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Src family kinases (SFKs) have been implicated in the pathogenesis of kidney fibrosis. However, the specific mechanism by which SFKs contribute to the progression of diabetic kidney disease (DKD) remains unclear. Our preliminary transcriptome analysis suggested that SFK expression was increased in diabetic kidneys and that the expression of Fyn (a member of the SFKs), along with genes related to unfolded protein responses from the endoplasmic reticulum (ER) stress signaling pathway, was upregulated in the tubules of human diabetic kidneys. Thus, we examined whether SFK-induced ER stress is associated with DKD progression. Mouse proximal tubular (mProx24) cells were transfected with Fyn or Lyn siRNA and exposed to high glucose and palmitate (HG-Pal). Streptozotocin-induced diabetic rats were treated with KF-1607, a novel pan-Src kinase inhibitor (SKI) with low toxicity. The effect of KF-1607 was compared to that of losartan, a standard treatment for patients with DKD. Among the SFK family members, the Fyn and Lyn kinases were upregulated under diabetic stress. HG-Pal induced p70S6 kinase and JNK/CHOP signaling and promoted tubular injury. Fyn knockdown but not Lyn knockdown inhibited this detrimental signaling pathway. In addition, diabetic rats treated with KF-1607 showed improved kidney function and decreased ER stress, inflammation, and fibrosis compared with those treated with losartan. Collectively, these findings indicate that Fyn kinase is a specific member of the SFKs implicated in ER stress activation leading to proximal tubular injury in the diabetic milieu and that pan-SKI treatment attenuates kidney injury in diabetic rats. These data highlight Fyn kinase as a viable target for the development of therapeutic agents for DKD., (© 2022. The Author(s).)

Diabetic kidney disease (DKD) involves various pathogenic processes during progression to end stage renal disease, and activated metabolic pathways might be changing based on major pathophysiologic mechanisms as DKD progresses. In this study, nuclear magnetic resonance spectroscopy (NMR)-based metabolic profiling was performed in db/db mice to suggest potential biomarkers for early detection and its progression. We compared concentrations of serum and urinary metabolites between db/m and db/db mice at 8 or 20 weeks of age and investigated whether changes between 8 and 20 weeks in each group were significant. The metabolic profiles demonstrated significantly increased urine levels of glucose and tricarboxylic acid cycle intermediates at both 8 and 20 weeks of age in db/db mice. These intermediates also exhibited strong positive associations with urinary albumin excretion, suggesting that they may be potential biomarkers for early diagnosis. On the contrary, branched chain amino acid and homocysteine-methionine metabolism were activated early in the disease, whereas ketone and fatty acid metabolism were significantly changed in the late phase of the disease. We demonstrated phase-specific alterations in metabolites during progression of DKD. This study provides insights into perturbed mechanisms during evolution of the disease and identifies potential novel biomarkers for DKD., (Copyright © 2018. Published by Elsevier Inc.)

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease, and the current pharmacological treatment for DKD is limited to renin-angiotensin system (RAS) inhibitors. Adenosine is detectable in the kidney and is significantly elevated in response to cellular damage. While all 4 known subtypes of adenosine receptors, namely, A 1 AR, A 2a AR, A 2b AR, and A 3 AR, are expressed in the kidney, our previous study has demonstrated that a novel, orally active, species-independent, and selective A 3 AR antagonist, LJ-1888, ameliorates unilateral ureteral obstruction-induced tubulointerstitial fibrosis. The present study examined the protective effects of LJ-2698, which has higher affinity and selectivity for A 3 AR than LJ-1888, on DKD. In experiment I, dose-dependent effects of LJ-2698 were examined by orally administering 1.5, 5, or 10 mg/kg for 12 weeks to 8-week-old db/db mice. In experiment II, the effects of LJ-2698 (10 mg/kg) were compared to those of losartan (1.5 mg/kg), which is a standard treatment for patients with DKD. LJ-2698 effectively prevented kidney injuries such as albuminuria, glomerular hypertrophy, tubular injury, podocyte injury, fibrosis, inflammation, and oxidative stress in diabetic mice as much as losartan. In addition, inhibition of lipid accumulation along with increases in PGC1α, a master regulator of mitochondrial biogenesis, were demonstrated in diabetic mice treated with either LJ-2698 or losartan. These results suggest that LJ-2698, a selective A 3 AR antagonist, may become a novel therapeutic agent against DKD.

