Early Features Of Diabetic Nephropathy Research Articles

Inhibition of miR-17~92 Cluster Ameliorates High Glucose-Induced Podocyte Damage.

The loss and damage of podocytes is an early feature of diabetic nephropathy (DN). The miR-17∼92 cluster was dysregulated in diabetic and polycystic kidney disease patients, but its role in DN is unclear. Hence, an in vitro study on the high glucose- (HG-) treated mouse podocytes (MPC5) was designed to elucidate the effect of miR-17∼92 cluster downregulation on cell viability, apoptosis, inflammation, fibrosis, and podocyte function. The results suggested that the miR-17∼92 cluster members miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a were upregulated in the renal biopsy tissue of DN patients and HG-treated MPC5. The downregulation of the miR-17∼92 cluster effectively suppressed the cell apoptosis, inflammation, fibrosis, and podocyte dysfunction in HG-stimulated MPC5 cells. The bioinformatics analysis and rescue experiments showed that ABCA1 (ATP-binding cassette transporter A1) is an effector of the miR-17~92 cluster. Silence of ABCA1 inhibited the protective effect of the miR-17∼92 cluster downregulation on podocyte damage. In summary, this research indicated that the downregulation of the miR-17∼92 cluster ameliorates HG-induced podocyte damage via targeting ABCA1.

ADMA elevation does not exacerbate development of diabetic nephropathy in mice with streptozotocin-induced diabetes mellitus

Background and aimsCardiovascular disease is nowadays the major cause of mortality and morbidity worldwide. The risk of developing cardiovascular disease is significantly increased in patients with diabetic nephropathy. It has been suggested that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthases (NOS), may play an important role in the pathogenesis of diabetic nephropathy. ADMA is mainly metabolized by dimethylarginine dimethylaminohydrolase 1 (DDAH1). The goal of this study was to test the hypothesis that elevation of systemic ADMA levels by knocking out DDAH1 would exacerbate functional and structural glomerular abnormalities in a murine model of diabetic nephropathy. MethodsStreptozotocin (STZ) was used to induce diabetes in adult DDAH1 knock-out and wild type mice. Healthy mice served as controls. Mice were sacrificed after 20 weeks of diabetes. Plasma ADMA levels were assessed by isotope-dilution tandem mass spectrometry and albumin by ELISA. Kidneys were used for FACS analysis and were also stained for markers of inflammation, cell proliferation, glomerular cells and cell matrix. ResultsSTZ led to development of diabetes mellitus in all injected animals. Deficiency of DDAH1 led to a significant increase in plasma ADMA levels in healthy and diabetic mice. The diabetic state itself did not influence systemic ADMA levels. Diabetic mice of both genotypes developed albuminuria and had increased glomerulosclerosis index. There were no changes in desmin expression, glomerular cell proliferation rate, matrix expansion and expression of Mac-2 antigen in the diabetic mice of both genotypes as compared to the healthy ones. ConclusionsIn summary, STZ-induced diabetes led to the development of early features of diabetic nephropathy. Deficiency of DDAH1 and subsequent increase in systemic ADMA levels did not exacerbate these changes, indicating that ADMA is not the major mediator of diabetic nephropathy in this experiment model.

ADMA reduction does not protect mice with streptozotocin-induced diabetes mellitus from development of diabetic nephropathy

Background and aimsCardiovascular disease is the major cause of morbidity and mortality in the world. Diabetes and its complications, such as diabetic nephropathy, dramatically increase cardiovascular risk. Association studies suggest that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthases, plays a role in the pathogenesis of diabetic nephropathy. The major pathway of ADMA catabolism is hydrolysis by dimethylarginine dimethylaminohydrolase 1 (DDAH1). The goal of this study was to test the hypothesis that lowering ADMA by overexpression of DDAH1 protects from development of diabetic nephropathy. MethodsDiabetes was induced with streptozotocin (STZ) in wild type and DDAH1 transgenic mice. Healthy mice served as controls. Mice were sacrificed after 20 weeks of diabetes. ADMA levels were assessed by isotope-dilution tandem mass spectrometry, creatinine by standard laboratory methods and albumin by ELISA. Kidney tissues were stained for markers of glomerular cells, cell matrix, inflammation and cell proliferation. ResultsSTZ led to development of diabetes in all injected mice. Transgenic overexpression of DDAH1 led to a decrease in plasma ADMA levels in healthy animals. Diabetic state itself did not lead to elevation of plasma ADMA levels. Diabetic mice of both genotypes developed albuminuria (27 and 25 vs. 9 and 6 μg albumin/mg creatinine) (p < 0.01). There were no changes in glomerular matrix expansion, podocyte injury, inflammatory or proliferative response. ConclusionsSTZ-induced diabetes led to the development of early features of diabetic nephropathy. Overexpression of DDAH1 and lowering of systemic ADMA levels did not prevent these changes, indicating that ADMA is not the major mediator of the early diabetic changes reflected by this experimental model.

