Development Of Experimental Diabetic Nephropathy Research Articles

Role of Brain Angiotensin-II in Development of Experimental Diabetic Nephropathy in Wistar Rats

Role of Brain Angiotensin-II in Development of Experimental Diabetic Nephropathy in Wistar Rats

Dietary Fiber Protects against Diabetic Nephropathy through Short-Chain Fatty Acid-Mediated Activation of G Protein-Coupled Receptors GPR43 and GPR109A.

Studies have reported "dysbiotic" changes to gut microbiota, such as depletion of gut bacteria that produce short-chain fatty acids (SCFAs) through gut fermentation of fiber, in CKD and diabetes. Dietary fiber is associated with decreased inflammation and mortality in CKD, and SCFAs have been proposed to mediate this effect. To explore dietary fiber's effect on development of experimental diabetic nephropathy, we used streptozotocin to induce diabetes in wild-type C57BL/6 and knockout mice lacking the genes encoding G protein-coupled receptors GPR43 or GPR109A. Diabetic mice were randomized to high-fiber, normal chow, or zero-fiber diets, or SCFAs in drinking water. We used proton nuclear magnetic resonance spectroscopy for metabolic profiling and 16S ribosomal RNA sequencing to assess the gut microbiome. Diabetic mice fed a high-fiber diet were significantly less likely to develop diabetic nephropathy, exhibiting less albuminuria, glomerular hypertrophy, podocyte injury, and interstitial fibrosis compared with diabetic controls fed normal chow or a zero-fiber diet. Fiber beneficially reshaped gut microbial ecology and improved dysbiosis, promoting expansion of SCFA-producing bacteria of the genera Prevotella and Bifidobacterium, which increased fecal and systemic SCFA concentrations. Fiber reduced expression of genes encoding inflammatory cytokines, chemokines, and fibrosis-promoting proteins in diabetic kidneys. SCFA-treated diabetic mice were protected from nephropathy, but not in the absence of GPR43 or GPR109A. In vitro, SCFAs modulated inflammation in renal tubular cells and podocytes under hyperglycemic conditions. Dietary fiber protects against diabetic nephropathy through modulation of the gut microbiota, enrichment of SCFA-producing bacteria, and increased SCFA production. GPR43 and GPR109A are critical to SCFA-mediated protection against this condition. Interventions targeting the gut microbiota warrant further investigation as a novel renoprotective therapy in diabetic nephropathy.

Vorapaxar treatment reduces mesangial expansion in streptozotocin-induced diabetic nephropathy in mice.

BackgroundTwenty years after the onset of diabetes, up to 40% of patients develop diabetic nephropathy. Protease-activated receptor-1 (PAR-1) has recently been shown to aggravate the development of experimental diabetic nephropathy. PAR-1 deficient mice develop less albuminuria and glomerular lesions and PAR-1 stimulation induces proliferation and fibronectin production in mesangial cells in vitro. Vorapaxar is a clinically available PAR-1 inhibitor which is currently used for secondary prevention of ischemic events.ObjectivesThe aim of this study was to investigate in a preclinical setting whether vorapaxar treatment may be a novel strategy to reduce diabetes-induced kidney damage.ResultsWhile control treated diabetic mice developed significant albuminuria, mesangial expansion and glomerular fibronectin deposition, diabetic mice on vorapaxar treatment did not show any signs of kidney damage despite having similar levels of hyperglycemia.ConclusionsThese data show that PAR-1 inhibition by vorapaxar prevents the development of diabetic nephropathy in this preclinical animal model for type I diabetes and pinpoint PAR-1 as a novel therapeutic target to pursue in the setting of diabetic nephropathy.Materials and Methods22 C57Bl/6 mice were made diabetic using multiple low-dose streptozotocin injections (50 mg/kg) and 22 littermates served as non-diabetic controls. Four weeks after the induction of diabetes, 11 mice of each group were assigned to control or vorapaxar treatment. Mice were sacrificed after 20 weeks of treatment and kidney damage was evaluated.

Endothelial Nitric Oxide Synthase Prevents Heparanase Induction and the Development of Proteinuria.

