Diabetic Glomeruli Research Articles

Chrysin Inhibits Advanced Glycation End Products-Induced Kidney Fibrosis in Renal Mesangial Cells and Diabetic Kidneys.

Advanced glycation end products (AGEs) play a causative role in the development of diabetic nephropathy via induction of matrix protein deposition in kidneys. This study investigated inhibitory effects of chrysin, present in bee propolis and herbs, on glomerulosclerosis in db/db mice and AGEs-exposed renal mesangial cells. The in vivo study explored the demoting effects of 10 mg/kg chrysin on glomerular fibrosis in a type 2 diabetic model. Oral supplementation of chrysin inhibited the collagen fiber accumulation and α-smooth muscle actin (α-SMA) induction in periodic acid schiff-positive renal tissues of db/db mice. Moreover, treating db/db mice with chrysin diminished the level of AGEs increased in diabetic glomeruli. The in vitro study employed human mesangial cells exposed to 100 μg/mL AGE-BSA for 72 h in the presence of 1–20 μM chrysin. Glucose increased mesangial AGE production via induction of receptor for AGEs. Chrysin suppressed the induction of collagens, α-SMA, fibroblast-specific protein-1 and matrix metalloproteinases enhanced by AGE-bovine serum albumin. Furthermore, chrysin blunted transforming growth factor-β1 induction and Smad 2/3 activation in AGEs-exposed mesangial cells. These results demonstrate that chrysin attenuated accumulation of myofibroblast-like cells and matrix proteins in AGEs-laden diabetic glomeruli. Therefore, chrysin may be a potential renoprotective agent targeting glucose-mediated AGEs-associated glomerulosclerosis and fibrosis.

Protein S Protects against Podocyte Injury in Diabetic Nephropathy.

Background Diabetic nephropathy (DN) is a leading cause of ESRD in the United States, but the molecular mechanisms mediating the early stages of DN are unclear.Methods To assess global changes that occur in early diabetic kidneys and to identify proteins potentially involved in pathogenic pathways in DN progression, we performed proteomic analysis of diabetic and nondiabetic rat glomeruli. Protein S (PS) among the highly upregulated proteins in the diabetic glomeruli. PS exerts multiple biologic effects through the Tyro3, Axl, and Mer (TAM) receptors. Because increased activation of Axl by the PS homolog Gas6 has been implicated in DN progression, we further examined the role of PS in DN.Results In human kidneys, glomerular PS expression was elevated in early DN but suppressed in advanced DN. However, plasma PS concentrations did not differ between patients with DN and healthy controls. A prominent increase of PS expression also colocalized with the expression of podocyte markers in early diabetic kidneys. In cultured podocytes, high-glucose treatment elevated PS expression, and PS knockdown further enhanced the high-glucose-induced apoptosis. Conversely, PS overexpression in cultured podocytes dampened the high-glucose- and TNF-α-induced expression of proinflammatory mediators. Tyro3 receptor was upregulated in response to high glucose and mediated the anti-inflammatory response of PS. Podocyte-specific PS loss resulted in accelerated DN in streptozotocin-induced diabetic mice, whereas the transient induction of PS expression in glomerular cells in vivo attenuated albuminuria and podocyte loss in diabetic OVE26 mice.Conclusions Our results support a protective role of PS against glomerular injury in DN progression.

