Tauroursodeoxycholic acid (TUDCA) abolishes chronic high salt-induced renal injury and inflammation.

AIM: Chronic high salt intake exaggerates renal injury and inflammation, especially with the loss of functional ETB receptors. Tauroursodeoxycholic acid (TUDCA) is a chemical chaperone and bile salt that is approved for the treatment of hepatic diseases. Our aim was to determine whether TUDCA is reno-protective in a model of ETB receptor deficiency with chronic high salt-induced renal injury and inflammation. METHODS: ETB -deficient and transgenic control rats were placed on normal (0.8% NaCl) or high salt (8% NaCl) diet for 3 weeks, receiving TUDCA (400 mg/kg/d; ip) or vehicle. Histological and biochemical markers of kidney injury, renal cell death and renal inflammation were assessed. RESULTS: In ETB -deficient rats, high salt diet significantly increased glomerular and proximal tubular histological injury, proteinuria, albuminuria, excretion of tubular injury markers KIM-1 and NGAL, renal cortical cell death and renal CD4+ T cell numbers. TUDCA treatment increased proximal tubule megalin expression as well as prevented high salt diet-induced glomerular and tubular damage in ETB -deficient rats, as indicated by reduced kidney injury markers, decreased glomerular permeability and proximal tubule brush border restoration, as well as reduced renal inflammation. However, TUDCA had no significant effect on blood pressure. CONCLUSIONS: TUDCA protects against the development of glomerular and proximal tubular damage, decreases renal cell death and inflammation in the renal cortex in rats with ETB receptor dysfunction on a chronic high salt diet. These results highlight the potential use of TUDCA as a preventive tool against chronic high salt induced renal damage.

Renoprotective impact of estrogen receptor-α and its splice variants in female mice with type 1 diabetes.

Estrogen has been implicated in the regulation of growth and immune function in the kidney, which expresses the full-length estrogen receptor-α (ERα66), its ERα splice variants, and estrogen receptor-ß (ERß). Thus, we hypothesized that these splice variants may inhibit the glomerular enlargement that occurs early in type 1 diabetes (T1D). T1D was induced by streptozotocin (STZ) injection in 8- to 12-wk-old female mice lacking ERα66 (ERα66KO) or all ERα variants (αERKO), and their wild-type (WT) littermates. Basal renal ERα36 protein expression was reduced in the ERα66KO model and was downregulated by T1D in WT mice. T1D did not alter ERα46 or ERß in WT-STZ; however, ERα46 was decreased modestly in ERα66KO mice. Renal hypertrophy was evident in all diabetic mice. F4/80-positive immunostaining was reduced in ERα66KO compared with WT and αERKO mice but was higher in STZ than in Control mice across all genotypes. Glomerular area was greater in WT and αERKO than in ERα66KO mice, with T1D-induced glomerular enlargement apparent in WT-STZ and αERKO-STZ, but not in ERα66KO-STZ mice. Proteinuria and hyperfiltration were evident in ERα66KO-STZ and αERKO-STZ, but not in WT-STZ mice. These data indicate that ERα splice variants may exert an inhibitory influence on glomerular enlargement and macrophage infiltration during T1D; however, effects of splice variants are masked in the presence of the full-length ERα66, suggesting that ERα66 acts in opposition to its splice variants to influence these parameters. In contrast, hyperfiltration and proteinuria in T1D are attenuated via an ERα66-dependent mechanism that is unaffected by splice variant status.

Endothelin receptor-specific control of endoplasmic reticulum stress and apoptosis in the kidney.

