Beyond proteinuria: VDR activation reduces renal inflammation in experimental diabetic nephropathy

Local inflammation is thought to contribute to the progression of diabetic nephropathy. The vitamin D receptor (VDR) activator paricalcitol has an antiproteinuric effect in human diabetic nephropathy at high doses. We have explored potential anti-inflammatory effects of VDR activator doses that do not modulate proteinuria in an experimental model of diabetic nephropathy to gain insights into potential benefits of VDR activators in those patients whose proteinuria is not decreased by this therapy. The effect of calcitriol and paricalcitol on renal function, albuminuria, and renal inflammation was explored in a rat experimental model of diabetes induced by streptozotocin. Modulation of the expression of mediators of inflammation by these drugs was explored in cultured podocytes. At the doses used, neither calcitriol nor paricalcitol significantly modified renal function or reduced albuminuria in experimental diabetes. However, both drugs reduced the total kidney mRNA expression of IL-6, monocyte chemoattractant protein (MCP)-1, and IL-18. Immunohistochemistry showed that calcitriol and paricalcitol reduced MCP-1 and IL-6 in podocytes and tubular cells as well as glomerular infiltration by macrophages, glomerular cell NF-κB activation, apoptosis, and extracellular matrix deposition. In cultured podocytes, paricalcitol and calcitriol at concentrations in the physiological and clinically significant range prevented the increase in MCP-1, IL-6, renin, and fibronectin mRNA expression and the secretion of MCP-1 to the culture media induced by high glucose. In conclusion, in experimental diabetic nephropathy VDR activation has local renal anti-inflammatory effects that can be observed even when proteinuria is not decreased. This may be ascribed to decreased inflammatory responses of intrinsic renal cells, including podocytes, to high glucose.     

Berberine ameliorates renal injury in diabetic C57BL/6 mice: Involvement of suppression of SphK-S1P signaling pathway

Berberine (BBR) was previously found to have beneficial effects on renal injury in experimental diabetic rats. However, the mechanisms underlying the effects are not fully understood. Sphingosine kinase-Sphingosine 1-phosphate (SphK-S1P) signaling pathway has been implicated in the pathogenesis of diabetic nephropathy (DN). The aim of this study was to investigate the effects of BBR on renal injury and the activation of SphK-S1P signaling pathway in alloxan-induced diabetic mice with nephropathy. Alloxan-induced diabetic mice were treated orally with BBR (300 mg/kg/day) or vehicle for 12 weeks. BBR inhibited the increases in fasting blood glucose, kidney/body weight ratio, blood urea nitrogen, serum creatinine and 24-h albuminuria in diabetic mice. It also prevented renal hypertrophy, TGF-β1 synthesis, FN and Col IV accumulation. Moreover, BBR down-regulated the elevated staining, activity and levels of mRNA and protein of SphK1, and S1P production as well. These findings suggest that the inhibitory effect of BBR on the activation of SphK-S1P signaling pathway in diabetic mouse kidney is a novel mechanism by which BBR partly exerts renoprotective effects on DN.     

Intermittent administration of a sustained-release prostacyclin analog ONO-1301 ameliorates renal alterations in a rat type 1 diabetes model

Diabetic nephropathy is the most common pathological disorder predisposing end-stage renal disease. ONO-1301 is a novel sustained-release prostacyclin analog possessing thromboxane (TX) synthase inhibitory activity. Here, we aimed to investigate the therapeutic efficacies of ONO-1301 in a rat type 1 diabetic nephropathy model. Streptozotocin (STZ)-induced diabetic rats received injections of slow-release form of ONO-1301 (SR-ONO) every 3 weeks. Animals were sacrificed at Week 14. SR-ONO significantly suppressed albuminuria, glomerular hypertrophy, mesangial matrix accumulation, glomerular accumulation of monocyte/macrophage, increase in glomerular levels of pro-fibrotic factor transforming growth factor (TGF)-beta1 and the number of glomerular alpha-smooth muscle actin (SMA)(+) cells in diabetic animals. The glomerular levels of hepatocyte growth factor (HGF) were significantly increased in SR-ONO-treated diabetic animals. Taken together, these results suggest the potential therapeutic efficacy of intermittent administration of SR-ONO in treating diabetic nephropathy potentially via inducing HGF, thus counteracting the pro-fibrotic effects of TGF-beta1.     

