vasodilators 6 and that endothelial superoxide dismutase (SOD) activity significantly increased the half-life of the endothelium-dependent relaxing factor produced by acetylcholine. 5 Recent studies have provided direct evidence that inactivation of SOD activity with diethyldithiocarbamate (DETC) selectively in...

Studies have highlighted the importance of histone deacetylase (HDAC)-mediated epigenetic processes in the development of diabetic complications. Inhibitors of HDAC are a novel class of therapeutic agents in diabetic nephropathy, but currently available inhibitors are mostly nonselective inhibit multiple HDACs, and different HDACs serve very distinct functions. Therefore, it is essential to determine the role of individual HDACs in diabetic nephropathy and develop HDAC inhibitors with improved specificity. First, we identified the expression patterns of HDACs and found that, among zinc-dependent HDACs, HDAC2/4/5 were upregulated in the kidney from streptozotocin-induced diabetic rats, diabetic db/db mice, and in kidney biopsies from diabetic patients. Podocytes treated with high glucose, advanced glycation end products, or transforming growth factor-β (common detrimental factors in diabetic nephropathy) selectively increased HDAC4 expression. The role of HDAC4 was evaluated by in vivo gene silencing by intrarenal lentiviral gene delivery and found to reduce renal injury in diabetic rats. Podocyte injury was associated with suppressing autophagy and exacerbating inflammation by HDAC4-STAT1 signaling in vitro. Thus, HDAC4 contributes to podocyte injury and is one of critical components of a signal transduction pathway that links renal injury to autophagy in diabetic nephropathy.

Podocyte injury is a major determinant of proteinuric kidney disease and the identification of potential therapeutic targets for preventing podocyte injury has clinical importance. Here, we show that histone deacetylase Sirt6 protects against podocyte injury through epigenetic regulation of Notch signaling. Sirt6 is downregulated in renal biopsies from patients with podocytopathies and its expression correlates with glomerular filtration rate. Podocyte-specific deletion of Sirt6 exacerbates podocyte injury and proteinuria in two independent mouse models, diabetic nephropathy, and adriamycin-induced nephropathy. Sirt6 has pleiotropic protective actions in podocytes, including anti-inflammatory and anti-apoptotic effects, is involved in actin cytoskeleton maintenance and promotes autophagy. Sirt6 also reduces urokinase plasminogen activator receptor expression, which is a key factor for podocyte foot process effacement and proteinuria. Mechanistically, Sirt6 inhibits Notch1 and Notch4 transcription by deacetylating histone H3K9. We propose Sirt6 as a potential therapeutic target for the treatment of proteinuric kidney disease.

Hydrogen sulfide (H 2 S), a novel endogenous gaseous bioactive substance, has recently been implicated in the regulation of cardiovascular and neuronal functions. However, its role in the control of renal function is unknown. In the present study, incubation of renal tissue homogenates with L-cysteine (L-Cys) (as a substrate) produced H 2 S in a concentration-dependent manner. This H 2 S production was completely abolished by inhibition of both cystathionine ␤-synthetase (CBS) and cystathionine ␥-lyase (CGL), two major enzymes for the production of H 2 S, using amino-oxyacetic acid (AOAA), an inhibitor of CBS, and propargylglycine (PPG), an inhibitor of CGL. However, inhibition of CBS or CGL alone induced a small decrease in H 2 S production. In anesthetized Sprague-Dawley rats, intrarenal arterial infusion of an H 2 S donor (NaHS) increased renal blood flow, glomerular filtration rate (GFR), urinary sodium (U Na ⅐V), and potassium (U K ⅐V) excretion. Consistently, infusion of both AOAA and PPG to inhibit the endogenous H 2 S production decreased GFR, U Na ⅐V, and U K ⅐V, and either one of these inhibitors alone had no significant effect on renal functions. Infusion of L-Cys into renal artery to increase the endogenous H 2 S production also increased GFR, U Na ⅐V, and U K ⅐V, which was blocked by AOAA plus PPG. It was shown that H 2 S had both vascular and tubular effects and that the tubular effect of H 2 S might be through inhibition of Na ϩ /K ϩ /2Cl Ϫ cotransporter and Na ϩ /K ϩ /ATPase activity. These results suggest that H 2 S participates in the control of renal function and increases urinary sodium excretion via both vascular and tubular actions in the kidney.

ANG converting enzyme (ACE) 2 (ACE2) is a homologue of ACE, which is not blocked by conventional ACE inhibitors. ACE2 converts ANG 1-10 (ANG I) to ANG 1-9, which can be hydrolyzed by ACE to form the biologically active peptide ANG 1-7. ACE2 is expressed in the kidney, but its precise intrarenal localization is unclear, and the role of intrarenal ACE2 in the production of ANG 1-7 is unknown. The present studies determined the relative distribution of ACE2 in the rat kidney and defined its role in the generation of ANG 1-7 in proximal tubule. In microdissected rat nephron segments, semiquantitative RT-PCR revealed that ACE2 mRNA was widely expressed, with relatively high levels in proximal straight tubule (PST). Immunohistochemistry demonstrated ACE2 protein in tubular segments, glomeruli, and endothelial cells. Utilizing mass spectrometry, incubation of isolated PSTs with ANG I (10(-6) M) led to generation of ANG 1-7 (sensitivity of detection > 1 x 10(-9) M), accompanied by the formation of ANG 1-8 (ANG II) and ANG 1-9. The ACE2 inhibitor DX600 completely blocked ANG I-mediated generation of ANG 1-7. Incubation of PSTs with ANG 1-9 also led to generation of ANG 1-7, an effect blocked by the ACE inhibitor captopril or enalaprilat, but not by DX600. Incubation of PSTs with ANG II or luminal perfusion of ANG II did not result in detection of ANG 1-7. The results indicate that ACE2 is widely expressed in rat nephron segments and contributes to the production of ANG 1-7 from ANG I in PST. ANG II may not be a major substrate for ACE2 in isolated PST. The data suggest that ACE2-mediated production of ANG 1-7 represents an important component of the proximal tubular renin-ANG system.