Background: NADPH oxidases (Nox) is a major enzyme system contributing to oxidative stress, which plays an important role in the pathogenesis of diabetic kidney disease (DKD). We have shown an elevation of renal Nox1, Nox2, and Nox4 in diabetic mice. APX-115, a pan-Nox inhibitor, attenuated the progression of DKD in mice. As the standard diabetic mice cannot fully mimic human DKD, the present study was aimed to show the dose-dependent effect and to provide a confirmatory evidence of APX-115 in attenuating DKD in diabetic rats., Method: Type 1 diabetes was induced by a single 60 mg/kg intraperitoneal injection of streptozotocin in Sprague-Dawley rats. 0.5, 5, or 30 mg APX-115/kg/day or losartan 1 mg/kg/day were administered orally to diabetic rats for 8 weeks., Results: APX-115 treatment showed an improvement in kidney function and tubular and podocyte -injury, as well as attenuation of inflammation, fibrosis, and oxidative stress as much as losartan, a comparative drug and mainstay treatment in DKD. Therapeutic effect of APX-115 was exhibited in a dose-dependent manner; a dose of 30 mg/kg displayed a superior efficacy., Conclusion: This finding verified the pre-clinical data of APX-115 in protecting against DKD, which is important to bring APX-115 toward the next stage of drug development., (© 2018 S. Karger AG, Basel.)

Fenofibrate activates not only peroxisome proliferator-activated receptor-α (PPARα) but also adenosine monophosphate-activated protein kinase (AMPK). AMPK-mediated cellular responses protect kidney from high-fat diet (HFD)-induced injury, and autophagy resulting from AMPK activation has been regarded as a stress-response mechanism. Thus the present study examined the role of AMPK and autophagy in the renotherapeutic effects of fenofibrate. C57BL/6J mice were divided into three groups: normal diet (ND), HFD, and HFD + fenofibrate (HFD + FF). Fenofibrate was administered 4 wk after the initiation of the HFD when renal injury was initiated. Mouse proximal tubule cells (mProx24) were used to clarify the role of AMPK. Feeding mice with HFD for 12 wk induced insulin resistance and kidney injury such as albuminuria, glomerulosclerosis, tubular injury, and inflammation, which were effectively inhibited by fenofibrate. In addition, fenofibrate treatment resulted in the activation of renal AMPK, upregulation of fatty acid oxidation (FAO) enzymes and antioxidants, and induction of autophagy in the HFD mice. In mProx24 cells, fenofibrate activated AMPK in a concentration-dependent manner, upregulated FAO enzymes and antioxidants, and induced autophagy, all of which were inhibited by treatment of compound C, an AMPK inhibitor. Fenofibrate-induced autophagy was also significantly blocked by AMPKα1 siRNA but not by PPARα siRNA. Collectively, these results demonstrate that delayed treatment with fenofibrate has a therapeutic effect on HFD-induced kidney injury, at least in part, through the activation of AMPK and induction of subsequent downstream effectors: autophagy, FAO enzymes, and antioxidants., (Copyright © 2017 the American Physiological Society.)

Diabetic nephropathy is the leading cause of end-stage renal disease worldwide, but no effective therapeutic strategy is available. Because plasminogen activator inhibitor-1 (PAI-1) is increasingly recognized as a key factor in extracellular matrix (ECM) accumulation in diabetic nephropathy, this study examined the renoprotective effects of TM5275 and TM5441, two novel orally active PAI-1 inhibitors that do not trigger bleeding episodes, in streptozotocin (STZ)-induced diabetic mice. TM5275 (50 mg/kg) and TM5441 (10 mg/kg) were administered orally for 16 weeks to STZ-induced diabetic and age-matched control mice. Relative to the control mice, the diabetic mice showed significantly increased (p < 0.05) plasma glucose and creatinine levels, urinary albumin excretion, kidney-to-bodyweight ratios, glomerular volume, and fractional mesangial area. Markers of fibrosis and inflammation along with PAI-1 were also upregulated in the kidney of diabetic mice, and treatment with TM5275 and TM5441 effectively inhibited albuminuria, mesangial expansion, ECM accumulation, and macrophage infiltration in diabetic kidneys. Furthermore, in mouse proximal tubular epithelial (mProx24) cells, both TM5275 and TM5441 effectively inhibited PAI-1-induced mRNA expression of fibrosis and inflammation markers and also reversed PAI-1-induced inhibition of plasmin activity, which confirmed the efficacy of the TM compounds as PAI-1 inhibitors. These data suggest that TM compounds could be used to prevent diabetic kidney injury.