In Vivo Knockdown of I‐mfa Using The Targeted Nanoparticle Delivery System Ameliorated Renal Injury in Diabetic Mice

Accumulation of extracellular matrix (ECM) proteins in glomerulus is one of early features of diabetic nephropathy. Glomerular mesangial cell (MC) is the major source of ECM. Inhibitor of Myogenic Family isoform a (I‐mfa) is a cytosolic protein interacting with components of Wnt/β‐catenin pathway and of store‐operated Ca2+ signaling pathway, both of which are involved in the development of diabetic nephropathy. The present study was carried out to determine if I‐mfa in MCs contributed to renal injury in diabetic mice. Twelve male mice were randomly divided into 4 groups (3 mice each group): non‐diabetes, diabetes, diabetes treated with scramble siRNA, and diabetes treated with siRNA against I‐mfa. Diabetes was generated by multiple low‐dose streptozotocin (STZ, 50 mg/kg) injections (intraperitoneal, once one day for 5 days) at an age of 6 weeks. Mice in group of non diabetes were injected with 10 mM citrate buffer (vehicle). Diabetes was indicated by a fasting blood glucose level of &gt; 300 mg/dl. Scramble or I‐mfa siRNA treatment started 24 weeks after STZ injection and the treatment lasted 2 weeks. All siRNAs (10 mg/kg body weight) were delivered by our recently established‐targeted nanoparticle/siRNA delivery system which is glomerular mesangial cell specific. We found that STZ treatment significantly increased blood glucose level after 4 weeks of injection and reduced body weight 26 weeks after treatment. I‐mfa siRNA treatment did not improve hyperglycemia, but lessened renal hypertrophy in STZ mice. Furthermore, diabetic mice showed significantly greater 24 hour urine output and this diabetic uresis was compromised by I‐mfa siRNA treatment. Although knocking down I‐mfa did not mitigate albuminuria, it lowered the level of blood urea nitrogen in diabetic mice. Consistent with the functional data, histological examination showed improved mesangial expansion and glomerular fibrosis in I‐mfa siRNA‐treated STZ mice. Immunohistochemical examination showed a significant reduction of protein abundance of glomerular fibronectin and collagen IV in the siRNA‐treated diabetic mice. Taken together, our data indicate that I‐mfa in MCs is involved in the development of diabetic kidney disease and thus, could be a potential therapeutic target for diabetic nephropathy.Support or Funding InformationAHA Southwestern Affiliate; Harry S. Moss Heart Trust; University Outstanding Talent Cultivated Projects of Anhui Province, China (gxfxZD2016118 and gxfxZD2016162)

An endoplasmic reticulum stress-regulated lncRNA hosting a microRNA megacluster induces early features of diabetic nephropathy.

It is important to find better treatments for diabetic nephropathy (DN), a debilitating renal complication. Targeting early features of DN, including renal extracellular matrix accumulation (ECM) and glomerular hypertrophy, can prevent disease progression. Here we show that a megacluster of nearly 40 microRNAs and their host long non-coding RNA transcript (lnc-MGC) are coordinately increased in the glomeruli of mouse models of DN, and mesangial cells treated with transforming growth factor-β1 (TGF- β1) or high glucose. Lnc-MGC is regulated by an endoplasmic reticulum (ER) stress-related transcription factor, CHOP. Cluster microRNAs and lnc-MGC are decreased in diabetic Chop−/− mice that showed protection from DN. Target genes of megacluster microRNAs have functions related to protein synthesis and ER stress. A chemically modified oligonucleotide targeting lnc-MGC inhibits cluster microRNAs, glomerular ECM and hypertrophy in diabetic mice. Relevance to human DN is also demonstrated. These results demonstrate the translational implications of targeting lnc-MGC for controlling DN progression.