Increased podocyte Sirtuin-1 function attenuates diabetic kidney injury

Podocyte injury and loss contribute to the progression of glomerular diseases, including diabetic kidney disease. We previously found that the glomerular expression of Sirtuin-1 (SIRT1) is reduced in human diabetic glomeruli and that the podocyte-specific loss of SIRT1 aggravated albuminuria and worsened kidney disease progression in diabetic mice. SIRT1 encodes an NAD-dependent deacetylase that modifies the activity of key transcriptional regulators affected in diabetic kidneys, including NF-κB, STAT3, p53, FOXO4, and PGC1-α. However, whether the increased glomerular SIRT1 activity is sufficient to ameliorate the pathogenesis of diabetic kidney disease has not been explored. We addressed this by inducible podocyte-specific SIRT1 overexpression in diabetic OVE26 mice. The induction of SIRT1 overexpression in podocytes for six weeks in OVE26 mice with established albuminuria attenuated the progression of diabetic glomerulopathy. To further validate the therapeutic potential of increased SIRT1 activity against diabetic kidney disease, we developed a new, potent and selective SIRT1 agonist, BF175. In cultured podocytes BF175 increased SIRT1-mediated activation of PGC1-α and protected against high glucose-mediated mitochondrial injury. Invivo, administration of BF175 for six weeks in OVE26 mice resulted in a marked reduction in albuminuria and in glomerular injury in a manner similar to podocyte-specific SIRT1 overexpression. Both podocyte-specific SIRT1 overexpression and BT175 treatment attenuated diabetes-induced podocyte loss and reduced oxidative stress in glomeruli of OVE26 mice. Thus, increased SIRT1 activity protects against diabetes-induced podocyte injury and effectively mitigates the progression of diabetic kidney disease.

Urinary Exosomal mRNA of WT1 as Diagnostic and Prognostic Biomarker for Diabetic Nephropathy.

Diabetic nephropathy (DN) is the major cause of end-stage renal failure and is associated with increased morbidity and mortality as compared to other causes of renal disease. Albuminuria is often the first clinical indicator of the presence of DN. However, albuminuria or proteinuria is a common symptom in patients with various renal disorders. Therefore, specific biomarkers for the diagnosis of DN are required. A primary hallmark of DN is the progressive damage and death of glomerular podocytes, resulting in the leaking of proteins into the urine. Urinary exosomes released by podocytes are microvesicles containing information of the originated cells. Podocyte-derived signal transduction factors (PDSTFs) are good candidates to assess podocyte injuries. The profile of PDSTFs in urinary exosomes from patients with DN is different from that from patients with minimal change nehrotic syndrome. In addition, PDSTFs molecules in exosomes were derived from primary murine podocytes under high glucose conditions. Among PDSTFs in urinary exosomes, Wilms tumor 1 (WT1) levels reflected damage of diabetic glomeruli in the patients. Urinary exosomal WT1 can predict the decline in eGFR for the following several years. In conclusion, urinary exosomal WT1 is a useful biomarker to improve risk stratification in patients with DN. J. Med. Invest. 65:208-215, August, 2018.

Chrysin ameliorates podocyte injury and slit diaphragm protein loss via inhibition of the PERK-eIF2α-ATF-CHOP pathway in diabetic mice.

Glomerular epithelial podocytes are highly specialized cells that play a crucial role in maintaining normal function of the glomerular filtration barrier via their foot processes. Chrysin (5,7-dihydroxyflavone) is a natural flavonoid found in propolis and mushrooms that has anti-inflammatory, antioxidant and anticancer properties. This study aimed to evaluate the renoprotective effects of chrysin on podocyte apoptotic loss and slit diaphragm protein deficiency in high glucose-exposed podocytes and in db/db mouse kidneys. Exposure to high glucose (33 mmol/L) caused glomerular podocyte apoptosis in vitro, which was dose-dependently attenuated by nontoxic chrysin (1-20 μmol/L) through reduction of DNA fragmentation. Chrysin treatment dose-dependently restored the increased Bax/Bcl-2 ratio, and suppressed Apaf-1 induction and the elevated cytochrome c release in high glucose-exposed renal podocytes. In diabetic db/db mice, oral administration of chrysin (10 mg·kg-1·d-1, for 10 weeks) significantly attenuated proteinuria, and alleviated the abnormal alterations in glomerular ultrastructure, characterized by apoptotic podocytes and foot process effacement. In addition, this compound improved the induction of slit diaphragm proteins podocin/nephrin in the diabetic glomeruli. Exposure to high glucose elevated the unfolded protein response (UPR) to ER stress in renal podocytes, evidenced by up-regulation of PERK-eIF2α-ATF4-CHOP. Chrysin treatment blocked such ER stress responses pertinent to podocyte apoptosis and reduced synthesis of slit diaphragm proteins in vitro and in vivo. These observations demonstrate that targeting ER stress is an underlying mechanism of chrysin-mediated amelioration of diabetes-associated podocyte injury and dysfunction.

