Podocyte-specific overexpression of the antioxidant metallothionein reduces diabetic nephropathy

Podocytes are critical components of the selective filtration barrier of the glomerulus and are susceptible to oxidative damage. For investigation of the role of oxidative stress and podocyte damage in diabetic nephropathy, transgenic mice that overexpress the antioxidant protein metallothionein (MT) specifically in podocytes (Nmt mice) were produced. MT expression was increased six- and 18-fold in glomeruli of two independent lines of Nmt mice, and podocyte-specific overexpression was confirmed. Glomerular morphology and urinary albumin excretion were normal in Nmt mice. OVE26 transgenic mice, a previously reported model of diabetic nephropathy, were crossed with Nmt mice to determine whether an antioxidant transgene targeted to podocytes could reduce diabetic nephropathy. Double-transgenic OVE26Nmt mice developed diabetes similar to OVE26 mice, but MT overexpression reduced podocyte damage, indicated by more podocytes, less glomerular cell death, and higher density of podocyte foot processes. In addition, expansion of glomerular and mesangial volume were significantly less in OVE26Nmt mice compared with OVE26 mice. Four-month-old OVE26Nmt mice had a 70 to 90% reduction in 24-h albumin excretion, but this protection does not seem to be permanent. These results provide evidence for the role of oxidative damage to the podocyte in diabetic mice and show that protection of the podocyte can reduce or delay primary features of diabetic nephropathy.     

Catalase deficiency accelerates diabetic renal injury through peroxisomal dysfunction

Mitochondrial reactive oxygen species (ROS) play an important role in diabetes complications, including diabetic nephropathy (DN). Plasma free fatty acids (FFAs) as well as glucose are increased in diabetes, and peroxisomes and mitochondria participate in FFA oxidation in an interconnected fashion. Therefore, we investigated whether deficiency of catalase, a major peroxisomal antioxidant, accelerates DN through peroxisomal dysfunction and abnormal renal FFA metabolism. Diabetes was induced by multiple injections of low-dose streptozotocin into catalase knock-out (CKO) and wild-type (WT) C57BL/6 mice. Murine mesangial cells (MMCs) transfected with catalase small interfering RNA followed by catalase overexpression were used to further elucidate the role of endogenous catalase. Despite equivalent hyperglycemia, parameters of DN, along with markers of oxidative stress, were more accelerated in diabetic CKO mice than in diabetic WT mice up to 10 weeks of diabetes. CKO mice and MMCs showed impaired peroxisomal/mitochondrial biogenesis and FFA oxidation. Catalase deficiency increased mitochondrial ROS and fibronectin expression in response to FFAs, which were effectively restored by catalase overexpression or N-acetylcysteine. These data provide unprecedented evidence that FFA-induced peroxisomal dysfunction exacerbates DN and that endogenous catalase plays an important role in protecting the kidney from diabetic stress through maintaining peroxisomal and mitochondrial fitness.     

Lipid peroxidation and activities of antioxidant enzymes in erythrocytes of patients with non-insulin dependent diabetes with or without diabetic nephropathy

Background: Diabetes is known to involve oxidative stress. However, data on oxidative stress in diabetic nephropathy is scant. The aim of this study was to examine lipid peroxidation and activities of key antioxidant enzymes in non-insulin dependent diabetes (NIDDM) without nephropathy and in those with diabetic nephropathy. Methods: Twenty-one NIDDM outpatients with obvious diabetic nephropathy (persistent proteinuria, over 0.5 g/day and albumin excretion over 200 &mgr;g/min), 14 NIDDM outpatients with a long diabetic history but without hypertension and with normal urinary albumin excretion rate (<20 &mgr;g/min) and 19 healthy persons were studied. Malondialdehyde (MDA) content of erythrocytes was quantified by HPLC according to Young et al. In erythrocytes superoxide dismutase (SOD) activity was determined by adrenaline method of Misra and Fridovich and catalase was estimated according to Beers and Sizer. Results: MDA content was significantly elevated in erythrocytes of NIDDM patients without nephropathy vs the control group and even higher in erythrocytes of NIDDM patients with diabetic nephropathy. SOD and catalase activities were lower in erythrocytes of NIDDM patients without nephropathy than in the control group and lowest in erythrocytes of NIDDM patients with nephropathy. Conclusions: These results confirm oxidative stress in erythrocytes of NIDDM patients with nephropathy. The intensity of oxidative stress appeared to be greater than in NIDDM patients without nephropathy.     