Protective effects of plant-derived natural products on renal complications

Diabetic nephropathy is the leading cause of renal failure worldwide. This debilitating disorder has several underlying pathophysiologic mechanisms, and therefore a variety of pharmacologic agents have been developed to prevent or treat diabetic nephropathy; however, synthetic drugs may possess unfavorable side effects. In response to this, the global use of herbal-based pharmacologic agents is increasing among diabetic patients. Numerous studies have reported therapeutic benefits of herbal-based compounds against diabetes-induced renal dysfunction. These agents can prevent renal dysfunction and improve renal function by blocking or suppressing deleterious pathways such as oxidative stress, inflammation, apoptosis, necrosis, and nitric oxide deprivation that lead to vascular injuries. In the current study, we have reviewed the beneficial properties of the most common herbal agents used in renal complications and diabetic nephropathy.     

CS-886, a new angiotensin II type 1 receptor antagonist,ameliorates glomerular anionic site loss and prevents progression of diabetic nephropathy in Otsuka Long-Evans Tokushima fatty rats

BACKGROUND: Diabetic nephropathy is a leading cause of end-stage renal disease in industrialized countries. Previous studies have documented that angiotensin converting enzyme (ACE) inhibitors consistently reduce albuminuria and retard the progression of diabetic nephropathy. However, the involvement of angiotensin II in diabetic nephropathy is not fully understood. MATERIALS AND METHODS: In this study we compared the effects of CS-866, a new angiotensin II type 1 receptor antagonist, to that of an ACE inhibitor, temocapril hydrochloride, on the development and progression of diabetic nephropathy using Otsuka Long-Evans Tokushima fatty rats, a type II diabetes mellitus model animal. RESULTS: High doses of CS-866 or temocapril treatment were found to significantly improve urinary protein and beta(2)-microglobulin excretions in diabetic rats. In electron microscopic analysis, loss of glomerular anionic sites, one of the causes of glomerular hyperpermeability in diabetic nephropathy, was found to be significantly prevented by CS-866 treatment. Light microscopic examinations revealed that both treatments ameliorated glomerular sclerosis and tubulointerstitial injury in diabetic rats. Furthermore, high doses of CS-866 or temocapril treatment significantly reduced the positive stainings for transforming growth factor-beta (TGF-beta), vascular endothelial growth factor, and type IV collagen in glomeruli of diabetic rats. CONCLUSIONS: These results indicate that intrarenal angiotensin II type 1 receptor activation plays a dominant role in the development and progression of diabetic nephropathy. Our study suggests that CS-866 represents a valuable new drug for the treatment of diabetic patients with nephropathy.     

G protein-coupled receptors: potential therapeutic targets for diabetic nephropathy

Diabetic nephropathy, a lethal microvascular complication of diabetes mellitus, is characterized by progressive albuminuria, excessive deposition of extracellular matrix, thickened glomerular basement membrane, podocyte abnormalities, and podocyte loss. The G protein-coupled receptors (GPCRs) have attracted considerable attention in diabetic nephropathy, but the specific effects have not been elucidated yet. Likewise, abnormal signaling pathways are closely interrelated to the pathologic process of diabetic nephropathy, despite the fact that the mechanisms have not been explored clearly. Therefore, GPCRs and its mediated signaling pathways are essential for priority research, so that preventative strategies and potential targets might be developed for diabetic nephropathy. This article will give us comprehensive overview of predominant GPCR types, roles, and correlative signaling pathways in diabetic nephropathy.     

Dehydrozingerone inhibits renal lipotoxicity in high-fat diet–induced obese mice

Ectopic fat accumulation in the kidneys causes oxidative stress, inflammation and cell death. Dehydrozingerone (DHZ) is a curcumin analog that exhibits antitumour, antioxidant and antidiabetic effects. However, the efficacy of DHZ in diabetic nephropathy (DN) is unknown. Here, we verified the efficacy of DHZ on DN. We divided the experimental animals into three groups: regular diet, 60% high-fat diet (HFD) and HFD with DHZ for 12 weeks. We analysed levels of renal triglycerides and urinary albumin and albumin-creatinine ratio, renal morphological changes and molecular changes via real-time polymerase chain reaction and immunoblotting. Furthermore, high glucose (HG)- or palmitate (PA)-stimulated mouse mesangial cells or mouse podocytes were treated with DHZ for 24 h. As a result, DHZ markedly reduced renal glycerol accumulation and albuminuria excretion through improvement of thickened glomerular basement membrane, podocyte loss and slit diaphragm reduction. In the renal cortex in the HFD group, phospho-AMPK and nephrin expression reduced, whereas arginase 2 and CD68 expression increased; however, these changes were recovered after DHZ administration. Increased reactive oxygen species (ROS) stimulated by HG or PA in podocytes was inhibited by DHZ treatment. Collectively, these findings indicate that DHZ ameliorates DN via inhibits of lipotoxicity-induced inflammation and ROS formation.     

