Glomerulosclerosis In Diabetic Nephropathy Research Articles

Importance of frequency and morphological characteristics of nodular diabetic glomerulosclerosis in diabetic nephropathy

The Renal Pathology Society proposed a pathological classification for diabetic nephropathy (DN) (RPS 2010). We retrospectively examined the renal structural-functional relationships using the RPS 2010 classification in 49 DN cases. We also evaluated the importance of the percentage of glomeruli with nodular diabetic glomerulosclerosis and their morphological characteristics (cellular, cellular and extracellular matrix [ECM] or ECM types) in the pathology of DN. The classes of DN (RPS 2010) were significantly correlated with the duration of diabetes mellitus (DM), degree of proteinuria, a decreased estimated glomerular filtration rate (eGFR), and the stages of Japanese clinical DM and chronic kidney disease (CKD). When the percentage of glomeruli with nodular glomerulosclerosis (IIIA <25%, IIIB 25-50%, IIIC 50-75%, and IIID >75%) was added to class III in this classification, the classes of DN had a greater correlation with the levels of proteinuria. The morphological characteristics of nodular glomerulosclerosis such as cellular, cellular and ECM, or ECM type were associated with several clinical parameters including the duration of DM, degree of proteinuria, a decreased eGFR, and/or the stages of clinical DM and CKD. Mesangial red blood cell fragments that is indicative of microvascular injury was found in cellular or cellular and ECM types of nodular glomerulosclerosis. The RPS 2010 classification is useful as a DN pathological classification that indicates a good correlation with the clinical characteristics of DN. In addition, the frequency and morphological characteristics of nodular diabetic glomerulosclerosis is important for the evaluation of the pathology in DN.

Osteomeles schwerinae Extract Prevents Diabetes-Induced Renal Injury in Spontaneously Diabetic Torii Rats.

Mesangial cell proliferation contributes to the development of glomerulosclerosis in diabetic nephropathy. This study was aimed at determining whether Osteomeles schwerinae (OSSC) extract can ameliorate renal damage in Spontaneously Diabetic Torii (SDT) rats. OSSC extract (100 and 250 mg/kg/day) was administered to the SDT rats through oral gavage for 17 weeks. At the end of the experiment, glucose, HbA1c, and albuminuria were measured. In addition, the levels of mesangial proliferation-related proteins were determined by western blotting and immunohistochemistry. Our results show that albuminuria, accumulation of the extracellular matrix (ECM), and renal expansion were markedly restored by OSSC extract administration. The OSSC treatment also inhibited α-smooth muscle actin and transforming growth factor-β1 protein expression. In addition, OSSC and its bioactive compounds hyperoside and quercitrin inhibited the platelet-derived growth factor-BB (PDGF-BB)/platelet-derived growth factor-B receptor (PDGFR-β) ligand binding in an in vitro assay. Taken together, these results indicate that OSSC inhibits ECM accumulation and mesangial proliferation of the glomeruli in SDT rats through inhibition of the interaction between PDGF-BB and PDGFR-β. OSSC has ameliorating effects on the initiation and progression of diabetes complications and can be used for the treatment of early diabetic renal dysfunction.

NEFROPROTECTIVE EFFECTS OF ACTOVEGIN IN PATIENTS WITH DIABETIC NEPHROPATHY

Apoptosis, programmed cell death, regarded as one of the main mechanisms of glomerulosclerosis in diabetic nephropathy (DN).&#x0D; The aim of the present researchto study the effects of actovegin on cell apoptosis in patients with DN. Materials and methods: we examined 80 type 2 diabetic patients with DN I - IVstages and 10 healthy subjects. CD95+cells number was evaluated by indirect immunohis tochemistry.Results: CD95+ - cells number in the peripheral blood of patients with DN was significantly higher than in healthy donors. The additional actovegin treatment was accompanied by a significant decrease of the investigated parameter when compared with the base therapy.

Hyperoside ameliorates glomerulosclerosis in diabetic nephropathy by downregulating miR-21.