Effects of Aerobic Exercise on Microalbuminuria and Enzymuria in Type 2 Diabetic Patients

Increased urinary albumin excretion is a strong predictor for the development of overt diabetic nephropathy and overall cardiovascular morbidity and mortality in patients with type 2 diabetes. In a previous study, regular aerobic physical activity in overweight/obese patients with type 2 diabetes mellitus was found to have significant beneficial effects on glycemic control, insulin resistance, cardiovascular risk factors, and oxidative stress. The aim of the present study was to investigate the effects of aerobic exercise in the same cohort of type 2 diabetic patients on urinary albumin excretion, serum levels and urinary excretion of enzymes, tubular damage, and metabolic control markers in type 2 diabetic patients. Changes from baseline to 3 and 6 months of aerobic exercise were assessed for urinary albumin excretion, serum activities, and urinary excretion of N-acetyl-β-D-glucosaminidase (NAGA), plasma cell glycoprotein 1 (PC-1) and aminopeptidase N (APN), as well as their association with insulin resistance, cardiovascular risk factors, and oxidative stress parameters in 30 male type 2 diabetic patients (aged 54.8 ± 7.3 years, with a mean BMI of 30.8 ± 3.0 kg/m²). Microalbuminuria was found in six (20%) diabetic patients at baseline, three of them (10%) after three months, and only one patient (3.33%) at the end of the study period. A significant correlation was found for urinary albumin excretion at baseline both with sulfhydryl-groups and catalase, but not for urinary albumin excretion with MDA and glutathione. The prevalence of microalbuminuria tended to decrease after six months of aerobic exercise in type 2 diabetic patients, independently of any improvement in insulin resistance and oxidative stress parameters. Neither between-group nor within-group changes were found for urinary PC-1, APN, and NAGA activity. Serum NAGA was significantly increased (p < 0.05) over the control level in diabetic patients at baseline, but it decreased to the normal level after six months of exercise. This study has shown that a six-month aerobic exercise, without any change in the medication, tended to decrease microalbuminuria without changing enzymuria. However, further studies are needed not only to confirm those findings, but to elucidate potential mechanisms that would clarify the beneficial effects of exercise.     

Effects of aerobic exercise on microalbuminuria and enzymuria in type 2 diabetic patients

Increased urinary albumin excretion is a strong predictor for the development of overt diabetic nephropathy and overall cardiovascular morbidity and mortality in patients with type 2 diabetes. In a previous study, regular aerobic physical activity in overweight/obese patients with type 2 diabetes mellitus was found to have significant beneficial effects on glycemic control, insulin resistance, cardiovascular risk factors, and oxidative stress. The aim of the present study was to investigate the effects of aerobic exercise in the same cohort of type 2 diabetic patients on urinary albumin excretion, serum levels and urinary excretion of enzymes, tubular damage, and metabolic control markers in type 2 diabetic patients. Changes from baseline to 3 and 6 months of aerobic exercise were assessed for urinary albumin excretion, serum activities, and urinary excretion of N-acetyl-beta-D-glucosaminidase (NAGA), plasma cell glycoprotein 1 (PC-1) and aminopeptidase N (APN), as well as their association with insulin resistance, cardiovascular risk factors, and oxidative stress parameters in 30 male type 2 diabetic patients (aged 54.8 +/- 7.3 years, with a mean BMI of 30.8 +/- 3.0 kg/m2). Microalbuminuria was found in six (20%) diabetic patients at baseline, three of them (10%) after three months, and only one patient (3.33%) at the end of the study period. A significant correlation was found for urinary albumin excretion at baseline both with sulfhydryl-groups and catalase, but not for urinary albumin excretion with MDA and glutathione. The prevalence of microalbuminuria tended to decrease after six months of aerobic exercise in type 2 diabetic patients, independently of any improvement in insulin resistance and oxidative stress parameters. Neither between-group nor within-group changes were found for urinary PC-1, APN, and NAGA activity. Serum NAGA was significantly increased (p < 0.05) over the control level in diabetic patients at baseline, but it decreased to the normal level after six months of exercise. This study has shown that a six-month aerobic exercise, without any change in the medication, tended to decrease microalbuminuria without changing enzymuria. However, further studies are needed not only to confirm those findings, but to elucidate potential mechanisms that would clarify the beneficial effects of exercise.     