Plasmin reduces fibronectin deposition by mesangial cells in a protease-activated receptor-1 independent manner

BackgroundProtease-activated receptor-1 (PAR-1) potentiates diabetic nephropathy (DN) as evident from reduced kidney injury in diabetic PAR-1 deficient mice. Although thrombin is the prototypical PAR-1 agonist, anticoagulant treatment does not limit DN in experimental animal models suggesting that thrombin is not the endogenous PAR-1 agonist driving DN. ObjectivesTo identify the endogenous PAR-1 agonist potentiating diabetes-induced nephropathy. MethodsUnbiased protease expression profiling in glomeruli from human kidneys with DN was performed using publically available microarray data. The identified prime candidate PAR-1 agonist was subsequently analysed for PAR-1-dependent induction of fibrosis in vitro. ResultsOf the 553 proteases expressed in the human genome, 247 qualified as potential PAR-1 agonists of which 71 were significantly expressed above background in diabetic glomeruli. The recently identified PAR-1 agonist plasmin(ogen), together with its physiological activator tissue plasminogen activator, were among the highest expressed proteases. Plasmin did however not induce mesangial proliferation and/or fibronectin deposition in vitro. In a PAR-1 independent manner, plasmin even reduced fibronectin deposition. ConclusionExpression profiling identified plasmin as potential endogenous PAR-1 agonist driving DN. Instead of inducing fibronectin expression, plasmin however reduced mesangial fibronectin deposition in vitro. Therefore we conclude that plasmin may not be the endogenous PAR-1 agonist potentiating DN.

Antiangiogenic Therapy for Diabetic Nephropathy.

Angiogenesis has been shown to be a potential therapeutic target for early stages of diabetic nephropathy in a number of animal experiments. Vascular endothelial growth factor (VEGF) is the main mediator for abnormal angiogenesis in diabetic glomeruli. Although beneficial effects of anti-VEGF antibodies have previously been demonstrated in diabetic animal experiments, recent basic and clinical evidence has revealed that the blockade of VEGF signaling resulted in proteinuria and renal thrombotic microangiopathy, suggesting the importance of maintaining normal levels of VEGF in the kidneys. Therefore, antiangiogenic therapy for diabetic nephropathy should eliminate excessive glomerular angiogenic response without accelerating endothelial injury. Some endogenous antiangiogenic factors such as endostatin and tumstatin inhibit overactivation of endothelial cells but do not specifically block VEGF signaling. In addition, the novel endothelium-derived antiangiogenic factor vasohibin-1 enhances stress tolerance and survival of the endothelial cells, while inhibiting excess angiogenesis. These factors have been demonstrated to suppress albuminuria and glomerular alterations in a diabetic mouse model. Thus, antiangiogenic therapy with promising candidates will possibly improve renal prognosis in patients with early stages of diabetic nephropathy.

Septin 7 reduces nonmuscle myosin IIA activity in the SNAP23 complex and hinders GLUT4 storage vesicle docking and fusion

Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex, as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane.

Crucial genes associated with diabetic nephropathy explored by microarray analysis.