Endothelial nitric oxide synthase (eNOS) deficiency exacerbates proteinuria and renal injury in several glomerular diseases, but the underlying mechanism is not fully understood. We recently showed that heparanase is essential for the development of experimental diabetic nephropathy and glomerulonephritis, and hypothesize that heparanase expression is regulated by eNOS. Here, we demonstrate that induction of adriamycin nephropathy (AN) in C57BL/6 eNOS-deficient mice leads to an increased glomerular heparanase expression accompanied with overt proteinuria, which was not observed in the AN-resistant wild type counterpart. In vitro, the eNOS inhibitor asymmetric dimethylarginine (ADMA) induced heparanase expression in cultured mouse glomerular endothelial cells. Moreover, ADMA enhanced transendothelial albumin passage in a heparanase-dependent manner. We conclude that eNOS prevents heparanase induction and the development of proteinuria.

The Critical Role of Methylglyoxal and Glyoxalase 1 in Diabetic Nephropathy

The discovery of increased formation of methylglyoxal (MG) by cell metabolism in high glucose concentration in vitro suggested possible relevance to diabetes and diabetes complications (1,2). MG is the precursor of quantitatively important advanced glycation end products (AGEs) of protein and DNA- and MG-derived AGEs increase in experimental and clinical diabetes (3,4). Increased MG and its metabolism by glyoxalase 1 (Glo1) was linked to clinical microvascular complications (nephropathy, retinopathy, and neuropathy) (5). Current clinical treatment decreasing MG and MG-derived AGEs, such as insulin lispro (6,7), has some clinical benefit in diabetic nephropathy (8), although the decrease in MG-derived AGE exposure is minor—∼17% (7). Greater benefits may be achieved with specific and effective anti-MG targeted therapy. An outstanding research problem is to gain unequivocal evidence that MG glycation is a key mediator of vascular complications and, if possible, provide some pointers as to how MG glycation could be effectively countered. In this issue, the study by Giacco et al. (9) provides key evidence by a functional genomic approach manipulating expression of Glo1 to increase or decrease endogenous MG glycation. The outcomes show that development of experimental diabetic nephropathy is driven by increased levels of MG glycation and increasing renal expression of Glo1 prevents this. Recent research has shown Glo1 expression may be increased …

Preventive effects of rutin on the development of experimental diabetic nephropathy in rats

AimsDiabetic nephropathy (DN) is an important microvascular complication and one of the main causes of end-stage renal disease. In this study, the preventive effect and mechanism of rutin on the development of DN in streptozotocin (STZ)-induced diabetic rats were investigated. Main methodsAfter an early DN model was induced by STZ, rats were orally administered rutin at 3 doses for 10weeks. Fasting blood glucose, creatinine (Cr), blood urea nitrogen (BUN), urine protein, kidney index, antioxidase, advanced glycosylation end products (AGEs), extracellular matrix (ECM) including collagen IV and laminin, connective tissue growth factor (CTGF), phosphorylated Smad 2/3 (p-Smad 2/3) and Smad 7 (p-Smad 7), and transforming growth factor-β1 (TGF-β1) were determined by different methods, respectively. The ultrastructural morphology was observed by a transmission electron microscope. Key findingsCompared with the DN group, rutin decreased the levels of fasting blood glucose, Cr, BUN, urine protein, the intensity of oxidative stress and p-Smad 7 significantly. The expression of AGEs, collagen IV and laminin, TGF-β1, p-Smad 2/3 and CTGF was inhibited by rutin significantly. Moreover, rutin was observed to inhibit proliferation of mesangial cells and decrease thickness of glomerular basement membrane (GBM) by electron microscopy. SignificanceThe preventive effect of rutin on the development of DN is closely related to oxidative stress and the TGF-β1/Smad/ECM and TGF-β1/CTGF/ECM signaling pathways. Those results suggest that rutin can prevent the development of experimental DN in rats.