Oxidative DNA damage and tubulointerstitial injury in diabetic nephropathy

BACKGROUND: Oxidative stress is an important pathogenetic factor in underlying diabetic complications. Recently, 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been reported to serve as a new sensitive biomarker of the oxidative DNA damage in vivo. We studied the relationship between oxidative DNA damage and tubulointerstitial injury in patients with diabetic nephropathy. METHODS: Type 2 diabetic patients (n = 25) and healthy control subjects (n = 20) were studied. The urine concentrations of 8-OHdG were measured by a competitive ELISA. The severity of the glomerular changes was graded using Gellman's criteria, and the severity of the tubulointerstitial lesions was determined by a semiquantitative estimate of the space occupied by the fibrous tissue and/or interstitial infiltrates. RESULTS: The urinary 8-OHdG excretion were significantly higher in the diabetics than in the healthy controls, and tended to increase with severity of the glomerular diffuse lesion, but it was not significant. The urinary 8-OHdG excretion significantly increased with severity of the tubulointerstitial lesion. CONCLUSIONS: Oxidative stress may contribute to the progression of tubulointerstitial injury in patients with diabetic nephropathy.     

Decreased renal α-Klotho expression in early diabetic nephropathy in humans and mice and its possible role in urinary calcium excretion

Hypercalciuria is one of the early manifestations of diabetic nephropathy. We explored here the role of α-Klotho, a protein expressed predominantly in distal convoluted tubules that has a role in calcium reabsorption. We studied 31 patients with early diabetic nephropathy and compared them with 31 patients with IgA nephropathy and 7 with minimal change disease. Renal α-Klotho expression was significantly lower and urinary calcium excretion (UCa/UCr) significantly higher in diabetic nephropathy than in IgA nephropathy or minimal change disease. Multiple regression analyses indicated that α-Klotho mRNA was inversely correlated with calcium excretion. We next measured these parameters in a mouse model of streptozotocin (STZ)-induced diabetic nephropathy, characterized by glomerular hyperfiltration, as seen in early diabetic nephropathy. We also confirmed a reduction of renal α-Klotho mRNA down to almost 50% and enhanced calcium excretion in mice with STZ-induced diabetic nephropathy in comparison with nondiabetic mice. Hypercalciuria was exacerbated in heterozygous α-Klotho knockout mice in comparison with wild-type mice, each with STZ-induced diabetic nephropathy. Thus, α-Klotho expression was decreased in distal convoluted tubules in diabetic nephropathy in humans and mice. Renal loss of α-Klotho may affect urinary calcium excretion in early diabetic nephropathy.     

Protective roles of thioredoxin,a redox-regulating protein,in renal ischemia/reperfusion injury

BACKGROUND: Thioredoxin (TRX) is a small protein with redox-regulating functions. Although TRX is known to be induced in response to various forms of oxidative stress, including ischemia/reperfusion injury, the induction and the specific role of this protein in the kidney have not been fully investigated. METHODS: Renal ischemia/reperfusion was induced by the clipping and release of renal arteries in C57BL/6 and human thioredoxin-overexpressing transgenic (hTRX-Tg) mice. TRX protein was detected by immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). TRX mRNA was detected by in situ hybridization and Northern blotting. Renal functions were evaluated by measuring the levels of blood urea nitrogen and serum creatinine in these mice. RESULTS: With ischemia/reperfusion, endogenous murine TRX was rapidly depleted from the cytosol in the cortical proximal tubuli and detected in the urinary lumen, whereas it was spread diffusely in all segments of the tubular epithelial cells in sham-operated mice. The urinary excretion of TRX increased transiently after ischemia/reperfusion and recovered to the control level in 72 hours. In the medullary thick ascending limb (mTAL), however, TRX was specifically retained in the cytosol. A similar distribution change of transgenic hTRX was observed in the kidney of hTRX-Tg. These hTRX-Tg mice were more resistant to the injury to the mTAL and functional deterioration caused by ischemia/reperfusion, compared with wild-type mice. CONCLUSION: The present findings suggest that TRX is retained in mTAL and secreted from proximal tubuli into urine during renal ischemia/reperfusion. The mTAL-specific retention of TRX may have a protective effect against renal ischemia/reperfusion injury.     