BackgroundThis study sought to investigate crucial genes correlated with diabetic nephropathy (DN), and their potential functions, which might contribute to a better understanding of DN pathogenesis.MethodsThe microarray dataset GSE1009 was downloaded from Gene Expression Omnibus, including 3 diabetic glomeruli samples and 3 healthy glomeruli samples. The differentially expressed genes (DEGs) were identified by LIMMA package. Their potential functions were then analyzed by the GO and KEGG pathway enrichment analyses using the DAVID database. Furthermore, miRNAs and transcription factors (TFs) regulating DEGs were predicted by the GeneCoDis tool, and miRNA-DEG-TF regulatory network was visualized by Cytoscape. Additionally, the expression of DEGs was validated using another microarray dataset GSE30528.ResultsTotally, 14 up-regulated DEGs and 430 down-regulated ones were identified. Some DEGs (e.g. MTSS1, CALD1 and ACTN4) were markedly relative to cytoskeleton organization. Besides, some other ones were correlated with arrhythmogenic right ventricular cardiomyopathy (e.g. ACTN4, CTNNA1 and ITGB5), as well as complement and coagulation cascades (e.g. C1R and C1S). Furthermore, a series of miRNAs and TFs modulating DEGs were identified. The transcription factor LEF1 regulated the majority of DEGs, such as ITGB5, CALD1 and C1S. Hsa-miR-33a modulated 28 genes, such as C1S. Additionally, 143 DEGs (one upregulated gene and 142 downregulated genes) were also differentially expressed in another dataset GSE30528.ConclusionsThe genes involved in cytoskeleton organization, cardiomyopathy, as well as complement and coagulation cascades may be closely implicated in the progression of DN, via the regulation of miRNAs and TFs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12882-016-0343-2) contains supplementary material, which is available to authorized users.

Persistent Insulin Resistance in Podocytes Caused by Epigenetic Changes of SHP-1 in Diabetes.

Poor glycemic control profoundly affects protein expression and the cell signaling action that contributes to glycemic memory and irreversible progression of diabetic nephropathy (DN). We demonstrate that SHP-1 is elevated in podocytes of diabetic mice, causing insulin unresponsiveness and DN. Thus, sustained SHP-1 expression caused by hyperglycemia despite systemic glucose normalization could contribute to the glycemic memory effect in DN. Microalbuminuria, glomerular filtration rate, mesangial cell expansion, and collagen type IV and transforming growth factor-β expression were significantly increased in diabetic Ins2+/C96Y mice compared with nondiabetic Ins2+/+ mice and remained elevated despite glucose normalization with insulin implants. A persistent increase of SHP-1 expression in podocytes despite normalization of systemic glucose levels was associated with sustained inhibition of the insulin signaling pathways. In cultured podocytes, high glucose levels increased mRNA, protein expression, and phosphatase activity of SHP-1, which remained elevated despite glucose concentration returning to normal, causing persistent insulin receptor-β inhibition. Histone posttranslational modification analysis showed that the promoter region of SHP-1 was enriched with H3K4me1 and H3K9/14ac in diabetic glomeruli and podocytes, which remained elevated despite glucose level normalization. Hyperglycemia induces SHP-1 promoter epigenetic modifications, causing its persistent expression and activity and leading to insulin resistance, podocyte dysfunction, and DN.

Coagulation Factor Xa and Protease-Activated Receptor 2 as Novel Therapeutic Targets for Diabetic Nephropathy.

The role of hypercoagulability in the pathogenesis of diabetic nephropathy (DN) remains elusive. We recently reported the increased infiltration of macrophages expressing tissue factor in diabetic kidney glomeruli; tissue factor activates coagulation factor X (FX) to FXa, which in turn stimulates protease-activated receptor 2 (PAR2) and causes inflammation. Here, we demonstrated that diabetes mellitus increased renal FX mRNA, urinary FXa activity, and FX expression in glomerular macrophages. Administration of an oral FXa inhibitor, edoxaban, ameliorated DN with concomitant reductions in the expression of PARs (Par1 and Par2) and of proinflammatory and profibrotic genes. Diabetes mellitus induced PAR2, and lack of Par2 ameliorated DN. FXa or PAR2 agonist increased inflammatory cytokines in endothelial cells and podocytes in vitro. We conclude that enhanced FXa and PAR2 exacerbate DN and that both are promising targets for preventing DN. Alleviating inflammation is probably more important than inhibiting coagulation per se when treating kidney diseases using anticoagulants.