A review of the anti-inflammatory properties of antidiabetic agents providing protective effects against vascular complications in diabetes

The global prevalence of Type 2 diabetes mellitus and its associated complications are growing rapidly. Although the role of hyperglycemia is well recognized in the pathophysiology of diabetic complications, its exact underlying mechanisms are not fully understood. In this regard, accumulating evidence suggests that the role of inflammation appears pivotal, with studies showing that most diabetic complications are associated with an inflammatory response. Several classes of antidiabetic agents have been introduced for controlling glycemia, with evidence that these pharmacological agents may have modulatory effects on inflammation beyond their glucose-lowering activity. Here we review the latest evidence on the anti-inflammatory effects of commonly used antidiabetic medications and discuss the relevance of these effects on preventing diabetic complications.     

Endocannabinoids and the renal proximal tubule: An emerging role in diabetic nephropathy

Diabetic nephropathy is a leading cause for the development of end-stage renal disease. In diabetes mellitus, a number of structural changes occur within the kidney which leads to a decline in renal function. Damage to the renal proximal tubule cells (PTCs) in diabetic nephropathy includes thickening of the basement membrane, tubular fibrosis, tubular lesions and hypertrophy. A clearer understanding of the molecular mechanisms involved in the development of diabetic kidney disease is essential for the understanding of the role cellular pathways play in its pathophysiology. The endocannabinoid system is an endogenous lipid signalling system which is involved in lipogenesis, adipogenesis, inflammation and glucose metabolism. Recent studies have demonstrated that in diabetic nephropathy, there is altered expression of the endocannabinoid system. Future investigations should clarify the role of the endocannabinoid system in the development of diabetic nephropathy and within this system, identify potential therapeutics to reduce the burden of this disease.     

The proximal tubule in the pathophysiology of the diabetic kidney

Diabetic nephropathy is a leading cause of end-stage renal disease. A better understanding of the molecular mechanism involved in the early changes of the diabetic kidney may permit the development of new strategies to prevent diabetic nephropathy. This review focuses on the proximal tubule in the early diabetic kidney, particularly on its exposure and response to high glucose levels, albuminuria, and other factors in the diabetic glomerular filtrate, the hyperreabsorption of glucose, the unique molecular signature of the tubular growth phenotype, including aspects of senescence, and the resulting cellular and functional consequences. The latter includes the local release of proinflammatory chemokines and changes in proximal tubular salt and fluid reabsorption, which form the basis for the strong tubular control of glomerular filtration in the early diabetic kidney, including glomerular hyperfiltration and odd responses like the salt paradox. Importantly, these early proximal tubular changes can set the stage for oxidative stress, inflammation, hypoxia, and tubulointerstitial fibrosis, and thereby for the progression of diabetic renal disease.     

Inhibitory effect of activin A on activation of lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells

Several works in the setting of early experimental diabetic nephropathy using anti-inflammatory drugs, such as mycophenolate mofetil (MMF), have shown that prevention of the development or amelioration of renal injury including proteinuria. The exact mechanisms by which anti-inflammatory drugs lower the albuminuria have no still to clarify well. In this study, diabetes was induced by injection of streptozotocin after uninephrectomy. Rats were randomly divided into three groups: control group, diabetic group and diabetic group treated with MMF. Elevated 24h urinary albumin excretion rate was markedly attenuated by MMF treatment. In diabetic rats receiving no treatment, there were increase in ED-1+ cells in the glomeruli, which were effectively suppressed by MMF treatment. The expression of nephrin and podocin protein was reduced in the glomeruli from diabetic rats, and MMF treatment significantly increased the expression of nephrin and podocin. The expression of IL-1, TNF-alpha and 3-NT protein in the glomeruli were significantly increased in diabetic rats, which were all significantly inhibited by MMF treatment. Our results show that MMF could decrease urinary albumin excretion, which mechanism may be at least partly correlated with upregulated expression of nephrin and podocin in the glomeruli of diabetic rat.     