Lack of A1 adenosine receptors augments diabetic hyperfiltration and glomerular injury

Intraglomerular hypertension and glomerular hyperfiltration likely contribute to the pathogenesis of diabetic nephropathy, and tubuloglomerular feedback (TGF) has been suggested to play a role in diabetic hyperfiltration. A1 adenosine receptor (A1AR) null mice lack a TGF response, so this model was used to investigate the contribution of TGF to hyperfiltration in diabetic Ins2(+/-) Akita mice. TGF responses in Ins2(+/-) A1AR(-/-) double mutants were abolished, whereas they were attenuated in Ins2(+/-) mice. GFR, assessed at 14, 24, and 33 wk, was approximately 30% higher in Ins2(+/-) than in wild-type (WT) mice and increased further in Ins2(+/-) A1AR(-/-) mutants (P < 0.01 versus both WT and Ins2(+/-) mice at all ages). Histologic evidence of glomerular injury and urinary albumin excretion were more pronounced in double-mutant than single-mutant or WT mice. In summary, the marked elevation of GFR in diabetic mice that lack a TGF response indicates that TGF is not required to cause hyperfiltration in the Akita model of diabetes. Rather, an A1AR-dependent mechanism, possibly TGF, limits the degree of diabetic hyperfiltration and nephropathy.     

Targeted therapies in diabetic nephropathy: an update

Diabetic nephropathy is the most common cause of end-stage renal disease worldwide. Various pathways in addition to the renin-angiotensinogen system have been implicated in the pathogenesis of diabetic nephropathy. Strategies to interrupt these pathophysiological pathways are a key to the development of new targeted therapies to prevent progression of diabetic nephropathy and are on the horizon. The various pharmacological drugs tried include aliskiren, a direct renin inhibitor blocking the first step in the renin pathway, and pentoxifylline and mammalian target of rapamycin inhibitors, which have antiinflammatory properties and have shown some promising results in management of diabetic nephropathy. Others include endothelin antagonists and vitamin D analogs which have been shown to decrease urinary albumin excretion. Inhibitors of advanced glycation and oxidative stress and plasminogen activator inhibitor-1 have proved useful in animal models of diabetic nephropathy. Others include ruboxistaurin, which blocks protein kinase C overexpression. Such targeted therapies would halt and might even reverse progression of diabetic nephropathy.     

Klotho attenuates renal hypertrophy and glomerular injury in Ins2Akita diabetic mice

Background

Expression of klotho, the renoprotective anti-aging gene, is decreased in diabetic model kidneys. We hypothesized that klotho protein attenuates renal hypertrophy and glomerular injury in a mouse model of diabetic nephropathy.

Methods

Klotho transgenic (KLTG) mice were crossed with spontaneously diabetic Ins2Akita (AKITA) mice. Glomerular morphology, macrophage infiltration, urinary albumin excretion and urinary 8-hydroxy-2-deoxy guanosine excretion were examined. In vitro, human glomerular endothelial cells were stimulated with high glucose with or without recombinant klotho, and calpain activity and proinflammatory cytokine expressions were measured.

Results

We found that klotho protein overexpression attenuates renal hypertrophy and glomerular injury in this mouse model of diabetic nephropathy. Klotho overexpression attenuated renal hypertrophy, albuminuria, glomerular mesangial expansion, and endothelial glycocalyx loss in the AKITA mice. AKITA mice exhibit high levels of urinary 8-hydroxy-2-deoxy guanosine excretion. In the presence of klotho overexpression, this effect was reversed. In addition, the glomerular macrophage infiltration characteristic of AKITA mice was attenuated in KLTG-AKITA mice. In human glomerular endothelial cells, high glucose induced calpain activity. This effect was suppressed by expression of recombinant klotho, which also suppressed the induction of proinflammatory cytokines.

Conclusion

Our data suggest klotho protein protects against diabetic nephropathy through multiple pathways.