Cyclin-dependent kinase 2 protects podocytes from apoptosis.

Loss of podocytes is an early feature of diabetic nephropathy (DN) and predicts its progression. We found that treatment of podocytes with sera from normoalbuminuric type 1 diabetes patients with high lipopolysaccharide (LPS) activity, known to predict progression of DN, downregulated CDK2 (cyclin-dependent kinase 2). LPS-treatment of mice also reduced CDK2 expression. LPS-induced downregulation of CDK2 was prevented in vitro and in vivo by inhibiting the Toll-like receptor (TLR) pathway using immunomodulatory agent GIT27. We also observed that CDK2 is downregulated in the glomeruli of obese Zucker rats before the onset of proteinuria. Knockdown of CDK2, or inhibiting its activity with roscovitine in podocytes increased apoptosis. CDK2 knockdown also reduced expression of PDK1, an activator of the cell survival kinase Akt, and reduced Akt phosphorylation. This suggests that CDK2 regulates the activity of the cell survival pathway via PDK1. Furthermore, PDK1 knockdown reduced the expression of CDK2 suggesting a regulatory loop between CDK2 and PDK1. Collectively, our data show that CDK2 protects podocytes from apoptosis and that reduced expression of CDK2 associates with the development of DN. Preventing downregulation of CDK2 by blocking the TLR pathway with GIT27 may provide a means to prevent podocyte apoptosis and progression of DN.

High glucose induces rat mesangial cells proliferation and MCP-1 expression via ROS-mediated activation of NF-κB pathway, which is inhibited by eleutheroside E

Glomerular hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy (DN). High glucose-induced oxidative stress is implicated in the etiology of DN. This study aims to investigate the effect of eleutheroside E (EE) on high glucose mediated rat mesangial cells (MCs) proliferation and monocyte chemoattractant protein-1 (MCP-1) expression and the underlying mechanism. MCs proliferation was assessed by MTT assay. Reactive oxygen species (ROS) level and MCP-1 expression were evaluated by ELISA kit. The protein expression of p47, NF-κB p65, p-NF-κB p65, IκBα, p-IκBα, IKKβ and p-IKKβ were determined by Western blot. The results showed that treatment with EE markedly attenuated high glucose induced MCs proliferation and in a dose-dependent manner. Intervention with EE also significantly blocked high glucose induced intracellular ROS production by decreasing NADPH oxidase activity. Meanwhile, EE administration could effectively alleviate the high glucose-stimulated activation of NF-κB, the degradation of IκBα and the expression of MCP-1. These results demonstrate that high glucose enhances MCs proliferation and MCP-1 expression by activating the ROS/NF-κB pathway and can be inhibited by EE. Our findings provide a new perspective for the clinical treatment of DN.

Store-Operated Ca2+ Channels in Mesangial Cells Inhibit Matrix Protein Expression.

Accumulation of extracellular matrix derived from glomerular mesangial cells is an early feature of diabetic nephropathy. Ca(2+) signals mediated by store-operated Ca(2+) channels regulate protein production in a variety of cell types. The aim of this study was to determine the effect of store-operated Ca(2+) channels in mesangial cells on extracellular matrix protein expression. In cultured human mesangial cells, activation of store-operated Ca(2+) channels by thapsigargin significantly decreased fibronectin protein expression and collagen IV mRNA expression in a dose-dependent manner. Conversely, inhibition of the channels by 2-aminoethyl diphenylborinate significantly increased the expression of fibronectin and collagen IV. Similarly, overexpression of stromal interacting molecule 1 reduced, but knockdown of calcium release-activated calcium channel protein 1 (Orai1) increased fibronectin protein expression. Furthermore, 2-aminoethyl diphenylborinate significantly augmented angiotensin II-induced fibronectin protein expression, whereas thapsigargin abrogated high glucose- and TGF-β1-stimulated matrix protein expression. In vivo knockdown of Orai1 in mesangial cells of mice using a targeted nanoparticle siRNA delivery system resulted in increased expression of glomerular fibronectin and collagen IV, and mice showed significant mesangial expansion compared with controls. Similarly, in vivo knockdown of stromal interacting molecule 1 in mesangial cells by recombinant adeno-associated virus-encoded shRNA markedly increased collagen IV protein expression in renal cortex and caused mesangial expansion in rats. These results suggest that store-operated Ca(2+) channels in mesangial cells negatively regulate extracellular matrix protein expression in the kidney, which may serve as an endogenous renoprotective mechanism in diabetes.