Apoptosis induced by endoplasmic reticulum stress involved in diabetic kidney disease

Endoplasmic reticulum stress has been suggested to play a crucial role in the pathogenesis of diabetic complications. However, whether it is involved in the renal injury of diabetic nephropathy is still not known. We investigated the involvement of ER-associated apoptosis in kidney disease of streptozocin (STZ)-induced diabetic rats. We used albuminuria examination, hematoxylin & eosin (H&E) staining and TUNEL analysis to identify the existence of diabetic nephropathy and enhanced apoptosis. We performed immunohistochemistry, Western blot, and real-time PCR to analyze indicators of ER molecule chaperone and ER-associated apoptosis. GRP78, the ER chaperone, was up-regulated significantly in diabetic kidney compared to control. Furthermore, three hallmarks of ER-associated apoptosis, C/EBP homologous protein (CHOP), c-JUN NH2-terminal kinase (JNK) and caspase-12, were found to have activated in the diabetic kidney. Taken together, those results suggested that apoptosis induced by ER stress occurred in diabetic kidney, which may contribute to the development of diabetic nephropathy.     

TLR4 Activation Promotes Podocyte Injury and Interstitial Fibrosis in Diabetic Nephropathy

Toll like receptor (TLR) 4 has been reported to promote inflammation in diabetic nephropathy. However the role of TLR4 in the complicated pathophysiology of diabetic nephropathy is not understood. In this study, we report elevated expression of TLR4, its endogenous ligands and downstream cytokines, chemokines and fibrogenic genes in diabetic nephropathy in WT mice with streptozotocin (STZ) diabetes. Subsequently, we demonstrated that TLR4^−/− mice were protected against the development of diabetic nephropathy, exhibiting less albuminuria, inflammation, glomerular hypertrophy and hypercellularity, podocyte and tubular injury as compared to diabetic wild-type controls. Marked reductions in interstitial collagen deposition, myofibroblast activation (α-SMA) and expression of fibrogenic genes (TGF-β and fibronectin) were also evident in TLR4 deficient mice. Consistent with our in vivo results, high glucose directly promoted TLR4 activation in podocytes and tubular epithelial cells in vitro, resulting in NF-κB activation and consequent inflammatory and fibrogenic responses. Our data indicate that TLR4 activation may promote inflammation, podocyte and tubular epithelial cell injury and interstitial fibrosis, suggesting TLR4 is a potential therapeutic target for diabetic nephropathy.     

Increased renal GLUT1 abundance and urinary TGF-beta 1 in streptozotocin-induced diabetic rats: implications for the development of nephropathy complicating diabetes.

Increased expression of transforming growth factor beta-1 (TGF-beta 1) and glucose transporter (GLUT1) has been implicated in the genesis of diabetic nephropathy. The aim of this study was to evaluate GLUT1 protein levels in the renal cortex of a rat model of diabetes as well as its relationship to urinary albumin and TGF-beta1. Streptozotocin-injected rats (n = 13) and controls (n = 13) were compared for their urinary albumin, and TGF-beta 1 and for renal cortical and medullar GLUT1 protein abundance. GLUT1 protein content was determined by optical densitometry after Western blotting using an anti-GLUT1 antibody; urinary albumin was measured using electroimmunoassay, urinary TGF-beta 1 using ELISA. Forty-five days of diabetes resulted in increased albuminuria (p < 0.05), urinary TGF-beta 1 (p < 0.05) and GLUT1 protein abundance (p < 0.05). There was a positive correlation between urinary TGF-beta 1 and plasma glucose levels (r = 0.65, p < 0.05) and albuminuria (r = 0.72, p < 0.05). We concluded that 45 days of diabetes result in incipient diabetic nephropathy and increased cortical GLUT1 protein abundance. We speculate that the higher cortical GLUT1 protein levels in diabetes may amplify the effects of hyperglycemia in determining higher intracellular glucose in mesangial cells, thereby contributing to diabetes-related kidney damage.     

Periostin as a Tissue and Urinary Biomarker of Renal Injury in Type 2 Diabetes Mellitus

Background

Improving the early detection of diabetic nephropathy remains a great challenge in disease management. Periostin is a marker of renal tubular injury and related to progressive kidney injury in animal models of chronic kidney disease. The clinical implications of urinary periostin activities in patients with type 2 diabetes have not been evaluated.

Methods

Urine samples were obtained from 30 healthy volunteers and 328 type 2 diabetic patients with normoalbuminuria (n=114), microalbuminuria (n=100) and macroalbuminuria (n=114). The excretion levels of urinary periostin were quantified with enzyme-linked immunosorbent assay. Immunohistochemical periostin expression was determined in kidney tissues from overt diabetic nephropathy.