Aims: The present study examined renoprotective effect of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) in diabetes. NRK-52E cells were utilized to determine the paracrine effect of hUCB-MSC., Methods: hUCB was harvested with the mother's consent. MSC obtained from the hUCB were injected through the tail vein. Growth arrested and synchronized NRK-52E cells were stimulated with transforming growth factor-β1 (TGF-β1) in the presence of hUCB-MSC conditioned media., Results: At 4 weeks after the streptozotocin (STZ) injection, diabetic rats showed significantly increased urinary protein excretion, renal and glomerular hypertrophy, fractional mesangial area, renal expression of TGF-β1 and α-smooth muscle actin, and collagen accumulation but decreased renal E-cadherin and bone morphogenic protein-7 expression, confirming diabetic renal injury. hUCB-MSC effectively prevented diabetic renal injury except renal and glomerular hypertrophy without a significant effect on blood glucose. CM-DiI-labeled hUCB-MSC and immunostaining of PKcs, a human nuclei antigen, confirmed a few engraftment of hUCB-MSC in diabetic kidneys. hUCB-MSC conditioned media inhibited TGF-β1-induced extracellular matrix upregulation and epithelial-to-mesenchymal transition in NRK-52E cells in a concentration-dependent manner., Conclusions: These results demonstrate the renoprotective effect of hUCB-MSC in STZ-induced diabetic rats possibly through secretion of humoral factors and suggest hUCB-MSC as a possible treatment modality for diabetic renal injury., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

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.

Background: Despite the beneficial effects of alagebrium (ALA), a putative advanced glycation end-product (AGE) breaker, on diabetic nephropathy, its renoprotective mechanisms are incompletely understood. Since oxidative stress exacerbates diabetic renal injury through interaction with AGE, the present study examined the antioxidative property of ALA in db/db mice, mesangial cells cultured under high glucose or H(2)O(2) and a test tube., Methods: ALA (2 mg/kg/day) was administered intraperitoneally for 12 weeks to 8-week-old db/m and db/db (D(ALA)E) mice or for 4 weeks to 16-week-old db/db mice (D(ALA)L). Oxidative stress markers (nitrotyrosine accumulation, expression and translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, cellular DCF-DA fluorescence) together with urinary albumin excretion and histological changes including mesangial expansion were measured. The concentration of H(2)O(2) in the presence and absence of ALA was measured by iodometric analysis in a test tube., Results: ALA significantly reduced not only urinary albumin excretion and renal pathological changes but also accumulation of pentosidine and nitrotyrosine and expression of NADPH oxidase subunits in db/db mice regardless of treatment protocol. In mesangial cells, ALA effectively prevented not only high glucose- but also H(2)O(2)-induced membrane translocation of NADPH oxidase subunit (p47 phox, p67 phox and rac1) and protein kinase C isoform (α, βI and βII) and Nox4 messenger RNA expression concomitant with cellular reactive oxygen species. Furthermore, ALA directly decreased H(2)O(2) in a test tube., Conclusion: ALA has both direct and indirect antioxidant effects that may play important roles in ALA's renoprotective effect in diabetic kidneys.