The purpose of this study was to investigate the therapeutic effects of hyperoside (Hyp) on glomerulosclerosis in diabetic nephropathy and its underlying mechanisms. Blood glucose, kidney mass, and renal function of mice were measured. Renal morphology was observed using hematoxylin and eosin, periodic acid - Schiff's, and Masson's trichrome stain. Fibronectin (FN) and collagen IV (COL IV) in kidney were determined by Western blot and immunohistochemical studies. Matrix metalloproteinases (MMP)-2 and -9 and tissue inhibitors of metalloproteinase (TIMP)-1 in renal tissues were detected on both the mRNA and protein levels. miRNA expression and artificial alterations by miRNA agomir transfection were evaluated to investigate the protective mechanism of Hyp in mesangial cells. Hyp effectively improved renal function and physiologic features of db/db mice. Hyp also ameliorated glomerulosclerosis by suppressing FN, COL IV, and TIMP-1 expressions and promoting MMP-9 and MMP-2 expressions. The change in MMP-9 mRNA expression was inconsistent with that in protein levels in kidney, indicating that there was a post-transcriptional regulation. Further exploration in vitro showed that miR-21 was downregulated by Hyp, increasing expression of its target, MMP-9. These results suggest that Hyp can ameliorate glomerulosclerosis in diabetic nephropathy by downregulating miR-21 to increase expression of its target, MMP-9.

Detection of Glomerulosclerosis in Diabetic Nephropathy Using Contour-based Segmentation

We have proposed a method for detection of glomerulosclerosis in diabetic nephropathy using Contour-Based Segmentation. Pathological images of the glomerulosclerosis were acquired from various patients. It is a challenging task as 100% detection of Nephropathy disease with regular pathological procedure is not possible. We propose a solution to the problem of segmentation of the glomerulosclerosis images for the analysis of abnormalities. The proposed method is the modification of the original Chan-Vese algorithm, with the varied values of parameters. The proposed method achieved better segmentation and the results are encouraging.

Hepatocyte growth factor gene therapy enhances infiltration of macrophages and may induce kidney repair in db/db mice as a model of diabetes

Aims/hypothesisWe previously demonstrated hepatocyte growth factor (HGF) gene therapy was able to induce regression of glomerulosclerosis in diabetic nephropathy through local reparative mechanisms. The aim of this study was to test whether bone-marow-derived cells are also involved in this HGF-induced reparative process.MethodsWe have created chimeric db/db mice as a model of diabetes that produce enhanced green fluorescent protein (EGFP) in bone marrow cells. We performed treatment with HGF gene therapy either alone or in combination with granulocyte-colony stimulating factor, in order to induce mobilisation of haematopoietic stem cells in these diabetic and chimeric animals.ResultsWe find HGF gene therapy enhances renal expression of stromal-cell-derived factor-1 and is subsequently associated with an increased number of bone-marrow-derived cells getting into the injured kidneys. These cells are mainly monocyte-derived macrophages, which may contribute to the renal tissue repair and regeneration consistently observed in our model. Finally, HGF gene therapy is associated with the presence of a small number of Bowman’s capsule parietal epithelial cells producing EGFP, suggesting they are fused with bone-marrow-derived cells and are contributing to podocyte repopulation.Conclusions/interpretationAltogether, our findings provide new evidence about the therapeutic role of HGF and open new opportunities for inducing renal regeneration in diabetic nephropathy.

Spironolactone ameliorates podocytic adhesive capacity via restoring integrin α3 expression in streptozotocin-induced diabetic rats

Podocyte responses to various injuries include detachment from the glomerular basement membrane (GBM) with impaired adhesion ability. Growing evidence suggests inappropriately enhanced aldosterone levels in glomeruli may contribute to podocytic injury and subsequently glomerulosclerosis in diabetic nephropathy (DN). In the present study, we aimed to investigate podocytic integrin alpha 3 expression and urinary podocyte excretion in streptozotocin (STZ)-induced diabetic rats, and to evaluate their responses to spironolactone (SPL). STZ-induced male diabetic Wistar rats were treated with vehicle (the STZ group, n=7), or spironolactone (the STZ+SPL group, n=6) for 12 weeks, six additional rats of similar body weight serving as control. Urine specimens were obtained for measurement of urine albumin concentration and urinary podocyte quantitation upon completion of the 12 weeks. Urinary podocyte excretion was quantified by immunofluorescence and expression of integrin alpha 3 was detected by immunohistochemistry and Western blotting. At 12 weeks, rats given STZ alone revealed an increase in blood glucose and were unaffected by spironolactone, whereas the STZ+SPL group showed considerable improvement in urine albumin and podocyte excretion, as well as up-regulation of integrin alpha 3. Our results suggest that spironolactone ameliorates impaired podocytic adhesion capacity and prevents STZ-induced DN progression.