Effect of angiotensin II type 1 receptor blocker on 12-lipoxygenase activity and slit diaphragm protein expression in type 2 diabetic rat glomeruli.

The 12-lipoxygenase (12-LO) and angiotensin II (Ang II) interaction plays an important role in diabetic nephropathy (DN). Proteinuria in DN is associated with decreased slit diaphragm proteins including nephrin and P-cadherin. Therefore, we investigated whether Ang II type 1 receptor (AT1) blocker (ARB) regulates 12-LO activity and slit diaphragm protein expression in diabetic rat glomeruli. Glomeruli were isolated with the sieving method, and classified into small glomeruli (SG; 75-μm sieve) and large glomeruli (LG; 125-μm sieve). 12(S)-HETE, a lipid product of 12-LO, was increased by Ang II in the glomeruli. Infusion of 12(S)-HETE and Ang II significantly decreased nephrin expression in LG, but increased it in SG compared to control. Glomerular P-cadherin expression was reduced after Ang II and 12(S)-HETE treatment without differences between LG and SG. ARB did not influence glycemic levels but completely abolished the increases in 12(S)-HETE, AT1 expression, and proteinuria in diabetic rats. Nephrin expression was significantly reduced in LG but increased in SG in diabetic rats compared to control. P-cadherin expression decreased in both diabetic LG and SG. The abnormalities of nephrin and P-cadherin were partially but significantly reversed by ARB. ARB potentially ameliorates DN via the up-regulation of glomerular nephrin and P-cadherin expression through the inhibition of 12-LO activation in the glomeruli of rats with DN.

Olmesartan Prevents Microalbuminuria in db/db Diabetic Mice Through Inhibition of Angiotensin II/p38/SIRT1-Induced Podocyte Apoptosis

Background/Aims: Blockage of the renin-angiotensin II system (RAS) prevents or delays albuminuria in diabetic patients. The aim of this study was to investigate the inhibitory mechanism of the angiotensin receptor blocker olmesartan on albuminuria in a murine model of diabetic nephropathy. Methods: Male db/db diabetic mice were fed with placebo or 20 mg/kg olmesartan by daily gavage for 12 weeks. Conditionally immortalized mouse podocytes were treated with glucose, angiotensin II, olmesartan or p38 inhibitor s8307 in different experimental conditions after differentiation. Results: Olmesartan reduced albuminuria in db/db mice without change in body weight and glycemia. The increase of apoptotic cells and decrease of podocytes in the diabetic glomerulus were prevented by olmesartan. Moreover, olmesartan restored silent mating type information regulation 1 (SIRT1) expression in diabetic glomeruli. Furthermore, olmesartan treatment suppressed p38 phosphorylation but did not restore adenosine 5‘-monophosphate-activated protein kinase (AMPK) phosphorylation in the diabetic glomerulus. In vitro study revealed that olmesartan prevented angiotensin II/p38/SIRT1 induced podocyte apoptosis, but it only slightly prevented high glucose/AMPK/SIRT1 induced podocyte apoptosis. In addition, the p38 inhibitor s8307 reversed SIRT1 expression and angiotensin II induced podocyte apoptosis. Conclusions: Olmesartan reduced albuminuria in diabetic nephropathy through inhibiting angiotensin II/p38/SIRT1 triggered podocyte apoptosis.

Involvement of Histone Lysine Methylation in p21 Gene Expression in Rat Kidney In Vivo and Rat Mesangial Cells In Vitro under Diabetic Conditions.