     

The thromboxane receptor antagonist S18886 attenuates renal oxidant stress and proteinuria in diabetic apolipoprotein E-deficient mice

Arachidonic acid metabolites, some of which may activate thromboxane A(2) receptors (TPr) and contribute to the development of diabetes complications, including nephropathy, are elevated in diabetes. This study determined the effect of blocking TPr with S18886 or inhibiting cyclooxygenase with aspirin on oxidative stress and the early stages of nephropathy in streptozotocin-induced diabetic apolipoprotein E(-/-) mice. Diabetic mice were treated with S18886 (5 mg . kg(-1) . day(-1)) or aspirin (30 mg . kg(-1) . day(-1)) for 6 weeks. Neither S18886 nor aspirin affected hyperglycemia or hypercholesterolemia. There was intense immunohistochemical staining for nitrotyrosine in diabetic mouse kidney. In addition, a decrease in manganese superoxide dismutase (MnSOD) activity was associated with an increase in MnSOD tyrosine-34 nitration. Tyrosine nitration was significantly reduced by S18886 but not by aspirin. Staining for the NADPH oxidase subunit p47(phox), inducible nitric oxide synthase, and 12-lipoxygenase was increased in diabetic mouse kidney, as were urine levels of 12-hydroxyeicosatetraenoic acid and 8-iso-prostaglandin F(2alpha). S18886 attenuated all of these markers of oxidant stress and inflammation. Furthermore, S18886 significantly attenuated microalbuminuria in diabetic mice and ameliorated histological evidence of diabetic nephropathy, including transforming growth factor-beta and extracellular matrix expression. Thus, in contrast to inhibiting cyclooxygenase, blockade of TPr may have therapeutic potential in diabetic nephropathy, in part by attenuating oxidative stress.     

Thioredoxin-Interacting Protein Deficiency Protects against Diabetic Nephropathy

Expression of thioredoxin-interacting protein (TxNIP), an endogenous inhibitor of the thiol oxidoreductase thioredoxin, is augmented by high glucose (HG) and promotes oxidative stress. We previously reported that TxNIP-deficient mesangial cells showed protection from HG-induced reactive oxygen species, mitogen-activated protein kinase phosphorylation, and collagen expression. Here, we investigated the potential role of TxNIP in the pathogenesis of diabetic nephropathy (DN) in vivo. Wild-type (WT) control, TxNIP^−/−, and TxNIP^+/− mice were rendered equally diabetic with low-dose streptozotocin. In contrast to effects in WT mice, diabetes did not increase albuminuria, proteinuria, serum cystatin C, or serum creatinine levels in TxNIP^−/− mice. Whereas morphometric studies of kidneys revealed a thickened glomerular basement membrane and effaced podocytes in the diabetic WT mice, these changes were absent in the diabetic TxNIP^−/− mice. Immunohistochemical analysis revealed significant increases in the levels of glomerular TGF-β1, collagen IV, and fibrosis only in WT diabetic mice. Additionally, only WT diabetic mice showed significant increases in oxidative stress (nitrotyrosine, urinary 8-hydroxy-2-deoxy-guanosine) and inflammation (IL-1β mRNA, F4/80 immunohistochemistry). Expression levels of Nox4-encoded mRNA and protein increased only in the diabetic WT animals. A significant loss of podocytes, assessed by Wilms’ tumor 1 and nephrin staining and urinary nephrin concentration, was found in diabetic WT but not TxNIP^−/− mice. Furthermore, in cultured human podocytes exposed to HG, TxNIP knockdown with siRNA abolished the increased mitochondrial O₂⁻ generation and apoptosis. These data indicate that TxNIP has a critical role in the progression of DN and may be a promising therapeutic target.     

Polymorphism of the aldosterone synthase gene is not associated with progression of diabetic nephropathy, but associated with hypertension in type 2 diabetic patients

Aim: Inhibition of aldosterone system is beneficial in diabetic nephropathy, and aldosterone synthesis is regulated at the gene-encoding aldosterone synthase (CYP11B2). Considering the role of aldosterone in diabetic nephropathy, genetic polymorphism of this gene may contribute to the development and progression of diabetic nephropathy. In this study, we investigated whether it is associated with diabetic nephropathy in type 2 diabetic patients. Methods: 197 type 2 diabetic patients and 71 healthy controls were enrolled. The study subjects were divided into four groups: healthy controls with normoalbuminuria (n = 71), normoalbuminuric diabetic group (n = 71), microalbuminuric diabetic group (n = 51) and overt proteinuria group (n = 51). Polymorphism of aldosterone synthase gene was determined using standard PCR technique. Results: Higher frequency of TT genotype and T allele in steroidogenic factor-1 (SF-1) binding site and wild type (WT) in intronic conversion (IC) in CYP11B2 was observed in diabetic patients than controls. However, there was no significant difference in SF-1 and IC genotype among diabetic patients according to the state of diabetic nephropathy. Subgroup analysis based on SF-1 polymorphism demonstrated that TT genotype is associated with higher systolic and diastolic blood pressure and higher plasma aldosterone level. In addition, WT in IC genotype showed a significantly higher urinary albumin excretion rate. Plasma aldosterone level was significantly related with systolic blood pressure. Conclusion: Our study suggests that aldosterone synthase gene polymorphism is not associated with progression of diabetic nephropathy, but it may contribute to the development of hypertension associated with increased aldosterone secretion in type 2 diabetic patients.     