Endothelin-1 (ET-1) promotes renal damage during cardiovascular disease; yet, the molecular mechanisms involved remain unknown. Endoplasmic reticulum (ER) stress, triggered by unfolded protein accumulation in the ER, contributes to apoptosis and organ injury. These studies aimed to determine whether the ET-1 system promotes renal ER stress development in response to tunicamycin. ETB deficient (ETB def) or transgenic control (TG-con) rats were used in the presence or absence of ETA receptor antagonism. Tunicamycin treatment similarly increased cortical ER stress markers in both rat genotypes; however, only ETB def rats showed a 14-24 fold increase from baseline for medullary GRP78, sXBP-1, and CHOP. Pre-treatment of TG-con rats with the ETA blocker ABT-627 for 1 week prior to tunicamycin injection significantly reduced the ER stress response in cortex and medulla, and also inhibited renal apoptosis. Pre-treatment with ABT-627 failed to decrease renal ER stress and apoptosis in ETB def rats. In conclusion, the ET-1 system is important for the development of tunicamycin-induced renal ER stress and apoptosis. ETA receptor activation induces renal ER stress genes and apoptosis, while functional activation of the ETB receptor has protective effects. These results highlight targeting the ETA receptor as a therapeutic approach against ER stress-induced kidney injury.

Free radical scavenging decreases endothelin-1 excretion and glomerular albumin permeability during type 1 diabetes.

Increased renal endothelin-1 (ET-1) production and an ETA receptor-dependent increase in glomerular albumin permeability (Palb) accompany type 1 diabetes mellitus (T1D). We hypothesized that T1D-induced oxidative stress contributes to renal ET-1 production and glomerular Palb Male rats with streptozotocin-induced T1D were provided free access to drinking water without additives (T1D rats) or containing the free radical scavenger tempol (1 mmol/L; T1D+Tempol). After 3 weeks, T1D+Tempol rats displayed lower urinary excretion of thiobarbituric acid reactive substances and glomerular superoxide production (dihydroethidium staining) compared to T1D rats. Urinary ET-1 excretion and inner medullary (but not cortical or outer medullary) prepro-ET-1 mRNA expression were lower in the T1D+Tempol group than in the T1D group. Palb, measured as the change in volume of isolated glomeruli upon exposure to oncotic gradients of albumin, was significantly lower in the T1D+Tempol group than in the T1D group. Tempol treatment did not alter protein excretion or creatinine clearance. These data support the postulate that oxidative stress contributes to glomerular Palb and renal ET-1 production during the early phase of type 1 diabetes.

Mechanisms of renal microvascular dysfunction in type 1 diabetes: potential contribution to end organ damage.

The mechanisms underlying initiation and progression of diabetic nephropathy are not well understood, despite the fact that diabetes represents the chief underlying cause of end-stage renal disease. The onset of diabetic hyperglycemia is now known to evoke functional alterations in the renal microvasculature, glomeruli and tubular epithelium. Although the scope of these effects is not yet fully recognized, the renal vascular dysfunction evident early after onset of T1D likely encompasses impaired electromechanical coupling in preglomerular vascular smooth muscle and altered interactions between tubular transport and vascular function. These changes, which arise in environment conducive to oxidative stress and inflammation, are thought to either initiate or facilitate the eventual development of diabetic nephropathy in susceptible individuals.

Classical estrogen receptors and ERα splice variants in the mouse.

Estrogens exert a variety of effects in both reproductive and non-reproductive tissues. With the discovery of ERα splice variants, prior assumptions concerning tissue-specific estrogen signaling need to be re-evaluated. Accordingly, we sought to determine the expression of the classical estrogen receptors and ERα splice variants across reproductive and non-reproductive tissues of male and female mice. Western blotting revealed that the full-length ERα66 was mainly present in female reproductive tissues but was also found in non-reproductive tissues at lower levels. ERα46 was most highly expressed in the heart of both sexes. ERα36 was highly expressed in the kidneys and liver of female mice but not in the kidneys of males. ERß was most abundant in non-reproductive tissues and in the ovaries. Because the kidney has been reported to be the most estrogenic non-reproductive organ, we sought to elucidate ER renal expression and localization. Immunofluorescence studies revealed ERα66 in the vasculature and the glomerulus. It was also found in the brush border of the proximal tubule and in the cortical collecting duct of female mice. ERα36 was evident in mesangial cells and tubular epithelial cells of both sexes, as well as podocytes of females but not males. ERß was found primarily in the podocytes in female mice but was also present in the mesangial cells in both sexes. Within the renal cortex, ERα46 and ERα36 were mainly located in the membrane fraction although they were also present in the cytosolic fraction. Given the variability of expression patterns demonstrated herein, identification of the specific estrogen receptors expressed in a tissue is necessary for interpreting estrogenic effects. As this study revealed expression of the ERα splice variants at multiple sites within the kidney, further studies are warranted in order to elucidate the contribution of these receptors to renal estrogen responsiveness.