Diabetic nephropathy (DN), a common complication associated with type 1 and type 2 diabetes mellitus (DM), characterized by glomerular mesangial expansion, inflammation, accumulation of extracellular matrix (ECM) protein, and hypertrophy, is the major cause of end-stage renal disease (ESRD). Increasing evidence suggested that p21-dependent glomerular and mesangial cell (MC) hypertrophy play key roles in the pathogenesis of DN. Recently, posttranscriptional modifications (PTMs) have uncovered novel molecular mechanisms involved in DN. However, precise regulatory mechanism of histone lysine methylation (HKme) mediating p21 related hypertrophy associated with DN is not clear. We evaluated the roles of HKme and histone methyltransferase (HMT) SET7/9 in p21 gene expression in glomeruli of diabetic rats and in high glucose- (HG-) treated rat mesangial cells (RMCs). p21 gene expression was upregulated in diabetic rats glomeruli; chromatin immunoprecipitation (ChIP) assays showed decreased histone H3-lysine9-dimethylation (H3K9me2) accompanied with enhanced histone H3-lysine4-methylation (H3K4me1/3) and SET7/9 occupancies at the p21 promoter. HG-treated RMCs exhibited increased p21 mRNA, H3K4me level, SET7/9 recruitment, and inverse H3K9me, which were reversed by TGF-β1 antibody. These data uncovered key roles of H3Kme and SET7/9 responsible for p21 gene expression in vivo and in vitro under diabetic conditions and confirmed preventive effect of TGF-β1 antibody on DN.

Inhibition of calcineurin/NFAT pathway plays an essential role in renoprotective effect of tropisetron in early stage of diabetic nephropathy

Recent studies have shown that calcineurin plays a central role in hypertrophy and extracellular matrix (ECM) accumulation in glomeruli at the early stages of diabetic nephropathy. Tropisetron is an effective antiemetic drug which also can potently inhibit calcineurin. The aim of this study was to investigate whether tropisetron can prevent glomerular hypertrophy and ECM expansion in early diabetic nephropathy. Streptozotocin (STZ)-induced diabetic rats were treated with tropisetron and cyclosporine A, a pharmacological calcineurin inhibitor, and the renal function and the expression of calcineurin and fibronectin were then assessed as well as nuclear localization of nuclear factor of activated T-cell c1 (NFATc1). 2 weeks after diabetes induction, all STZ-treated rats showed hyperglycemia, polyuria, body weight loss and renal dysfunction, as evidenced by increased glomerular filtration rate (GFR), along with a marked pathological changes in kidney. Calcineurin expression was increased in association with increased nuclear localization of the calcineurin substrate NFATc1 and fibronectin expression in glomeruli of diabetic rats. In parallel, the diabetic glomeruli became hypertrophic with an increase in kidney weight. Tropisetron, as potent as cyclosporine A, significantly ameliorated the early nephropathy symptoms, potentially through suppression of calcineurin expression, nuclear localization of NFATc1 and accumulation of fibronectin, and thereby reduced hypertrophy in glomeruli of diabetic rats.In conclusion, our results showed that tropisetron could ameliorate kidney injury in the early stage of diabetic nephropathy in rats. The renoprotective effects of tropisetron can be attributed, at least in part, to the suppression of diabetes-induced increases in calcineurin expression in kidney tissue.

ATP synthase subunit-β down-regulation aggravates diabetic nephropathy.