Mechanism of Chinese herbal medicine delaying glomerulosclerosisin diabetic nephropathy

The pathomechanisms of glomerulosclerosis in diabetic nephropathy (DN) are considered to be related with glycometabolism disorder, podocyte injury, intra-renal hemodynamics abnormality, fibrogenic cytokines over-expression, oxidative stress and inflammatory reaction. Chinese herbal medicine could delay the progression of glomerulosclerosis in DN by ameliorating the harmful factors of these pathological changes. Therefore, it is possible to postpone the progress of DN to end-stage renal disease through the treatment with Chinese herbal medicine.

Adenosine mediates transforming growth factor-beta 1 release in kidney glomeruli of diabetic rats

Up regulation of the transforming growth factor-beta 1 (TGF-β1) axis has been recognized as a pathogenic event for progression of glomerulosclerosis in diabetic nephropathy. We demonstrate that glomeruli isolated from diabetic rats accumulate up to sixfold more extracellular adenosine than normal rats. Both decreased nucleoside uptake activity by the equilibrative nucleoside transporter 1 and increased AMP hydrolysis contribute to raise extracellular adenosine. Ex vivo assays indicate that activation of the low affinity adenosine A 2B receptor subtype (A 2BAR) mediates TGF-β1 release from glomeruli of diabetic rats, a pathogenic event that could support progression of glomerulopathy when the bioavailability of adenosine is increased.

From Fibrosis to Sclerosis

Progression of diabetic nephropathy to end-stage kidney disease is mediated by a host of processes, but none is as important as the gradual, inexorable scarring of the renal glomerulus, known as glomerulosclerosis. Hence, a host of studies over the decades have attempted to elucidate the molecular mechanisms that lead to this chronic sclerosing condition so that effective therapies and preventative strategies can be developed. Over the past several years, the general understanding of the pathogenic factors that lead to this important feature of diabetic nephropathy has improved considerably. Glomerulosclerosis in diabetic nephropathy is caused by accumulation of extracellular matrix (ECM) proteins in the mesangial interstitial space, resulting in fibrosis manifested by either diffuse or nodular changes (1). The most common matrix proteins detected are collagen types I, III, and IV and fibronectin (2). These accumulate both due to increased synthesis by mesangial cells and reduced degradation by mesangial matrix metalloproteinases (3). Over 20 years ago, Mauer et al. (4) established the clear link between mesangial matrix expansion and progression of diabetic kidney disease by demonstrating that measures of mesangial expansion strongly predicted the clinical manifestations of diabetic nephropathy. Since then, the critical charge to investigators has been to elucidate the mechanisms that promote glomerulosclerosis in diabetic nephropathy. In this brief perspective, we will review pathogenic processes that appear to be critical in the development of diabetic glomerulosclerosis, emphasizing newer findings and insights. During the 1990s, a general consensus emerged about major signaling mechanisms involved in stimulating mesangial cell synthesis of ECM proteins (Fig. 1). In this consensus view, high extracellular glucose induces an increase in glucose uptake via increased expression of the facilitative glucose transporter GLUT1 (5,6). The resultant enhancement in glucose metabolic flux leads to activation of a number of metabolic pathways that result in increased advanced …

Urinary Smad1 Is a Novel Marker to Predict Later Onset of Mesangial Matrix Expansion in Diabetic Nephropathy