Angiotensin-converting enzyme inhibition curbs tyrosine nitration of mitochondrial proteins in the renal cortex during the early stage of diabetes mellitus in rats.

Experiments were performed to evaluate the hypothesis that ACE (angiotensin-converting enzyme) inhibition (enalapril) suppresses 3-NT (3-nitrotyrosine) production in the renal cortex during the early stage of Type 1 DM (diabetes mellitus) in the rat. Enalapril was administered chronically for 2 weeks to subsets of STZ (streptozotocin)-induced DM and vehicle-treated sham rats. O(2)(-) (superoxide anion) and NO(x) (nitrate+nitrite) levels were measured in the media bathing renal cortical slices after 90 min incubation in vitro. SOD (superoxide dismutase) activity and 3-NT content were measured in the renal cortex homogenate. Renal cortical nitrated protein was identified by proteomic analysis. Renal cortical production of O(2)(-) and 3-NT was increased in DM rats; however, enalapril suppressed these changes. DM rats also exhibited elevated renal cortical NO(x) production and SOD activity, and these changes were magnified by enalapril treatment. 2-DE (two-dimensional gel electrophoresis)-based Western blotting revealed more than 20 spots with positive 3-NT immunoreactivity in the renal cortex of DM rats. Enalapril treatment blunted the DM-induced increase in tyrosine nitration of three proteins ACO2, GDH1 and MMSDH (aconitase 2, glutamate dehydrogenase 1 and methylmalonate-semialdehyde dehydrogenase), each of which resides in mitochondria. These data are consistent with enalapril preventing DM-induced tyrosine nitration of mitochondrial proteins by a mechanism involving suppression of oxidant production and enhancement of antioxidant capacity, including SOD activation.

Tempol prevents altered K(+) channel regulation of afferent arteriolar tone in diabetic rat kidney.

Experiments were performed to test the hypothesis that oxidative stress underlies the enhanced tonic dilator impact of inward-rectifier K(+) channels on renal afferent arterioles of rats with streptozotocin-induced diabetes mellitus. Sham and diabetic rats were left untreated or provided Tempol in their drinking water for 26±1 days, after which afferent arteriolar lumen diameter and its responsiveness to K(+) channel blockade were measured using the in vitro blood-perfused juxtamedullary nephron technique. Afferent diameter averaged 19.4±0.8 µm in sham rats and 24.4±0.8 µm in diabetic rats (P<0.05). The decrease in diameter evoked by Ba(2+) (inward-rectifier K(+) channel blocker) was 3 times greater in diabetic rats than in sham rats. Glibenclamide (K(ATP) channel blocker) and tertiapin-Q (Kir1.1/Kir3.x channel blocker) decreased afferent diameter in diabetic rats but had no effect on arterioles from sham rats. Chronic Tempol treatment prevented diabetes mellitus-induced increases in both renal vascular dihydroethidium staining and baseline afferent arteriolar diameter. Moreover, Tempol prevented the exaggeration of afferent arteriolar responses to Ba(2+), tertiapin-Q, and glibenclamide otherwise evident in diabetic rats. Preglomerular microvascular smooth muscle cells expressed mRNA encoding Kir1.1, Kir2.1, and Kir6.1. Neither diabetes mellitus nor Tempol altered Kir1.1, Kir2.1, Kir6.1, or SUR2B protein levels in renal cortical microvessels. To the extent that the effects of Tempol reflect its antioxidant actions, our observations indicate that oxidative stress contributes to the exaggerated impact of Kir1.1, Kir2.1, and K(ATP) channels on afferent arteriolar tone during diabetes mellitus and that this phenomenon involves posttranslational modulation of channel function.

NADPH oxidase and PKC contribute to increased Na transport by the thick ascending limb during type 1 diabetes.