Andrographolide suppresses high glucose-induced fibronectin expression in mesangial cells via inhibiting the AP-1 pathway

Mesangial cells (MCs) proliferation and accumulation of glomerular matrix proteins such as fibronectin (FN) are the early features of diabetic nephropathy, with MCs known to upregulate matrix protein synthesis in response to high glucose. Recently, it has been found that andrographolide has renoprotective effects on diabetic nephropathy. However, the molecular mechanism underlying these effects remains unclear. Cell viability and proliferation was evaluated by MTT. FN expression was examined by immunofluorescence and immunoblotting. Activator protein-1 (AP-1) activation was assessed by immunoblotting, luciferase reporter and electrophoretic mobility shift assays. Andrographolide significantly decreased high glucose-induced cell proliferation and FN expression in MCs. Exposure of MCs to high glucose markedly stimulated the expression of phosphorylated c-jun, whereas the stimulation was inhibited by andrographolide. Plasmid pAP-1-Luc luciferase reporter assay showed that andrographolide blocked high glucose-induced AP-1 transcriptional activity. EMSA assay demonstrated that increased AP-1 binding to an AP-1 binding site at -1,029 in the FN gene promoter upon high glucose stimulation, and the binding were disrupted by andrographolide treatment. These data indicate that andrographolide suppresses high glucose-induced FN expression by inhibiting AP-1-mediated pathway.

Wilm's Tumor-1 Protein Levels in Urinary Exosomes from Diabetic Patients with or without Proteinuria

BackgroundPodocyte injury is an early feature of diabetic nephropathy (DN). Recently, urinary exosomal Wilm's tumor-1 protein (WT1), shed by renal epithelial cells, has been proposed as a novel biomarker for podocyte injury. However, its usefulness as biomarker for early diabetic nephropathy has not been verified yet. We investigated urinary exosomal WT1 in type-1 diabetic patients to confirm its role as a non-invasive biomarker for predicting early renal function decline.MethodsThe expression of WT1 protein in urinary exosomes from spot urine samples of type-1 diabetes mellitus patients (n = 48) and healthy controls (n = 25) were analyzed. Patients were divided based on their urinary albumin excretion, ACR (mg/g creatinine) into non- proteinuria group (ACR<30 mg/g, n = 30) and proteinuria group (ACR>30 mg/g, n = 18). Regression analysis was used to assess the association between urinary exosomal levels of WT1 with parameters for renal function. Receiver Operating Characteristic (ROC) curve analysis was used to determine the diagnostic performance of exosomal WT-1.ResultsWT1 protein was detected in 33 out of 48 diabetic patients and in only 1 healthy control. The levels of urinary exosomal WT1 protein is significantly higher (p = 0.001) in patients with proteinuria than in those without proteinuria. In addition, all the patients with proteinuria but only half of the patients without proteinuria were positive for exosomal WT1. We found that the level of exosomal WT1 were associated with a significant increase in urine protein-to-creatinine ratio, albumin-to-creatinine ratio, and serum creatinine as well as a decline in eGFR. Furthermore, patients exhibiting WT1-positive urinary exosomes had decreased renal function compared to WT1-negative patients. ROC analysis shows that WT-1 effectively predict GFR<60 ml. min-1/1.73 m2.ConclusionThe predominant presence of WT1 protein in urinary exosomes of diabetic patients and increase in its expression level with decline in renal function suggest that it could be useful as early non-invasive marker for diabetic nephropathy.

Is it or is it not a pathogenic mutation? Is it or is it not the podocyte?

Is it or is it not a pathogenic mutation? Is it or is it not the podocyte?