We reported that Smad1 is a key transcriptional factor for mesangial matrix expansion in diabetic nephropathy. In this study, we examined whether urinary Smad1 in an early phase of diabetes can predict later development of glomerulosclerosis in diabetic nephropathy and how an angiotensin II type 1 receptor blocker (ARB) can modulate structural changes and urinary markers. Smad1 and albumin in the urine were examined 4 weeks after injection of streptozotocin in 48 rats or 6 weeks of diabetes in db/db mice. Their renal pathology was analyzed after 20 weeks in rats or 12 weeks in mice. Among 48 diabetic rats 7 rats were treated with olmesartan for 20 weeks. Urinary Smad1 of diabetic rats at 4 weeks was nicely correlated with mesangial matrix expansion at 24 weeks (r = 0.70, P < 0.001), while albuminuria showed a weaker association (r = 0.31, P = 0.043). Olmesartan treatment significantly ameliorated glomerulosclerosis and dramatically decreased urinary Smad1 (from 3.9 +/- 2.9 to 0.3 +/- 0.3 ng/mg creatinine, P < 0.05). In db/db mice, urinary Smad1 at 6 weeks was also significantly correlated with mesangial expansion at 18 weeks. In contrast, there was no change in urinary Smad1 in control diabetic rats or mice. The increase of urinary Smad1 in the early stages of diabetes is correlated with later development of glomerulosclerosis in two rodent models. These data indicate that urinary Smad1 could be a novel predictor for later onset of morphological changes and can be used to monitor the effect of ARBs in diabetic nephropathy.

Ginkgo biloba extract suppresses hypertrophy and extracellular matrix accumulation in rat mesangial cells

To observe the effects of Ginkgo biloba extract (EGb) on the hypertrophy of mesangial cells and the accumulation of extracellular matrix (ECM) in mesangial cells. Cultured mesangial cells were allotted into 7 groups: normal group, solvent control group, high glucose group, low dose of EGb group, moderate dose of EGb group, high dose of EGb group, and captopril group. Activities of cell antioxidases, S phase percentage and G(0)/G(1) phase percentage, collagen IV and laminin, Smad2/3 and Smad7, TGF-beta(1) mRNA were measured by different methods. For EGb-treated groups, when compared with high glucose group, the cell percentage of S phase was raised and the percentage of G(0)/G(1) was lowered. The intensity of oxidative stress was weakened. The expression of Smad2/3 was greatly decreased and Smad7 was increased. Collagen IV, laminin and TGF- beta(1)mRNA were also reduced. EGb can suppress cell hypertrophy and the accumulation of ECM in rat mesangial cells, which means it could play a vital role in the delay of glomerulosclerosis in diabetic nephropathy.

High glucose stimulates synthesis of fibronectin in human mesangial cells via serum and glucocorticoid induced protein kinase-1 signaling pathway

To investigate the role of serum and glucocorticoid induced kinase-1 (SGK(1)) pathways in fibronectin (FN) synthesis in human mesangial cell (HMC) under high glucose condition and the mechanism by which SGK(1) contributes to glomerulosclerosis in diabetic nephropathy (DN). HMCs were cultured and transfected with (P)IRES2-EGFP-(S422D) SGK(1) mutant (SD), plasmid containing SGK(1) dominant activation mutant, or blank plasmid. Non-transfected HMCs were used as control group. Then the HMCs were divided into 6 groups: transfected with SD + high glucose (SD-HG, 25 mmol/L D-glucose), transfected with FP + high glues (FP + HG), non-transfected + high glucose (NT-HG), transfeted with SD + normal glucose (SD-NG, 5.5 mmol/L D-glucose), transfected with FP + normal glues (FP + NG), and non-transfected + normal glucose (NT-NG). Eight hours after the glucose stimulation, RT-PCR was used to examine the SGK(1) mRNA expression and fibronectin (FN). Western blotting was used to detect the fibronectin (FN) protein expression. The SGK(1) mRNA expression of the SD + HG group was 0.709, significantly higher than those of the FP + HG and NT + HP groups (0.497 and 0.491, both P < 0.01). The SGK(1) protein expression of the SD + HG group was 1,178,497, significantly higher than those of the FP + HG and NT + HP groups (193,875 and 195,597 respectively, both P < 0.01). The FN mRNA expression of the SD + HG group was 0.749, significantly higher than those of the FP + HG and NT + HP groups (0.463 and 0.475 respectively, both P < 0.01). The FN protein expression of the SD + HG group was 659,550, significantly higher than those of the FP + HG and NT + HG groups (342,354 and 340,428 respectively, both P < 0.01). There were not significant differences in the expressions of FN mRNA and protein among different NG groups. SGK(1) may be involved in the signal transduction leading to the increase of fibronectin production in DN and therefore may play an active part in glomerulosclerosis in DN.