Oxidative stress defined as an excessive production of reactive oxygen species (ROS) surpassing existing antioxidative defense mechanisms plays a critical role in the development and progression of diabetic vascular complications including nephropathy. Over production of ROS in diabetic milieu is both a direct consequence of hyperglycemia and an indirect consequence through advanced glycation end products (AGEs) or mediators of glucotoxicity such as cytokines and growth factors. Among many pathways, nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase and mitochondrial dysfunction have been recognized as two major sources of ROS generation in diabetic kidneys, and NADPH oxidase-derived ROS has been shown to facilitate renal mitochondrial superoxide production in hyperglycemia. Low antioxidant bioavailability promotes cellular oxidative stress leading to additional cellular damage. Although large-scale clinical trials using classical antioxidants have failed to show a significant effect on the development of vascular complications in diabetes, new strategies targeting NF-E2-related factor 2, the primary transcription factor that controls the antioxidant response, mitochondrial dysfunction, or NADPH oxidase might provide a potential approach for the prevention and treatment of diabetic nephropathy., (Copyright © 2011 S. Karger AG, Basel.)

Excessive accumulation of extracellular matrix (ECM) in the kidneys and epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells contributes to the renal fibrosis that is associated with diabetic nephropathy. Histone deacetylase (HDAC) determines the acetylation status of histones and thereby controls the regulation of gene expression. This study examined the effect of HDAC inhibition on renal fibrosis induced by diabetes or transforming growth factor (TGF)-beta1 and determined the role of reactive oxygen species (ROS) as mediators of HDAC activation. In streptozotocin (STZ)-induced diabetic kidneys and TGF-beta1-treated normal rat kidney tubular epithelial cells (NRK52-E), we found that trichostatin A, a nonselective HDAC inhibitor, decreased mRNA and protein expressions of ECM components and prevented EMT. Valproic acid and class I-selective HDAC inhibitor SK-7041 also showed similar effects in NRK52-E cells. Among the six HDACs tested (HDAC-1 through -5 and HDAC-8), HDAC-2 activity significantly increased in the kidneys of STZ-induced diabetic rats and db/db mice and TGF-beta1-treated NRK52-E cells. Levels of mRNA expression of fibronectin and alpha-smooth muscle actin were decreased, whereas E-cadherin mRNA was increased when HDAC-2 was knocked down using RNA interference in NRK52-E cells. Interestingly, hydrogen peroxide increased HDAC-2 activity, and the treatment with an antioxidant, N-acetylcysteine, almost completely reduced TGF-beta1-induced activation of HDAC-2. These findings suggest that HDAC-2 plays an important role in the development of ECM accumulation and EMT in diabetic kidney and that ROS mediate TGF-beta1-induced activation of HDAC-2.

Background/aims: Plasminogen activator inhibitor (PAI)-1 is increasingly recognized as a profibrotic factor but the mechanisms are not entirely clear. The present study examined the profibrotic mechanism of PAI-1 focusing on its effect on transforming growth factor (TGF)-beta1 in experimental diabetes., Methods: PAI-1 knockout (KO) mesangial cells cultured under high glucose (HG) in addition to streptozotocin-induced diabetic PAI-1 KO mice were used., Results: PAI-1 deficiency did not affect plasma glucose significantly but reduced the fractional mesangial area, fibronectin and collagen I expression in the renal cortex after 20 weeks of diabetes as well as in HG-stimulated mesangial cells along with suppression of TGF-beta1 mRNA expression. PAI-1 deficiency also reduced HG-induced betaig-h3, a TGF-beta1-induced gene product, mRNA expression. All these losses-of-function in PAI-1 KO mesangial cells were effectively gained by recombinant PAI-1. Recombinant PAI-1-induced fibronectin and collagen I expression was abrogated by TGF-beta1 receptor inhibitor or anti-TGF-beta antibody suggesting that the effect of PAI-1 was mediated by TGF-beta1. In a similar context, recombinant PAI-1 stimulated TGF-beta1 promoter activity to the same extent as TGF-beta1 itself., Conclusion: Since TGF-beta1 is well known to stimulate the PAI-1 promoter, we suggest that TGF-beta1 and PAI-1 together constitute a positive feedback loop in the development of renal fibrosis in diabetes., (Copyright 2009 S. Karger AG, Basel.)