High glucose induces renal mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which is inhibited by resveratrol

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. Hyperglycemia-induced oxidative stress is implicated in the etiology of diabetic nephropathy. Resveratrol has potent antioxidative and protective effects on diabetic nephropathy. We aimed to examine whether high glucose (HG)-induced NADPH oxidase activation and reactive oxygen species (ROS) production contribute to glomerular mesangial cell proliferation and fibronectin expression and the effect of resveratrol on HG action in mesangial cells. By using rat mesangial cell line and primary mesangial cells, we found that NADPH oxidase inhibitor (apocynin) and ROS inhibitor (N-acetyl cysteine) both inhibited HG-induced mesangial cell proliferation and fibronectin expression. HG-induced elevation of NADPH oxidase activity and production of ROS in mesangial cells was inhibited by apocynin. These results suggest that HG induces mesangial cell proliferation and fibronectin expression through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunits p22phox and p47phox expression through JNK/NF-κB pathway, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced mesangial cell proliferation and fibronectin expression through inhibiting HG-induced JNK and NF-κB activation, NADPH oxidase activity elevation and ROS production. These results demonstrate that HG enhances mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide novel therapeutic targets for diabetic nephropathy.

Insulin in podocyte podiatry.

Insulin in podocyte podiatry.

Regulation of Type IV Collagen α Chains of Glomerular Epithelial Cells in Diabetic Conditions

An early feature of diabetic nephropathy is the alteration of the glomerular basement membrane (GBM), which may result in microalbuminuria, subsequent macroproteinuria, and eventual chronic renal failure. Although type IV collagen is the main component of thickened GBM in diabetic nephropathy, cellular metabolism of each α chains of type IV collagen has not been well studied. To investigate the regulation of α(IV) chains in diabetic conditions, we examined whether glucose and advanced glycosylation endproduct (AGE) regulate the metabolism of each α(IV) chains in the diabetic tissue and glomerular epithelial cells (GEpC). Glomerular collagen α3(IV) and α5(IV) chains protein were higher and more intense in immunofluorescence staining according to diabetic durations compared to controls. In vitro, mainly high glucose and partly AGE usually increased total collagen protein of GEpC by [3H]-proline incorporation assay and each α(IV) chain proteins including α1(IV), α3(IV), and α5(IV) in time-dependent and subchain-specific manners. However, the changes of each α(IV) chains mRNA expression was not well correlated to the those of each chain proteins. The present findings suggest that the metabolism of individual α(IV) chains of GBM is differentially regulated in diabetic conditions and those changes might be induced not only by transcriptional level but also by post-translational modifications.

Enhanced Expression of Janus Kinase–Signal Transducer and Activator of Transcription Pathway Members in Human Diabetic Nephropathy

OBJECTIVE—Glomerular mesangial expansion and podocyte loss are important early features of diabetic nephropathy, whereas tubulointerstitial injury and fibrosis are critical for progression of diabetic nephropathy to kidney failure. Therefore, we analyzed the expression of genes in glomeruli and tubulointerstitium in kidney biopsies from diabetic nephropathy patients to identify pathways that may be activated in humans but not in murine models of diabetic nephropathy that fail to progress to glomerulosclerosis, tubulointerstitial fibrosis, and kidney failure.RESEARCH DESIGN AND METHODS—Kidney biopsies were obtained from 74 patients (control subjects, early and progressive type 2 diabetic nephropathy). Glomerular and tubulointerstitial mRNAs were microarrayed, followed by bioinformatics analyses. Gene expression changes were confirmed by real-time RT-PCR and immunohistological staining. Samples from db/db C57BLKS and streptozotocin-induced DBA/2J mice, commonly studied murine models of diabetic nephropathy, were analyzed.RESULTS—In human glomeruli and tubulointerstitial samples, the Janus kinase (Jak)-signal transducer and activator of transcription (Stat) pathway was highly and significantly regulated. Jak-1, -2, and -3 as well as Stat-1 and -3 were expressed at higher levels in patients with diabetic nephropathy than in control subjects. The estimated glomerular filtration rate significantly correlated with tubulointerstitial Jak-1, -2, and -3 and Stat-1 expression (R2 = 0.30–0.44). Immunohistochemistry found strong Jak-2 staining in glomerular and tubulointerstitial compartments in diabetic nephropathy compared with control subjects. In contrast, there was little or no increase in expression of Jak/Stat genes in the db/db C57BLKS or diabetic DBA/2J mice.CONCLUSIONS—These data suggest a direct relationship between tubulointerstitial Jak/Stat expression and progression of kidney failure in patients with type 2 diabetic nephropathy and distinguish progressive human diabetic nephropathy from nonprogressive murine diabetic nephropathy.