Response of mesangial cells to low-density lipoprotein and angiotensin II in diabetic (OLETF) rats

Progression of diabetic nephropathy is closely associated with morphological changes in glomeruli, such as thickening of the glomerular basement membrane, mesangial expansion, and glomerulosclerosis. To elucidate early glomerular events, we compared the mitogenic activity and extracellular matrix production in mesangial cells (MC) isolated from diabetic rats prior to the manifestation of nephropathy and those showing overt nephropathy. This study may help to clarify the mechanisms underlying diabetic nephropathy and provide clues about early therapeutic interventions for preventing or slowing this process. Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a chronic model for human type 2 diabetes mellitus, and age-matched control (LETO) rats were used. Glomerular cell numbers, expression of immediate early genes (c-Fos and c-Myc) and proliferating cell nuclear antigen (PCNA), and low-density lipoprotein (LDL) deposition were determined in renal tissue sections from rats aged 15 to 75 weeks. Mesangial cells (MCs) from OLETF rats at two different stages of the disease, that is, young (12- to 14-week-old) OLETF rats (y-OLETF) prior to the manifestation of nephropathy and old (48- to 50-week-old) OLETF rats (o-OLETF) showing nephropathy, were isolated and cultured. After stimulation with native (n-) or oxidized (ox-) LDL or angiotensin II (Ang II), DNA synthesis and extracellular matrix (ECM) production were examined. Cellular expression of LDL/scavenger receptors was analyzed using fluorescence-labeled LDL and binding to 125I-labeled-LDL. The number of cells per glomerular cross section was significantly higher in OLETF rats than in LETO rats between 25 and 65 weeks of age. In OLETF glomeruli, c-Fos, c-Myc, and PCNA were transiently expressed in the early phase. Glomerular LDL deposition increased with the age of OLETF rats. Addition of a low dose of n-LDL (10 microg/mL) to the culture medium significantly stimulated DNA synthesis of y-OLETF MCs, as compared with o-OLETF MCs and LETO MCs (P < 0.05). A high dose of n-LDL (100 microg/mL) caused cytotoxic effects in all cells. Exposure to ox-LDL minimally affected DNA synthesis of OLETF or LETO MCs. LDL receptors and scavenger receptors were predominant in y-OLETF and o-OLETF, respectively. After stimulation with n-LDL and ox-LDL, expression of type I and type III collagen, along with transforming growth factor-beta (TGF-beta), was higher in o-OLETF MCs that in y-OLETF MCs or LETO MCs. Exposure to Ang II markedly induced DNA synthesis and ECM mRNA expression in y-OLETF MCs and o-OLETF MCs, respectively. These findings indicate that the cell proliferation process precedes the evolution of diabetic glomerulopathy. The responses of OLETF MCs to n-LDL/ox-LDL and Ang II differed depending on the stage of diabetes. In the early phase, MCs were prone to proliferate, whereas in the late stage, MCs, which expressed higher levels of TGF-beta, tended to synthesize ECM. A functional switch in MCs may contribute to the development of glomerulosclerosis in diabetic nephropathy.

HDL causes mesangial cell mitogenesis through a tyrosine kinase-dependent receptor mechanism.