Type 1 diabetes triggers protein kinase C (PKC)-dependent NADPH oxidase activation in the renal medullary thick ascending limb (mTAL), resulting in accelerated superoxide production. As acute exposure to superoxide stimulates NaCl transport by the mTAL, we hypothesized that diabetes increases mTAL Na(+) transport through PKC-dependent and NADPH oxidase-dependent mechanisms. An O(2)-sensitive fluoroprobe was used to measure O(2) consumption by mTALs from rats with streptozotocin-induced diabetes and sham rats. In sham mTALs, total O(2) consumption was evident as a 0.34±0.03 U change in normalized relative fluorescence (ΔNRF)/min per mg protein. Ouabain (2 mmol/L) reduced O(2) consumption by 69±4% and 500 µmol/L furosemide reduced O(2) consumption by 58±8%. Total O(2) consumption was accelerated in mTAL from diabetic rats (0.74±0.07 ΔNRF/min/mg protein; P<0.05 versus sham), reflecting increases in ouabain- and furosemide-sensitive O(2) consumption. NADPH oxidase inhibition (100 µmol/L apocynin) reduced furosemide-sensitive O(2) consumption by mTAL from diabetic rats to values not different from sham. The PKC inhibitor calphostin C (1 µmol/L) or the PKCα/ß inhibitor Gö6976 (1 µmol/L) decreased furosemide-sensitive O(2) consumption in both groups, achieving values that did not differ between sham and diabetic. PKCß inhibition had no effect in either group. Similar inhibitory patterns were evident with regard to ouabain-sensitive O(2) consumption. We conclude that NADPH oxidase and PKC (primarily PKCα) contribute to an increase in O(2) consumption by the mTAL during type 1 diabetes through effects on the ouabain-sensitive Na(+)-K(+)-ATPase and furosemide-sensitive Na(+)-K(+)-2Cl(-) cotransporter that are primarily responsible for active transport Na(+) reabsorption by this nephron segment.

The renal vascular response to diabetes.

PURPOSE OF REVIEW: Diabetes mellitus is the primary cause of end-stage renal disease, yet the mechanisms underlying diabetic nephropathy remain ill-defined. The widely accepted opinion holds that events occurring early during the course of diabetes engender the eventual decline in renal function. This review will summarize recent advances (published January 2008 through June 2009) regarding the renal vascular and glomerular functional changes that occur during the early stage of diabetes. RECENT FINDINGS: Reduced C-peptide levels and increased cyclooxygenase-2 activity both seem to promote diabetic hyperfiltration, presumably via effects on afferent arteriolar tone. In addition, exaggerated tonic influences of K+ channels on afferent arteriolar function likely act in concert with impaired Ca2+ influx responses to changes in membrane potential to promote vasodilation. Mechanisms underlying these changes remain largely speculative. Diabetes may also alter autoregulation of renal blood flow and glomerular filtration rate, as well as provoke afferent arteriolar dilation secondary to alterations in proximal tubular reabsorption; however, conflicting evidence continues to flood the literature concerning these events. SUMMARY: New evidence has expanded our appreciation of the complexity of events that promote preglomerular vasodilation during the early stage of diabetes; however, it seems that the more we know, the less we understand.

Protein kinase C-dependent NAD(P)H oxidase activation induced by type 1 diabetes in renal medullary thick ascending limb.