Connective tissue growth factor is associated with the early renal hypertrophy in uninephrectomized diabetic rats

Renal hypertrophy has been regarded as the early feature of diabetic nephropathy (DN), which may eventually lead to proteinuria and renal fibrosis. However, the exact mechanism of renal hypertrophy is still unclear. The aim of this study was to investigate the possible association of connective tissue growth factor (CTGF) with renal hypertrophy in uninephrectomized diabetic rats. Seventy-two Sprague-Dawley (SD) rats were randomly divided into two groups: control group (group C, n = 32) and diabetic nephropathy (group DN, n = 40). Each group was re-divided into 4 subgroups according to the experimental period. The rats were sacrificed at 1, 2, 4, and 8 weeks respectively after induction of diabetes. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) after rats had received uninephrectomy. Blood glucose (BG), body weight (BW), 24-h urinary albumin excretion (24hUalb), kidney weight (KW), KW/BW, glomerular tuft area (AG), glomerular tuft volume (VG), proximal tubular area (AT) at each time point, the width of glomerular basement membrane (GBM) and tubular basement membrane (TBM) at week 8 were measured when the rats were sacrificed. Renal expression of CTGF and p27kip1 were detected by immunohistochemical staining. The relationship between CTGF expression and increasing of VG and AT was analyzed. There was a significant increase of 24hUalb, KW, and KW/BW from week 1 onward in diabetic rats compared to those in group C (P < 0.05, respectively), diabetic rats also had a significant increase of AG, VG, and AT from week 1 onward. It was also shown that diabetic rats had a thickening of GBM [(245.7 +/- 103.0) nm vs (121.8 +/- 19.1) nm, P < 0.01] and TBM [(767.7 +/- 331.1) nm vs (293.0 +/- 110.5) nm, P < 0.01] at week 8. There was a weak expression for CTGF and p27kip1 in normal glomeruli and tubuli, while a significant increasing expression of CTGF and p27kip1 was found in glomeruli and tubuli in diabetic kidney from week 1 onward (P < 0.05, respectively), and the extent of CTGF expression was positively correlated with AG (r = 0.92, P < 0.05), VG (r = 0.86, P < 0.05), AT (r = 0.94, P < 0.01) and positively correlated with the expression of p27kip1 (r = 0.96, P < 0.01). The expression of CTGF increases in diabetic rat kidney at the early stage, which might be an important mediator of renal hypertrophy through arresting cell cycling.

Glucose-Induced Reactive Oxygen Species Cause Apoptosis of Podocytes and Podocyte Depletion at the Onset of Diabetic Nephropathy

Diabetic nephropathy is the most common cause of end-stage renal disease in the U.S. Recent studies demonstrate that loss of podocytes is an early feature of diabetic nephropathy that predicts its progressive course. Cause and consequences of podocyte loss during early diabetic nephropathy remain poorly understood. Here, we demonstrate that podocyte apoptosis increased sharply with onset of hyperglycemia in Ins2(Akita) (Akita) mice with type 1 diabetes and Lepr(db/db) (db/db) mice with obesity and type 2 diabetes. Podocyte apoptosis coincided with the onset of urinary albumin excretion (UAE) and preceded significant losses of podocytes in Akita (37% reduction) and db/db (27% reduction) mice. Increased extracellular glucose (30 mmol/l) rapidly stimulated generation of intracellular reactive oxygen species (ROS) through NADPH oxidase and mitochondrial pathways and led to activation of proapoptotic p38 mitogen-activated protein kinase and caspase 3 and to apoptosis of conditionally immortalized podocytes in vitro. Chronic inhibition of NADPH oxidase prevented podocyte apoptosis and ameliorated podocyte depletion, UAE, and mesangial matrix expansion in db/db mice. In conclusion, our results demonstrate for the first time that glucose-induced ROS production initiates podocyte apoptosis and podocyte depletion in vitro and in vivo and suggest that podocyte apoptosis/depletion represents a novel early pathomechanism(s) leading to diabetic nephropathy in murine type 1 and type 2 diabetic models.