There is an increasing evidence that reactive oxygen species (ROS) play a major role in the development of diabetic complications. Oxidative stress is increased in diabetes and the overproduction of ROS in diabetes is a direct consequence of hyperglycemia. Various types of vascular cells including renal cells are able to produce ROS under hyperglycemic condition. Both NADPH oxidase and mitochondrial electron gradient play roles in hyperglycemia-induced ROS generation. In addition to their ability to directly inflict macromolecular damage, ROS can function as signaling molecules. ROS mediate hyperglycemia-induced activation of signal transduction cascades and transcription factors leading to transcriptional activation of profibrotic genes in the kidney. Furthermore, ROS-activated signaling molecules generate and signal through ROS and thus ROS act as a signal amplifier. Intensive glycemic control and inhibition of angiotensin II delay the onset and progression of diabetic nephropathy, in part, through prevention of overproduction of ROS. Conventional and catalytic antioxidants have been shown to prevent or delay the onset of diabetic nephropathy. Combination of strategies to prevent overproduction of ROS and to increase the removal of preformed ROS may prove to be effective in preventing the development and progression of diabetic nephropathy.

Lithospermic acid B (LAB), an active component isolated from Salvia miltiorrhizae, has been reported to have renoprotective effects in type 1 diabetic animal models. In the present study we investigated the effects of LAB on the prevention of diabetic nephropathy in type 2 diabetic Otsuka Long-Evans-Tokushima Fatty (OLETF) rats. LAB (20 mg/kg) was given orally once daily to 10-week-old male OLETF rats for 28 weeks. Treatment of OLETF rats with LAB had little effects on body weight and blood glucose levels. Treatment with LAB resulted in significant reduction in blood pressure. LAB markedly attenuated albuminuria and significantly lowered levels of lipid peroxidation, monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-beta (TGF-beta1) expression in renal tissues of OLETF rats. In addition, LAB inhibited the progression of glomerular hypertrophy, mesangial expansion, and expansion of the extracellular matrix in the renal cortex. Collectively, these results suggest that LAB has beneficial effects on the diabetic nephropathy in OLETF rats by decreasing blood pressure, oxidative stress, and MCP-1 expression. Our results suggest that LAB might be a new therapeutic agent for the prevention of nephropathy in type 2 diabetes.

Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) in the kidney. Decreased ECM degradation as well as increased ECM synthesis plays an important role in ECM remodeling that favours tissue fibrosis. Plasminogen activator (PA)/plasmin/PA inhibitor (PAI) system is involved in ECM degradation and PAI-1 plays a critical role in ECM remodeling in the kidney. Normal human kidneys do not express PAI-1 but PAI-1 is overexpressed in pathologic conditions associated with renal fibrosis including diabetic nephropathy. Reactive oxygen species mediate PAI-1 up-regulation in renal cells cultured under high glucose, hypoxia, and TGF-beta1. Recent studies utilizing PAI-1 deficient mice suggest that PAI-1 induce ECM deposition in diabetic kidney through increased ECM synthesis by TGF-beta1 up-regulation as well as through decreased ECM degradation by suppression of plasmin and MMP-2 activity.

Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) in the kidney. Reactive oxygen species (ROS) play a central role in the ECM synthesis and degradation in the glomeruli and tubulointerstitium leading to renal fibrosis. High glucose (HG) induces cellular ROS through protein kinase C (PKC)-dependent activation of NADPH oxidase and through mitochondrial metabolism. ROS thus generated activate signal transduction cascade (PKC, mitogen-activated protein kinases, and janus kinase/signal transducers and activators of transcription) and transcription factors (nuclear factor-kappaB, activated protein-1, and specificity protein-1), up-regulate transforming growth factor-beta1 (TGF-beta1), angiotensin II (Ang II), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) gene and protein expression, and promote formation of advanced glycation end-products (AGE). PKC, TGF-beta1, Ang II, and AGE also induce cellular ROS and signal through ROS leading to enhanced ECM synthesis. NF-kappaB-MCP-1 pathway is activated by ROS and promotes monocyte recruitment and profibrotic process in the kidney. HG- and TGF-beta1-induced PAI-1 up-regulation is mediated by ROS and contribute to ECM accumulation via suppression of plasmin ativity. TGF-beta1-induced myofibroblast transformation of renal tubular epithelial cells (epithelial-mesenchymal transition) is also mediated by ROS and contribute to tubulointerstitial fibrosis. In summary, ROS transduce and amplify glucose signalling in renal cells under high glucose environment and play a critical role in excessive ECM deposition in the diabetic kidney. A better understanding of ROS production and removal will allow more effective therapeutic strategies in diabetic renal and other vascular complications.