Type 1 diabetes provokes a protein kinase C (PKC)-dependent accumulation of superoxide anion in the renal medullary thick ascending limb (mTAL). We hypothesized that this phenomenon involves PKC-dependent NAD(P)H oxidase activation. The validity of this hypothesis was explored using mTAL suspensions prepared from rats with streptozotocin-induced diabetes and from sham (vehicle-treated) rats. Superoxide production was 5-fold higher in mTAL suspensions from diabetic rats compared with suspensions from sham rats. The NAD(P)H oxidase inhibitor apocynin caused an 80% decrease in superoxide production by mTAL from diabetic rats (P<0.05 vs untreated) without altering superoxide production by sham mTAL. NAD(P)H oxidase activity was >2-fold higher in mTAL from diabetic rats than in sham mTAL (P<0.05). Pretreatment with calphostin C (broad-spectrum PKC inhibitor) or rottlerin (PKCdelta inhibitor) reduced NAD(P)H oxidase activity by approximately 80% in both groups; however, PKCalpha/beta or PKCbeta inhibition did not alter NAD(P)H oxidase activity in either group. Protein levels of Nox2, Nox4, and p47phox were significantly higher in diabetic mTAL than in mTAL from sham rats. In summary, elevated superoxide production by mTAL from diabetic rats was normalized by NAD(P)H oxidase inhibition. PKC-dependent, PKCdelta-dependent, and total NAD(P)H oxidase activity was greater in mTAL from diabetic rats compared with sham. Protein levels of Nox2, Nox4, and p47phox were increased in mTAL from diabetic rats. We conclude that increased superoxide production by the mTAL during diabetes involves a PKCdelta-dependent increase in NAD(P)H oxidase activity in concert with increased protein levels of catalytic and regulatory subunits of the enzyme.

PKC-dependent superoxide production by the renal medullary thick ascending limb from diabetic rats.

Type 1 diabetes (T1D) is a state of oxidative stress accompanied by PKC activation in many tissues. The primary site of O2*- production by the normal rat kidney is the medullary thick ascending limb (mTAL). We hypothesized that T1D increases O2*- production by the mTAL through a PKC-dependent mechanism involving increased expression and translocation of one or more PKC isoforms. mTAL suspensions were prepared from rats with streptozotocin-induced T1D (STZ mTALs) and from normal or sham rats (normal/sham mTALs). O2*- production by STZ mTALs was fivefold higher than normal/sham mTALs (P < 0.05). PMA (30 min) mimicked the effect of T1D on O2*- production. Exposure to calphostin C or chelerythrine (PKC inhibitors), Gö6976 (PKCalpha/beta inhibitor), or rottlerin (PKCdelta inhibitor) decreased O2*- production to <20% of untreated baseline in both normal/sham and STZ mTALs. PKCbeta inhibitors had no effect. PKC activity was increased in STZ mTALs (P < 0.05 vs. normal/sham mTALs) and was unaltered by antioxidant exposure (tempol). PKCalpha protein levels were increased by 70% in STZ mTALs, with a approximately 30% increase in the fraction associated with the membrane (both P < 0.05 vs. sham). PKCbeta protein levels were elevated by 29% in STZ mTALs (P < 0.05 vs. sham) with no change in the membrane-bound fraction. Neither PKCdelta protein levels nor its membrane-bound fraction differed between groups. Thus STZ mTALs display PKC activation, upregulation of PKCalpha and PKCbeta protein levels, increased PKCalpha translocation to the membrane, and accelerated O2*- production that is eradicated by inhibition of PKCalpha or PKCdelta (but not PKCbeta). We conclude that increased PKCalpha expression and activity are primarily responsible for PKC-dependent O2*- production by the mTAL during T1D.

PP2B-dependent NO production in the medullary thick ascending limb during diabetes.

Calcineurin (PP2B) has recently been shown to be upregulated in the medullary thick ascending limb (mTAL) during diabetes. The mTAL expresses all three isoforms of nitric oxide synthase (NOS), which are subject to phosphoregulation and represent substrates for PP2B. Therefore, we hypothesized that diabetes induces PP2B-dependent upregulation of NOS activity and NO production in the mTAL. Three weeks after injection of streptozotocin (STZ rats) or vehicle (sham rats), mTAL suspensions were prepared for use in functional and biochemical assays. PP2B activity and expression were increased in mTALs from STZ rats compared with sham. Nitrite production was significantly reduced in mTALs from STZ rats compared with sham. However, incubation with the free radical scavenger, tempol, unmasked a significant increase in nitrite production by mTALs from STZ rats. Inhibition of PP2B attenuated the increase in nitrite production and NOS activity evident in mTALs from STZ rats. Analysis of specific NOS isoform activity revealed increased NOS1 and NOS2 activities in mTALs from STZ rats. All three NOS isoform activities were regulated in a PP2B-dependent manner. Western blot analysis detected no differences in NOS isoform expression, although phosphorylation of pThr(495)-NOS3 was significantly reduced in mTALs from STZ rats. Phosphorylation of pSer(852)-NOS1, pSer(633)-NOS3, and pSer(1177)-NOS3 was similar in mTALs from STZ and sham rats. Inhibition of PP2B did not alter the phosphorylation of NOS1 or NOS3 at known sites. In conclusion, while NO bioavailability in mTALs is reduced during diabetes, free radical scavenging with tempol unmasks increased NO production that involves PP2B-dependent activation of NOS1 and NOS2.