In this study, we investigated the role of ATP synthase subunit-β (ATP5b) in diabetic nephropathy. Histopathological changes, fibrosis, and protein expressions of α-smooth muscle actin (α-SMA), advanced glycation end-products (AGEs), and ATP5b were obviously observed in the kidneys of db/db diabetic mice as compared with the control db/m+ mice. The increased ATP5b expression was majorly observed in diabetic renal tubules and was notably observed to locate in cytoplasm of tubule cells, but no significant increase of ATP5b in diabetic glomeruli. AGEs significantly increased protein expression of ATP5b and fibrotic factors and decreased ATP content in cultured renal tubular cells via an AGEs-receptor for AGEs (RAGE) axis pathway. Oxidative stress was also induced in diabetic kidneys and AGEs-treated renal tubular cells. The increase of ATP5b and CTGF protein expression in AGEs-treated renal tubular cells was reversed by antioxidant N-acetylcysteine. ATP5b-siRNA transfection augmented the increased protein expression of α-SMA and CTGF and CTGF promoter activity in AGEs-treated renal tubular cells. The in vivo ATP5b-siRNA delivery significantly enhanced renal fibrosis and serum creatinine in db/db mice with ATP5b down-regulation. These findings suggest that increased ATP5b plays an important adaptive or protective role in decreasing the rate of AGEs-induced renal fibrosis during diabetic condition.

Potential biomarkers associated with diabetic glomerulopathy through proteomics

Diabetic nephropathy (DN) is characterized by the development of progressive glomerulosclerotic lesions gradually leading to an increasing loss of functioning kidney parenchyma. Relatively little proteomic research of isolated glomeruli of experimental animal models has been done so far. Isolated glomerular proteomics is an innovative tool that potentially detects simultaneous expressions of glomeruli in diabetic pathological contexts. We compared the isolated glomerular profiles of rats with and without diabetes. The proteins in the aliquots of glomeruli were subjected to two-dimensional gel electrophoresis. The protein spots were matched and quantified using an imaging analysis system. The peptide mass fingerprints were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and a bioinformation search. We found that diabetes increased collagen type I and collagen type IV levels in diabetic glomeruli when compared to normal control group using Dynabeads. We found that rats with diabetes had significantly higher abundance of the Protein disulfide isomerase associated 3, Aspartoacylase-3,3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase, Lactamase beta 2 and Agmat protein. However, diabetic glomeruli in rats had significantly lower levels of the Regucalcin, rCG52140, Aldo-keto reductase family 1, Peroxiredoxin 1, and l-arginine: glycine amidinotransferase. These proteins of interest were reported to modulate disturbances in the homeostasis of endoplasmic reticulum stress, disturbance of inflammatory and fibrinogenic activities, impairing endothelial function, and dysregulation in the antioxidation capacity/oxidative stress in several tissue types under pathological contexts. Taken together, our high-throughput isolated glomerular proteomic findings indicated that multiple pathological reactions presumably occurred in DN.

P16ink4a Expression Is Increased through 12-Lipoxygenase in High Glucose-Stimulated Glomerular Mesangial Cells and Type 2 Diabetic Glomeruli

Background/Aims: Arachidonic acid-metabolizing enzyme, 12-lipoxygenase (12-LO), is involved in the glomerular hypertrophy of diabetic nephropathy (DN), in which cyclin-dependent kinase inhibitors (CKIs) play important roles. However, it is unclear whether 12-LO regulates the expression of the CKI p16^ink4a in DN. Methods: Primary glomerular mesangial cells (MCs) and glomeruli isolated from rats were used in this study. The rats were fed a high-fat diet and given low-dose streptozotocin to induce type 2 diabetes. The 12-LO product, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), was infused through an osmotic minipump. Enzyme-linked immunosorbent assay, Western blot, and morphometric analyses were performed. Results: High glucose (HG) increased the p16^ink4a protein expression in MCs, but this increase was prevented by the 12-LO inhibitor, cinnamyl-3,​4-dihydroxy-α-cynanocinnamate (CDC). The levels of p-p38MAPK and p16^ink4a in MCs were significantly elevated after the 12(S)-HETE treatment, whereas the p38MAPK inhibitor SB203580 prevented these increases. Compared with levels in control MCs, marked increases in p38MAPK activation and p16^ink4a expression were observed in MCs plated on collagen IV, while the CDC treatment prevented these changes. Subcutaneous injection of CDC did not affect glucose levels, but completely attenuated the diabetes-related increases in the 12(S)-HETE content, p16^ink4a expression, p-p38MAPK levels, glomerular volume, and the kidney/body weight ratio. Compared with levels in controls, p16^ink4a and p-p38MAPK in the glomeruli derived from 12(S)-HETE-treated rats were significantly higher. Conclusions: 12-LO-p38MAPK mediates the upregulation of p16^ink4a in HG-stimulated MCs and type 2-diabetic glomeruli, and new therapies aimed at 12-LO inhibition may prove beneficial in ameliorating diabetes-induced glomerular hypertrophy.