Background: Plasminogen activator inhibitor-1 (PAI-1) plays an important role in remodeling of extracellular matrix (ECM) in the glomeruli. PAI-1 is up-regulated by high glucose and is overexpressed in diabetic kidney. Since reactive oxygen species (ROS) mediate ECM accumulation in diabetic glomeruli and was recently found to mediate transforming growth factor-beta1 (TGF-beta1)-induced PAI-1 up-regulation in glomerular mesangial cells, we examined the role of ROS in high glucose-induced PAI-1 expression in cultured glomerular mesangial cells and in streptozotocin-induced diabetic rat glomeruli., Methods: Growth arrested and synchronized primary rat mesangial cells were treated with different concentrations of glucose in the presence or absence of N-acetylcysteine (NAC) or trolox, or after cellular reduced form of glutathione (GSH) depleted with DL-buthionine-(S,R)-sulfoximine (BSO). Taurine was administered to diabetic rats from 2 days to 4 weeks after streptozotocin injection. Urinary protein excretion, glomerular volume, and fractional mesangial area were measured as markers of renal injury and lipid peroxide (LPO) as an oxidative stress marker. PAI-1 mRNA expression was measured by Northern blot analysis in mesangial cells and reverse transcription-polymerase chain reaction (RT-PCR) in glomeruli, PAI-1 protein by Western blot analysis and enzyme-linked immunosorbent assay (ELISA), and plasmin activity by fluorometry., Results: High glucose significantly increased PAI-1 mRNA and protein expression and decreased plasmin activity in mesangial cells. Equimolar concentrations of l-glucose or mannitol did not affect PAI-1 expression. BSO pretreatment significantly increased basal PAI-1 expression and amplified the response to high glucose. NAC effectively inhibited high glucose-induced, but not basal, PAI-1 expression. Reduced plasmin activity in mesangial cells by high glucose was rescued by antioxidants. Anti-TGF-beta antibody inhibited both high glucose- and H(2)O(2)-induced PAI-1 up-regulation. Taurine significantly reduced plasma LPO, glomerular PAI-1 expression, glomerular volume, fractional mesangial area, and proteinuria in streptozotocin-induced diabetic rats., Conclusion: These results demonstrate that ROS mediate high glucose-induced up-regulation of PAI-1 expression in cultured mesangial cells and in diabetic glomeruli. Since both high glucose and TGF-beta1 induce cellular ROS and ROS mediate both high glucose- and TGF-beta1-induced PAI-1, ROS appear to amplify TGF-beta1 signaling in high glucose-induced PAI-1 up-regulation. Antioxidants can prevent accumulation of ECM protein in diabetic glomeruli partly by abrogating up-regulation of PAI-1 and suppression of plasmin activity.

Excessive deposition of extracellular matrix (ECM) in the kidney is the hallmark of diabetic nephropathy. Although the amount of ECM deposited in the kidney depends on the balance between the synthesis and degradation of ECM, the role of ECM degradation in matrix remodeling has been less well appreciated. High glucose, advanced glycation end products, angiotensin II, and TGF-beta1 all increase intracellular reactive oxygen species (ROS) in renal cells and contribute to the development and progression of diabetic renal injury. The role of ROS in increased ECM synthesis has been well documented. ROS may also play a critical role in decreased ECM degradation by mediating high glucose- and TGF-beta1-induced inhibition of the proteolytic system, plasmin, and matrix metalloproteinases in the glomeruli. A recent observation suggests that ROS play an important role in tubulointerstitial fibrosis by mediating TGF-beta1-induced epithelial-mesenchymal transition (EMT). Accelerated ECM degradation is required to disrupt tubular basement membrane and complete EMT. ROS thus seem to be involved in both decreased and increased ECM degradation. It is not clear how cells determine when and where to increase or decrease ECM degradation in response to ROS. Precise definition of ROS-activated signaling pathways leading to ECM remodeling in the kidney will provide new strategies to prevent or treat diabetic renal injury.