Intercellular adhesion molecule-1-deficient mice are resistant against renal injury after induction of diabetes

Diabetic nephropathy is a leading cause of end-stage renal failure. Several mechanisms, including activation of protein kinase C, advanced glycation end products, and overexpression of transforming growth factor (TGF)-beta, are believed to be involved in the pathogenesis of diabetic nephropathy. However, the significance of inflammatory processes in the pathogenesis of diabetic microvascular complications is poorly understood. Accumulation of macrophages and overexpression of leukocyte adhesion molecules and chemokines are prominent in diabetic human kidney tissues. We previously demonstrated that intercellular adhesion molecule (ICAM)-1 mediates macrophage infiltration into the diabetic kidney. In the present study, to investigate the role of ICAM-1 in diabetic nephropathy, we induced diabetes in ICAM-1-deficient (ICAM-1(-/-)) mice and ICAM-1(+/+) mice with streptozotocin and examined the renal pathology over a period of 6 months. The infiltration of macrophages was markedly suppressed in diabetic ICAM-1(-/-) mice compared with that of ICAM-1(+/+) mice. Urinary albumin excretion, glomerular hypertrophy, and mesangial matrix expansion were significantly lower in diabetic ICAM-1(-/-) mice than in diabetic ICAM-1(+/+) mice. Moreover, expressions of TGF-beta and type IV collagen in glomeruli were also suppressed in diabetic ICAM-1(-/-) mice. These results suggest that ICAM-1 is critically involved in the pathogenesis of diabetic nephropathy.     

Increased urinary excretion of monocyte chemoattractant protein-1 in proteinuric renal diseases

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is produced mainly by tubular epithelial cells in kidney and contributes to renal interstitial inflammation and fibrosis. More recently, we have demonstrated that urinary MCP-1 excretion is increased in proportion to the degree of albuminuria (proteinuria) and positively correlated with urinary N-acetylglucosaminidase (NAG) levels in type 2 diabetic patients. Based on these findings, we have suggested that heavy proteinuria, itself, probably aggravates renal tubular damage and accelerates the disease progression in diabetic nephropathy by increasing the MCP-1 expression in renal tubuli. In the present study, to evaluate whether urinary MCP-1 excretion is increased in the proteinuric states not only in diabetic nephropathy but also in other renal diseases, we examined urinary MCP-1 levels in IgA nephropathy patients with macroalbuminuria (IgAN group; n = 6), and compared the results with the data obtained from type 2 diabetic patients with overt diabetic nephropathy (DN group; n = 23) and those without diabetic nephropathy (non-DN group; n = 27). Urinary MCP-1 excretion levels in non-DN, DN, IgAN groups were 157.2 (52.8-378.5), 346.1 (147.0-1276.7), and 274.4 (162.2-994.5) ng/g creatinine, median (range), respectively. Expectedly, urinary MCP-1 and NAG excretion levels in DN and IgAN groups were significantly elevated as compared with non-DN group. Therefore, we suggest that MCP-1 expression in renal tubuli is enhanced in proteinuric states,irrespective of the types of renal disease, and that increased MCP-1 expression probably contributes to renal tubular damage in proteinuric states.     