Hypercholesterolemia and mesangial cell proliferation have been proposed to play a role in the progression of glomerulosclerosis in diabetic nephropathy and other renal diseases. Although LDL is mitogenic for and cytotoxic to mesangial cells, the effect of HDL on these cells is unknown. HDL stimulates fibroblast mitogenesis and is the principal cholesterol-bearing lipoprotein in the rat, the experimental model for studying the effect of hyperlipidemia on renal disease. Insulin is mitogenic in several cell systems, and its levels are increased in serum in non-insulin-dependent diabetes mellitus. This study investigates whether HDL acts as a growth factor in mesangial cells and whether it functions in parallel with insulin. It was found that HDL at protein concentrations between 10 and 500 microg/ml, both alone and in the presence of 100 nM insulin, increased DNA synthesis in mesangial cells (129 to 165% of control for HDL alone; 140 to 235% for HDL plus insulin), whereas HDL at 1000 microg/ml and greater inhibited mesangial cell proliferation. Insulin alone at 100 nM stimulated [3H]thymidine incorporation in the same cell system (145% of control); the mitogenic effect of insulin was additive to that of HDL. Purified apo A-I had a similar effect, but at significantly lower concentrations. Specific binding of HDL to mesangial cells was demonstrated (B(max) [binding constant] of 5.19 +/- 0.70 x 10(-7) micromol of HDL bound/mg cell protein and K(b) of 2.83 +/- 0.22 nM). Tetranitromethane alters apo A-I, preventing binding to its cognate receptor. Tetranitromethane-modified HDL did not bind to mesangial cells and had no effect on [3H]thymidine incorporation. Addition of HDL to mesangial cells caused an immediate transient increase in free intracellular calcium in several representative mesangial cells, similar to the response seen with platelet-derived growth factor. The mitogenic effect of HDL was not altered after attenuation of cellular protein kinase C activity, but the stimulatory effect of HDL alone and in combination with insulin on DNA synthesis was completely eliminated after inhibition of cellular tyrosine kinases by 24-h pretreatment with 0.25 microM herbimycin A. Thus, HDL binds to a specific apo A-I-dependent receptor, promotes DNA synthesis, and initiates second-messenger events by a tyrosine kinase-dependent and protein kinase C-independent mechanism.

Effect of glucose and heparin on mesangial alpha 1(IV)COLL and MMP- 2/TIMP-2 mRNA expression

Mesangial cells are responsible for the synthesis of mesangial matrix as well as its degradation, which is mediated by a number of proteolytic activities, including metalloproteinases (MMPs). Imbalanced matrix protein metabolism may be responsible for mesangial expansion and glomerulosclerosis in diabetic nephropathy. Heparin prevents this complication. In human and murine mesangial cell cultures, RT-PCR was able to detect mRNA expression for a number of molecules involved in the mesangial extracellular matrix turnover: type IV collagen [alpha 1(IV)COLL], MMP-1, MMP-2, MMP-3, MMP-9 and MMP-10, and the tissue inhibitors TIMP-1 and TIMP-2. The expression of mRNA for alpha 1(IV)COLL and MMP-2/TIMP-2 balance was studied in human cells in the presence of high glucose and heparin. mRNAs for all the studied molecules were expressed at different levels. Interestingly, a shift in the balance of alpha 1(IV)COLL, MMP-2 and TIMP-2 was observed in high glucose, which was partially reversed by heparin supplementation. The new equilibrium was mostly due to the down-regulation of type IV collagen expression, rather than further reduction of potential proteolysis. Our data, while extending the list of potential mediators of mesangial matrix catabolism, highlight a molecular mechanism by which the pathogenesis of diabetic nephropathy may be sustained, and at the same time suggest that heparin may have the potential to correct this abnormality.

Vitamin E Prevents Glucose-Induced Lipid Peroxidation and Increased Collagen Production in Cultured Rat Mesangial Cells

High glucose directly promotes lipid peroxidation and stimulates collagen production by cultured mesangial cells. We conducted the following experiments to ascertain whether vitamin E, a lipophilic antioxidant agent, could reverse these effects in vitro. Rat mesangial cells were grown to confluence and then maintained for 7 additional days in control media (DME) containing 5.6 m M glucose or experimental media containing 33.3 m M glucose. The test condition resulted in a near doubling of mesangial cell lipid peroxidations, assessed by measuring the content of conjugated dienes. High-glucose media also enchanced collagen production by 28% above the basal level in control media. Addition of mitamin E (100 μ M) reversed both effects of the elevated ambient glucose level and prevented lipid peroxidation and normalized collagen production in cultured mesangial cells. These findings sugggest that vitamin E functions as an endogenous antioxidant agent in the kidney to limit the development of glomerulosclerosis in diabetic nephropathy.