Bone Morphogenetic Protein 4 and Smad1 Mediate Extracellular Matrix Production in the Development of Diabetic Nephropathy.

Diabetic nephropathy is the leading cause of end-stage renal disease. It is pathologically characterized by the accumulation of extracellular matrix in the mesangium, of which the main component is α1/α2 type IV collagen (Col4a1/a2). Recently, we identified Smad1 as a direct regulator of Col4a1/a2 under diabetic conditions in vitro. Here, we demonstrate that Smad1 plays a key role in diabetic nephropathy through bone morphogenetic protein 4 (BMP4) in vivo. Smad1-overexpressing mice (Smad1-Tg) were established, and diabetes was induced by streptozotocin. Nondiabetic Smad1-Tg did not exhibit histological changes in the kidney; however, the induction of diabetes resulted in an ∼1.5-fold greater mesangial expansion, consistent with an increase in glomerular phosphorylated Smad1. To address regulatory factors of Smad1, we determined that BMP4 and its receptor are increased in diabetic glomeruli and that diabetic Smad1-Tg and wild-type mice treated with a BMP4-neutralizing antibody exhibit decreased Smad1 phosphorylation and ∼40% less mesangial expansion than those treated with control IgG. Furthermore, heterozygous Smad1 knockout mice exhibit attenuated mesangial expansion in the diabetic condition. The data indicate that BMP4/Smad1 signaling is a critical cascade for the progression of mesangial expansion and that blocking this signal could be a novel therapeutic strategy for diabetic nephropathy.

Podocyte hypertrophy precedes apoptosis under experimental diabetic conditions.

Podocyte hypertrophy and apoptosis are two hallmarks of diabetic glomeruli, but the sequence in which these processes occur remains a matter of debate. Here we investigated the effects of inhibiting hypertrophy on apoptosis, and vice versa, in both podocytes and glomeruli, under diabetic conditions. Hypertrophy and apoptosis were inhibited using an epidermal growth factor receptor inhibitor (PKI 166) and a pan-caspase inhibitor (zAsp-DCB), respectively. We observed significant increases in the protein expression of p27, p21, phospho-eukaryotic elongation factor 4E-binding protein 1, and phospho-p70 S6 ribosomal protein kinase, in both cultured podocytes exposed to high-glucose (HG) medium, and streptozotocin-induced diabetes mellitus (DM) rat glomeruli. These increases were significantly inhibited by PKI 166, but not by zAsp-DCB. In addition, the amount of protein per cell, the relative cell size, and the glomerular volume were all significantly increased under diabetic conditions, and these changes were also blocked by treatment with PKI 166, but not zAsp-DCB. Increased protein expression of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, together with increased Bax/Bcl-2 ratios, were also observed in HG-stimulated podocytes and DM glomeruli. Treatment with either zAsp-DCB or PKI 166 resulted in a significant attenuation of these effects. Both PKI 166 and zAsp-DCB also inhibited the increase in number of apoptotic cells, as assessed by Hoechst 33342 staining and TUNEL assay. Under diabetic conditions, inhibition of podocyte hypertrophy results in attenuated apoptosis, whereas blocking apoptosis has no effect on podocyte hypertrophy, suggesting that podocyte hypertrophy precedes apoptosis.