Diabetic nephropathy is characterized by excessive deposition of extracellular matrix (ECM) in the kidney. TGF-beta1 has been identified as the key mediator of ECM accumulation in diabetic kidney. High glucose induces TGF-beta1 in glomerular mesangial and tubular epithelial cells and in diabetic kidney. Antioxidants inhibit high glucose-induced TGF-beta1 and ECM expression in glomerular mesangial and tubular epithelial cells and ameliorate features of diabetic nephropathy, suggesting that oxidative stress plays an important role in diabetic renal injury. High glucose induces intracellular reactive oxygen species (ROS) in mesangial and tubular epithelial cells. High glucose-induced ROS in mesangial cells can be effectively blocked by inhibition of protein kinase C (PKC), NADPH oxidase, and mitochondrial electron transfer chain complex I, suggesting that PKC, NADPH oxidase, and mitochondrial metabolism all play a role in high glucose-induced ROS generation. Advanced glycation end products, TGF-beta1, and angiotensin II can also induce ROS generation and may amplify high glucose-activated signaling in diabetic kidney. Both high glucose and ROS activate signal transduction cascade (PKC, mitogen-activated protein kinases, and janus kinase/signal transducers and activators of transcription) and transcription factors (nuclear factor-kappaB, activated protein-1, and specificity protein 1) and upregulate TGF-beta1 and ECM genes and proteins. These observations suggest that ROS act as intracellular messengers and integral glucose signaling molecules in diabetic kidney. Future studies elucidating various other target molecules activated by ROS in renal cells cultured under high glucose or in diabetic kidney will allow a better understanding of the final cellular responses to high glucose.

Oxidative stress has been known to play an important role in the development and progression of diabetic nephropathy, but the intracellular signal transduction pathways regulated by reactive oxygen species (ROS) have not been clearly defined. High glucose (HG) induces intracellular ROS directly via glucose metabolism and auto-oxidation and indirectly through the formation of advanced glycation end products and their receptor binding. ROS mimic the stimulatory effects of HG and upregulate transforming growth factor-beta 1, plasminogen activator inhibitor-1, and extracellular matrix (ECM) proteins by glomerular mesangial cells, thus leading to mesangial expansion. ROS activate other signaling molecules, such as protein kinase C and mitogen-activated protein kinases and transcription factors, such as nuclear factor-kappa B, activator protein-1, and specificity protein 1 leading to transcription of genes encoding cytokines, growth factors, and ECM proteins. Finally, various antioxidants inhibit mesangial cell activation by HG and ameliorate features of diabetic nephropathy. These findings qualify ROS as intracellular messengers and as integral glucose-signaling molecules in glomerular mesangial cells in diabetic nephropathy. With this new concept, ROS assume a greater importance in the pathogenesis of diabetic nephropathy. Future studies elucidating other downstream-signaling molecules activated by ROS in mesangial and other renal cells will allow us to understand the final cellular responses to HG, such as proliferation, differentiation, apoptosis, and ECM accumulation. With this new information, we should be able to develop strategies for a more rational treatment of diabetic nephropathy. [ABSTRACT FROM AUTHOR]

The aim of this study is to investigate the role of the proximal tubule in microalbuminuria in the early stage of diabetic nephropathy. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (50 mg/kg, i.v.). After 2 weeks, albumin delivery in the proximal tubule was measured using micropuncture and the endocytosis process of FITC-labeled albumin was evaluated with immunoelectron microscopy. Albumin was significantly reabsorbed in the proximal convoluted tubule (PCT) of controls (0.39±0.05 ng/min at early PCT to 0.17±0.08 at late PCT, P<0.05), whereas albumin reabsorption was inhibited in diabetic rats (0.27±0.05 to 0.21±0.08). Immunogold study revealed that FITC-albumin was significantly less reabsorbed in endosomes and lysosomes of S1 segments in diabetic rats than in controls (endosome: 1.20±0.10 vs 2.16±0.15 µm–1, P<0.0001; lysosome: 0.26±0.03 vs 0.83±0.07, P<0.0001). The expression of megalin, an endocytosis receptor, was decreased at the apical membrane of PCT in diabetic rats. The lipid peroxidation production in the proximal tubule was significantly increased in diabetic rats. In conclusion, albuminuria in early-stage diabetic rats can be partly explained by a decreased albumin endocytosis with reduced megalin expression and with increased lipid peroxidation in the proximal tubule. [ABSTRACT FROM AUTHOR]