Potassium channel contributions to afferent arteriolar tone in normal and diabetic rat kidney.

We previously reported an enhanced tonic dilator impact of ATP-sensitive K+ channels in afferent arterioles of rats with streptozotocin (STZ)-induced diabetes. The present study explored the hypothesis that other types of K+ channel also contribute to afferent arteriolar dilation in STZ rats. The in vitro blood-perfused juxtamedullary nephron technique was utilized to quantify afferent arteriolar lumen diameter responses to K+ channel blockers: 0.1-3.0 mM 4-aminopyridine (4-AP; KV channels), 10-100 microM barium (KIR channels), 1-100 nM tertiapin-Q (TPQ; Kir1.1 and Kir3.x subfamilies of KIR channels), 100 nM apamin (SKCa channels), and 1 mM tetraethylammonium (TEA; BKCa channels). In kidneys from normal rats, 4-AP, TEA, and Ba2+ reduced afferent diameter by 23 +/- 3, 8 +/- 4, and 18 +/- 2%, respectively, at the highest concentrations employed. Neither TPQ nor apamin significantly altered afferent diameter. In arterioles from STZ rats, a constrictor response to TPQ (22 +/- 4% decrease in diameter) emerged, and the response to Ba2+ was exaggerated (28 +/- 5% decrease in diameter). Responses to the other K+ channel blockers were similar to those observed in normal rats. Moreover, exposure to either TPQ or Ba2+ reversed the afferent arteriolar dilation characteristic of STZ rats. Acute surgical papillectomy did not alter the response to TPQ in arterioles from normal or STZ rats. We conclude that 1) KV, KIR, and BKCa channels tonically influence normal afferent arteriolar tone, 2) KIR channels (including Kir1.1 and/or Kir3.x) contribute to the afferent arteriolar dilation during diabetes, and 3) the dilator impact of Kir1.1/Kir3.x channels during diabetes is independent of solute delivery to the macula densa.

Prepubertal onset of diabetes prevents expression of renal cortical connective tissue growth factor.

Puberty unmasks or accelerates the nephropathy of diabetes mellitus (DM). We performed focused microarray analysis to test the hypothesis that one or more genes in the transforming growth factor beta (TGF-beta) signaling system would be differentially regulated in male rats depending on their age at onset of DM. Littermates were started on the 6-week protocol at 4 weeks or 14 weeks of age. Renal cortical RNA was isolated and analyzed using gene chips with more than 30,000 transcripts. Age-specific effects of DM were demonstrated for 1,760 transcripts. Analysis then focused on 89 genes involved in the TGF-beta signaling pathway. Three of these genes showed age-dependent responses to DM, confirmed by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Connective tissue growth factor (CTGF) mRNA and protein were both increased approximately 30% in the renal cortex 6 weeks after adult-onset DM, with no alteration in either parameter after juvenile onset. Follistatin and avian myelocytomatosis viral oncogene homolog mRNA both showed a similar age-related pattern of response to DM, but protein levels did not parallel mRNA for either of these gene products. Given the known roles of CTGF in progressive nephropathies, it is an attractive candidate to explain pubertal acceleration or unmasking of the kidney disease of diabetes.

Testosterone treatment promotes tubular damage in experimental diabetes in prepubertal rats.