Nox4 NAD(P)H Oxidase Mediates Hypertrophy and Fibronectin Expression in the Diabetic Kidney

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. We investigated the role of the NAD(P)H oxidase Nox4 in generation of reactive oxygen species (ROS), hypertrophy, and fibronectin expression in a rat model of type 1 diabetes induced by streptozotocin. Phosphorothioated antisense (AS) or sense oligonucleotides for Nox4 were administered for 2 weeks with an osmotic minipump 72 h after streptozotocin treatment. Nox4 protein expression was increased in diabetic kidney cortex compared with non-diabetic controls and was down-regulated in AS-treated animals. AS oligonucleotides inhibited NADPH-dependent ROS generation in renal cortical and glomerular homogenates. ROS generation by intact isolated glomeruli from diabetic animals was increased compared with glomeruli isolated from AS-treated animals. AS treatment reduced whole kidney and glomerular hypertrophy. Moreover, the increased expression of fibronectin protein was markedly reduced in renal cortex including glomeruli of AS-treated diabetic rats. Akt/protein kinase B and ERK1/2, two protein kinases critical for cell growth and hypertrophy, were activated in diabetes, and AS treatment almost abolished their activation. In cultured mesangial cells, high glucose increased NADPH oxidase activity and fibronectin expression, effects that were prevented in cells transfected with AS oligonucleotides. These data establish a role for Nox4 as the major source of ROS in the kidneys during early stages of diabetes and establish that Nox4-derived ROS mediate renal hypertrophy and increased fibronectin expression.

P27Kip1 knockout mice are protected from diabetic nephropathy: Evidence for p27Kip1 haplotype insufficiency

p27Kip1 knockout mice are protected from diabetic nephropathy: Evidence for p27Kip1 haplotype insufficiency

Rosiglitazone Improves Glomerular Hyperfiltration, Renal Endothelial Dysfunction, and Microalbuminuria of Incipient Diabetic Nephropathy in Patients

Microalbuminuria, an early feature of diabetic nephropathy, indicates intrarenal endothelial damage. In type 2 diabetes, microalbuminuria is strongly related to insulin resistance. We therefore investigated whether rosiglitazone, an insulin-sensitizing drug that is known to improve endothelial dysfunction, was able to improve intrarenal endothelial dysfunction and microalbuminuria. Nineteen type 2 diabetic patients participated in this double-blind cross-over trial. Nine patients with newly diagnosed disease without microalbuminuria were randomized to a treatment with rosiglitazone or nateglinide, each for 12 weeks. Ten patients with microalbuminuria were randomized to rosiglitazone or placebo, each for 12 weeks in addition to their previous antidiabetic medication. After each treatment, glomerular filtration rate (GFR), renal plasma flow, and filtration fraction were measured before and after blockade of nitric oxide (NO) by intravenous administration of N-monomethyl-L-arginine-acetate (L-NMMA). Ten healthy subjects served as control subjects. Type 2 diabetic patients at baseline showed glomerular hyperfiltration compared with healthy control subjects. Rosiglitazone reduced elevated GFR and filtration fraction toward control primarily in patients with microalbuminuria (GFR: 133.4 +/- 9.8 vs. 119.6 +/- 8.7 ml/min; filtration fraction: 23.2 +/- 1.7 vs. 20.5 +/- 1.6% before and after rosiglitazone, respectively; control subjects: GFR 111.7 +/- 8.6 ml/min, filtration fraction 20.4 +/- 1.5%). Rosiglitazone improved intrarenal NO bioavailability in type 2 diabetes toward control as shown by infusion of L-NMMA. Rosiglitazone reduced albumin excretion in type 2 diabetes with microalbuminuria from 116.5 +/- 31 to 40.4 +/- 12 mg/day. Rosiglitazone ameliorated glomerular hyperfiltration in early type 2 diabetes, improved NO bioavailability, and lessened renal end-organ damage in type 2 diabetes with microalbuminuria.