Neutrophil Proteinase 3 Induces Diabetes in a Mouse Model of Glucose Tolerance

Type 1 diabetes is considered to be an autoimmune disease in which T cells attack pancreatic islet cells. Impaired glucose tolerance with type 2 diabetes has been classified as an obesity-associated metabolic syndrome. However, recent studies have revealed that type 2 diabetes is an autoinflammatory disease due to an imbalance of inflammatory cytokine production and related molecular components that cause inflammation. Insulin-like growth factor (IGF) and the insulin-like growth factor-binding protein-3 (IGFBP3) system are known to be involved in the development of experimental diabetic nephropathy, and urinary IGFBP3 protease activity has been observed in patients with type 2 diabetes. A serine protease was found to be responsible for the proteolytic activity in diabetic urine; however, the identity of the precise enzyme remains unknown. We investigated neutrophil proteinase 3 (PR3) to see whether it has specific enzymatic activity associated with insulin-like growth factor-1 and IGFBP3. In our study, both molecules were sufficiently degraded, which leads us to believe that PR3 may induce insulin resistance in the mouse model utilized. In addition, we found that PR3 in the urine of diabetic patients similarly affects insulin resistance. Moreover, PR3-immunized mice had an increase in glucose clearance due to inhibition of PR3 activity. As such, PR3 can be considered as an inflammatory enzyme directly linking inflammation to type 2 diabetes through downregulation of insulin-like growth factor-1/IGFBP3.

Involvement of vascular endothelial nitric oxide synthase in development of experimental diabetic nephropathy in rats

Endothelial nitric-oxide synthase (eNOS) acts as a common pathogenic pathway in diabetic nephropathy (DN). However, its functional consequences are still not fully understood. Caveolin, a membrane protein, inhibits the eNOS by making caveolin-eNOS complex, and its expression is upregulated during diabetes mellitus (DM). This study was designed to determine the role of caveolin in eNOS-mediated NO synthesis and release in DN. DM in rat was induced by feeding of high-fat diet (HFD) for 2 weeks, followed by single dose of streptozotocin (STZ) (35 mg/kg, ip) further followed by HFD for further 8 weeks. Serum nitrite/nitrate ratio was measured to determine the plasma level of NO. Diabetic rat, after 6 weeks of STZ, developed elevated level of BUN, protein in urine, urinary output, serum creatinine, serum cholesterol, kidney weight, kidney weight/body weight, and renal cortical collagen content, while serum nitrite/nitrate concentration was significantly decreased as compared to normal control group. Treatment with sodium nitrite (NO donor), L: -arginine (NO precursor), daidzein (caveolin inhibitor), and combination of L: -arginine and daidzein for 2 weeks markedly attenuated these changes and increased serum nitrite/nitrate ratio. However, treatment with L-NAME, a eNOS inhibitor, significantly attenuated the L: -arginine-, daidzein-, or combination of L: -arginine and daidzein-induced ameliorative effects in DN. The finding of this study suggests that caveolin plays a vital role in the eNOS-mediated decrease in renal level of NO, which may be responsible for the development of DN in rats.

Minocycline with Aspirin: An Approach to Attenuate Diabetic Nephropathy in Rats

Degradation of extracellular matrix (ECM) by enhanced production of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in diabetes leads to nephropathy. Cyclooxygenases (COX) further increase levels of these MMPs. The objective of present study was to inhibit MMP-2 and MMP-9 by combination of minocycline and aspirin to treat diabetic nephropathy. Diabetes was induced in male Wistar rats by streptozotocin (STZ, 55 mg/kg i.p.). Four weeks after diabetes induction, the rats were treated with minocycline (50 mg/kg, p.o.), aspirin (50 mg/kg, p.o.), or minocycline (50 mg/kg, p.o.) plus aspirin (50 mg/kg, p.o.) for a period of 4 weeks. At the end of eighth week fluid input, urine output, and renal function tests were carried out for diagnosis of diabetic nephropathy. Renal hypertrophy was measured and histopathology was done to evaluate renal damage. Diabetes produced significant loss of body weight, polyuria, polydipsia, hyperglycemia, and increase in blood pressure. Serum creatinine, urea, and blood urea nitrogen levels were found to be increased significantly in the STZ group diabetic rats. Treatment with combination of minocycline and aspirin significantly prevented the rise in creatinine, urea, and blood urea nitrogen levels and increased creatinine clearance. Image analysis of kidneys revealed that collagen level was significantly decreased in combined treated group when compared with control. Results of present study suggest that MMP-2 and MMP-9 inhibition in presence of COX inhibitor prevents the development of experimental diabetic nephropathy in rats and can be a potential approach for the treatment.