Translocation of glomerular p47phox and p67phox by protein kinase C-beta activation is required for oxidative stress in diabetic nephropathy

Oxidative stress is implicated to play an important role in the development of diabetic vascular complications, including diabetic nephropathy. It is unclear whether oxidative stress is primarily enhanced in the diabetic glomeruli or whether it is merely a consequence of diabetes-induced glomerular injury. To address this issue, we examined diabetic glomeruli to determine whether oxidative stress is enhanced, as well as examined the role of protein kinase C (PKC)-beta activation in modulating NADPH oxidase activity. Urinary 8-hydroxydeoxyguanosine excretion and its intense immune-reactive staining in the glomeruli were markedly higher in diabetic than in control rats, and these alterations were ameliorated by a treatment with a selective PKC-beta inhibitor, ruboxistaurin (RBX; LY333531) mesylate, without affecting glycemia. NADPH oxidase activity, which was significantly enhanced in diabetic glomeruli and the source of reactive oxygen species (ROS) generation, was also improved by RBX treatment by preventing the membranous translocation of p47phox and p67phox from cytoplasmic fraction without affecting their protein levels. Adenoviral-mediated PKC-beta(2) overexpression enhanced ROS generation by modulating the membranous translocation of p47phox and p67phox in cultured mesangial cells. We now demonstrate that oxidative stress is primarily enhanced in the diabetic glomeruli due to a PKC-beta-dependent activation of NADPH oxidase resulting in ROS generation.     

The effects of dual blockade of the renin-angiotensin system on urinary protein and transforming growth factor-beta excretion in 2 groups of patients with IgA and diabetic nephropathy

AIMS: The therapeutic benefits of dual blockade of the renin-angiotensin system (RAS) have been inconsistent on renal function and proteinuria. To know the contribution of the heterogeneity of study subjects to such inconsistency, we evaluated the effects of dual blockade of RAS in 2 groups of selected renal diseases, IgA and diabetic nephropathy. To avoid confounding by the blood pressure-reducing effects, angiotensin II receptor antagonists (ATRAs) were added on the patients with long-term, optimally controlled blood pressure taking ACE inhibitors. Twenty-four-hour urinary protein excretion rate and urinary TGF-beta1 level were measured as surrogate markers of renal injury. METHODS: We conducted a prospective crossover trial with 14 IgA and 18 type-2-diabetic nephropathy patients showing moderate degree of proteinuria (> or = 1.0 g/day) and renal dysfunction (creatinine clearance 25 - 75/ml/min). Four to 8 mg once-daily dose of candesartan and placebo were alternatively added on ramipril dose of 5 - 7.5 mg/day for 16 weeks. RESULTS: All baseline data except for the age factor were statistically the same between the 2 disease groups. Twenty-four-hour mean arterial blood pressures were 91.2 +/- 1.6 and 92.3 +/- 1.8 mmHg in IgA and diabetic nephropathy patients respectively at baseline (p = NS). Mean arterial pressure did not change by the addition of candesartan or placebo in both groups. The addition of candesartan (combination) reduced 24-hour urinary protein excretion rate in IgA nephropathy patients with a mean change of -12.3 +/- 4.5%, which is significantly greater compared to a mean change of -0.1 +/- 3.3% after the addition of placebo (placebo) (mean difference 12.4 +/- 5.0, 95% CI 1.2 - 23.5; p < 0.05). Urinary TGF-beta1 level was reduced considerably by the combination therapy, with a -28.9 +/- 6.0% decrease, which was significantly different to that by the placebo, with +4.3 +/- 12.4% (33.3 +/- 13.5, 3.2 - 63.3; p < 0.05). In diabetic nephropathy patients, the addition of candesartan did not reduce 24-hour urinary protein excretion rate. Mean changes of 24-hour urinary protein excretion rate were -0.8 +/- 4.7% by the combination therapy and +0.5 +/- 6.1% by placebo (mean difference 1.3 +/- 4.7, 95% CI -6.8 - 13.5; p < NS). The level of urinary TGF-beta1 was reduced by the combination therapy, with -14.3 +/- 9.5% decrease, but it did not reach statistical significance compared to placebo of +0.7 +/- 15.5% (15.0 +/- 13.5, -14.4 - 44.5; p < NS). The changes in 24-hour urinary protein excretion rate and urinary TGF-beta1 level were neither correlated with each other, nor with the change in mean arterial pressure. Significant changes in the renal function were not detected during the study period. CONCLUSION: Definite beneficial effects of dual blockade of RAS on proteinuria and TGF-beta1 excretion were found in IgA nephropathy patients, which was independent of blood pressure-reducing effect. With our 16-week trial, such benefits were not observed in type 2 diabetic nephropathy. The reduction in urinary TGF-beta1 level suggests that the combination therapy may provide additional renoprotection through the antisclerosing effects. Based on our results, for a proper interpretation the therapeutic effects of the combination therapy should be evaluated separately according to the underlying renal disease.     

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.