Puberty unmasks or accelerates progressive kidney diseases, including diabetes mellitus (DM), perhaps through effects of sex steroids. To test the hypothesis that rising androgen levels at puberty permit diabetic kidney damage, we studied four groups of male rats with and without streptozocin-induced DM: adult onset (A), adult onset after castration (AC), juvenile onset (J), and juvenile onset with testosterone treatment (JT). Profibrotic markers were measured after 6 wk with blood glucose levels 300-450 mg/dl. JT permitted increased expression of mRNA for two isoforms of transforming growth factor-beta and connective tissue growth factor compared with J animals with DM; prior castration did not provide protection in adult-onset DM. JT also permitted greater tubular staining for alpha-smooth muscle actin and fibroblast-specific protein, two markers of cell damage and potential epithelial mesenchymal transition. Once again, castration was not protective for these effects of DM in the AC group. These data indicate that puberty permits detrimental effects in the tubulointerstitium in the diabetic kidney, an effect mimicked by testosterone treatment of juvenile animals and partially blunted by castration of adults, but damage does not correlate with testosterone levels, suggesting a less direct mechanism.

Endothelin A receptor blockade reduces diabetic renal injury via an anti-inflammatory mechanism.

Endothelin (ET) receptor blockade delays the progression of diabetic nephropathy; however, the mechanism of this protection is unknown. Therefore, the aim of this study was to test the hypothesis that ET(A) receptor blockade attenuates superoxide production and inflammation in the kidney of diabetic rats. Diabetes was induced by streptozotocin (diabetic rats with partial insulin replacement to maintain modest hyperglycemia [HG]), and sham rats received vehicle treatments. Some rats also received the ETA antagonist ABT-627 (sham+ABT and HG+ABT; 5 mg/kg per d; n = 8 to 10/group). During the 10-wk study, urinary microalbumin was increased in HG rats, and this effect was prevented by ET(A) receptor blockade. Indices of oxidative stress, urinary excretion of thiobarbituric acid reactive substances, 8-hydroxy--deoxyguanosine, and H2O2 and plasma thiobarbituric acid reactive substances were significantly greater in HG rats than in sham rats. These effects were not prevented by ABT-627. In addition, renal cortical expression of 8-hydroxy--deoxyguanosine and NADPH oxidase subunits was not different between HG and HG+ABT rats. ETA receptor blockade attenuated increases in macrophage infiltration and urinary excretion of TGF-beta and prostaglandin E2 metabolites in HG rats. Although ABT-627 did not alleviate oxidative stress in HG rats, inflammation and production of inflammatory mediators were reduced in association with prevention of microalbuminuria. These observations indicate that ETA receptor activation mediates renal inflammation and TGF-beta production in diabetes and are consistent with the postulate that ETA blockade slows progression of diabetic nephropathy via an anti-inflammatory mechanism.

Impact of angiotensin-converting enzyme inhibition on renal cortical nitrotyrosine content during increased extracellular glucose concentration.

OBJECTIVES: Experiments evaluated the hypothesis that angiotensin-converting enzyme (ACE) inhibition suppresses hyperglycemia-induced nitrotyrosine (NT) production in the renal cortex. DESIGN AND METHODS: Rats were untreated (UNTR, n = 6) or received the ACE inhibitor enalapril (20 mg/kg/day; ENAL, n = 6) for 2 weeks. Renal cortical slices were incubated for 90 min in media containing 5 (normal) or 20 mmol/L (high) glucose. Superoxide anion (O2*-) and nitrate + nitrite (NO(X)) levels were measured in the media. Superoxide dismutase (SOD) activity and NT content were measured in the tissue homogenate. RESULTS: In the UNTR group, high glucose increased O2*- and NO(X) production by the renal cortex (P < 0.05 vs. normal glucose). Likewise, NT content and SOD activity of the renal cortex augmented (P < 0.05 vs. normal glucose). In the ENAL group, O2*- production and NT content were glucose-insensitive, but high glucose exerted an exaggerated impact on NO(X) production and SOD activity (P < 0.01 vs. UNTR in high glucose). CONCLUSION: Accelerated NT content in the renal cortex during high-glucose conditions was prevented by ACE inhibitor treatment. It was suggested that, apart from its anti-hypertensive effect, the mechanism of suppressed NT degradation in the renal cortex by the ACE inhibitor enhances both O2*- degradation per se and antioxidative effects including SOD activation.