Effects of Ginkgo biloba on prevention of development of experimental diabetic nephropathy in rats

To observe the preventive and therapeutic effects of Ginkgo biloba extract (GbE) on early experimental diabetic nephropathy (DN) in rats. After an early DN model was induced by streptozotocin, rats were administered GbE at 3 doses for 12 weeks. Fasting blood glucose, creatinine (Cr), blood urea nitrogen (BUN), urine protein, kidney index, anti-oxidase, advanced glycosylation end products (AGE), collagen IV and laminin, matrix metalloproteinases-2 (MMP-2) and the tissue inhibitor of metalloproteinase-2 (TIMP-2), connective tissue growth factor (CTGF), and transforming growth factor-beta1 (TGF-beta1) mRNA were measured by different methods. The ultrastructural morphology and the thickness of glomerular base membrane (GBM) were observed by a transmission electron microscope. For the GbE-treated DN rats, when compared with the vehicle-treated DN rats, the fasting blood glucose level, Cr, BUN, urine protein level, and the intensity of oxidative stress were significantly decreased. The expression of MMP-2 greatly increased, and TIMP-2 decreased. Also, AGE, either in serum or in renal, the collagen IV, laminin, CTGF levels, and TGF-beta1 mRNA were reduced. Furthermore, both relative grades of mesangium hyperplasia by microscopical observation and the thickness of GBM by electron microscope measurement decreased significantly. GbE has protective effects on several pharmacological targets in the progress of DN and is a potential drug for the prevention of early DN.

Imatinib Attenuates Diabetic Nephropathy in Apolipoprotein E-Knockout Mice

In the diabetic kidney, clinical as well as experimental observations have shown an upregulation of growth factors such as PDGF. These studies, however, were not designed to address whether upregulation of PDGF is merely a manifestation of diabetic renal injury or whether PDGF plays an active role in the pathophysiology of diabetic nephropathy. The objectives of this study were first to assess whether PDGF-dependent pathways are involved in the development of diabetic nephropathy and second to determine the effects of PDGF receptor antagonism on this disorder and associated molecular and cellular processes. This study used the diabetic apolipoprotein E-knockout (apoE-KO) mouse, a recently described model of accelerated diabetic nephropathy. Diabetes was induced by injection of streptozotocin in 6-wk-old apoE-KO mice. Diabetic animals received treatment with a tyrosine kinase inhibitor that inhibits PDGF action, imatinib (STI-571, 10 mg/kg per d orally) or no treatment for 20 wk. Nondiabetic apoE-KO mice served as controls. This model of accelerated renal disease with albuminuria as well as glomerular and tubulointerstitial injury was associated with increased renal expression of PDGF-B, proliferating cells, and alpha-smooth muscle actin-positive cells. Furthermore, there was increased accumulation of type I and type IV collagen as well as macrophage infiltration. Imatinib treatment ameliorated both renal functional and structural parameters of diabetes as well as overexpression of a number of growth factors, collagens, proliferating cells, alpha-smooth muscle actin-positive cells, and macrophage infiltration within the kidney. Tyrosine kinase inhibition with imatinib seems to retard the development of experimental diabetic nephropathy.

Role of insulin and the IGF system in renal hypertrophy in diabetic Psammomys obesus (sand rat).

Diabetic nephropathy is caused by multiple factors related to the altered metabolic environment in diabetes mellitus (DM). Experimental diabetic kidney disease is characterized by renal hypertrophy associated with increased tissue concentrations of insulin-like growth factor I (IGF-I). To assess the specific roles of serum insulin and glucose in mediating the development of diabetic nephropathy, the effects of both hyperinsulinaemic and hypoinsulinaemic DM were studied in Psammomys obesus (sand rat), a model of type 2 DM. The IGF-I system was studied in normal Psammomys obesus gerbils and at 5, 15 and 70 days after the induction of either hyper- or hypoinsulinaemic DM. To induce hyperinsulinaemic DM, Psammomys were raised on a high-energy diet. Hypoinsulinaemic DM was induced by either administration of streptozotocin or a specially designed diet. Hyperinsulinaemic hyperglycaemic Psammomys did not exhibit renal hypertrophy (unchanged kidney/body-weight ratio) and renal IGF-I levels were in the normal range on days 5, 15 and 70. In contrast, Psammomys with hypoinsulinaemic hyperglycaemia induced either by streptozotocin injection or by pancreas exhaustion brought on by a long-term caloric excess diet, had significant increases in kidney/body-weight ratio which were associated with elevated renal IGF-I and mRNA and protein levels of kidney IGF binding protein I. This study shows that serum insulin levels in the presence of hyperglycaemia have an important role in the development of experimental diabetic nephropathy in the Psammomys model. The implication of this finding is that the pathophysiological mechanisms for diabetic kidney disease in experimental models may be different for type 1 and type 2 DM.

Effects of aminoguanidine in preventing experimental diabetic nephropathy are related to the duration of treatment

It has been postulated that the accumulation of advanced glycation end products (AGEs) in the kidney is important in the pathogenesis of diabetic nephropathy. Previously, aminoguanidine has been shown to inhibit the accumulation of renal AGEs and to retard the development of experimental diabetic nephropathy. The present study serially assessed the accumulation of AGEs in the aorta and kidney, as well as renal functional and structural parameters over 32 weeks of experimental diabetes in the absence and presence of aminoguanidine. In addition, it was determined if aminoguanidine was more effective if administered earlier or later in the evolution of diabetic nephropathy by treating diabetic rats with aminoguanidine in the first or second half of the 32-week study period. In the serial studies, glomerular and renal tubular fluorescence increased over the 32 week period and this increase was attenuated by aminoguanidine treatment. Concomitant with the effects of aminoguanidine on fluorescence, there was a retardation in the rise in urinary albumin excretion and prevention of mesangial expansion. Early or late administration of aminoguanidine in diabetic rats reduced tissue fluorescence in glomeruli and renal tubules. At 32 weeks, renal AGEs were increased in diabetic rats as assessed by tissue fluorescence. Using a specific RIA, renal AGEs were increased in diabetic rats and decreased by aminoguanidine treatment, administered over the entire 32 weeks or in the first or latter half of the 32-week study period. Aminoguanidine therapy for the entire 32-week study period retarded the rise in albuminuria in the diabetic rats and was more effective than 16 weeks of treatment either in the first or second half of the study. Early and late aminoguanidine administration were similar in their capacity to retard the development of albuminuria in diabetic rats. Similar effects were observed on mesangial expansion. The increased glomerular basement thickness in diabetic rats was not affected by aminoguanidine, irrespective of duration or timing of therapy. This study confirms that in vivo generation of AGEs in the kidney is time dependent and closely linked to the development of experimental diabetic nephropathy. The renoprotective effects of aminoguanidine in diabetes appear to be related to the duration but not to the timing of treatment.

The relative roles of advanced glycation, oxidation and aldose reductase inhibition in the development of experimental diabetic nephropathy in the Sprague-Dawley rat

Advanced glycation is an important pathogenic mechanism in the development of diabetic complications. However, other biochemical processes, such as the polyol pathway or lipid and protein oxidation which can interact with advanced glycation can also yield tissue fluorescence and may also be implicated in the genesis of diabetic microangiopathy. Aminoguanidine is an inhibitor of advanced glycation, but it is not known if all of its effects are mediated by this mechanism. The present study explores the relative contributions of aldose reductase, oxidative stress and advanced glycation on the development of aortic and renal fluorescence and urinary albumin excretion in streptozotocin diabetic rats. The study groups included non-diabetic (control), streptozotocin diabetic rats and diabetic rats receiving aminoguanidine, the anti-oxidants butylated hydroxytoluene and probucol and the aldose reductase inhibitor, ponalrestat. Serial measurements of glycaemic control and urinary albumin excretion were performed every 8 weeks. At 32 weeks, animals were killed, tissues removed and collagen extracted for measurement of fluorescence. Diabetic rats had increased fluorescence in aorta, glomeruli and renal tubules. Aminoguanidine prevented an increase in fluorescence at all three sites suggesting that diabetes-related tissue fluorescence is predominantly due to advanced glycation. Ponalrestat retarded fluorescence in aorta only and butylated hydroxytoluene attenuated fluorescence at the renal sites but not in the aorta. Diabetic rats had increased renal cortical sorbitol levels. Ponalrestat normalized renal cortical sorbitol levels but aminoguanidine did not affect this parameter. The only agent to decrease plasma thiobarbituric acid reactive substances was butylated hydroxytoluene. Diabetic rats developed albuminuria over the 32-week period. This increase in urinary albumin excretion was only attenuated significantly by aminoguanidine therapy, but not by probucol or ponalrestat. The effects of butylated hydroxytoluene on albuminuria were intermediate between aminoguanidine-treated and untreated diabetic rats. The failure of either antioxidants or aldose reductase inhibition to reproduce the renal effects of aminoguanidine suggest that aminoguanidine may act predominantly via inhibition of advanced glycation and not via the alternative biochemical processes evaluated in this study.