Results

Increased periostin expression in glomeruli and tubular epithelium in diabetic renal pathology was observed. Urinary periostin levels were significantly elevated in the patients of the normoalbuminuria [3.06 (IQR: 1.12, 6.77) ng/mgCr], microalbuminuria [8.71 (IQR: 5.09, 19.29) ng/mgCr] and macroalbuminuria [13.58 (IQR: 3.99, 16.19) ng/mgCr] compared with healthy controls [1.15 (IQR: 0.60, 1.63) ng/mgCr] (P<0.01).Increased urine periostin level significantly correlated with aging, high albuminuria and decline of GFR. Urine periostin ELISA also demonstrated high performance for the diagnosis of established normoalbuminuric, microalbuminuric and macroalbuminuric type 2 diabetes (AUC 0.78 (95%CI, 0.71 to 0.86), 0.99 (95%CI, 0.98 to 1.00) and 0.95 (95%CI, 0.91 to 0.98), respectively).

Conclusion

The study indicates that increased urine periostin levels can be detected in patients with type 2 diabetes before the onset of significant albuminuria. Urinary periostin is an associated renal derangement in patients with established diabetic nephropathy and it may be used as an early marker of diabetic renal injury.     

A search for association between the polymorphic markers of PON1 and PON2 genes and diabetic nephropathy in patients with type I diabetes mellitus

Alleles and genotypes of polymorphic markers of paraoxonase 1 and paraoxonase 2 genes (PON1 and PON2) encoding enzymes of the body antioxidative defense were compared in type 1 diabetes mellitus patients with or without diabetic nephropathy. The patients with nonoverlapping ("polar") phenotypes constituted different groups. The first group contained patients with diabetic nephropathy (DN+, n = 62), clinical proteinuria (albuminuria above 300 mg per day), and at least 15-year disease duration. In control group, the patients had no diabetic nephropathy (DN-, n = 68), their albuminuria was below 200 mg per day, and disease duration was at least 20 years. Comparative analysis with exact Fisher's test revealed no significant differences in frequencies of alleles and genotypes of the PON1 gene polymorphic marker Gln192Arg and of PON2 gene polymorphic markers Ala148Gly and Cys311Ser. Our results suggest that the polymorphic markers studied are not associated with diabetic nephropathy among Russian patients in Moscow.     

Hinokitiol, a metal chelator derived from natural plants, suppresses cell growth and disrupts androgen receptor signaling in prostate carcinoma cell lines

Diabetic patients are often treated with a lipid lowering statin and an ACE inhibitor or angiotensin receptor antagonist against hypertension or albuminuria. These drugs may also improve glucose tolerance, but the mechanism for this remains elusive. We now studied whether these drugs and the fatty acid palmitate influence insulin secretion in vivo in rats through effects on islet blood perfusion. Whole pancreatic blood flow was markedly increased by captopril and irbesartan, and decreased by palmitate. Islet blood flow was significantly and preferentially enhanced by captopril, irbesartan, and pravastatin, and suppressed by palmitate. Both captopril and irbesartan raised serum insulin concentrations significantly. However, glycemia was not affected in any group. In conclusion, the present study suggests that a local pancreatic RAS and pravastatin may be selectively controlling pancreatic islet blood flow and thereby influencing insulin secretion. The antidiabetic actions of statins and RAS inhibitors might in part occur through the beneficial direct islet effects shown here. Conversely, free fatty acids that are elevated in type 2 diabetic patients may contribute to an impaired nutritive islet blood flow and thereby further aggravate the diabetic state by limiting the supply of insulin needed to curb hyperglycemia.     

Advanced glycation end products and diabetic nephropathy: a comparative study using diabetic and normal rats with methylglyoxal-induced glycation

Hyperglycemia-related advanced glycation end product (AGE) formation is a key mechanism in diabetic nephropathy. Since methylglyoxal (MG) is a potent AGE precursor, we aimed to assess the role of MG-related AGE formation in the progression of renal damages. A comparative study between Wistar (W, normal) and Goto-Kakizaki (GK, nonobese type 2 diabetic) rats was performed at 6 and 14 months old and after 14 weeks of MG administration to 6-month-old rats. Diabetic rats showed progressive structural, biochemical, and functional alterations, including AGE, albuminuria, and tissue hypoxia, which were partially mimicked by MG administration to young GK rats. Aged Wistar rats had an impairment of some parameters, whereas MG administration caused a phenotype similar to young GK rats, including oxidative stress, impaired apoptotic and angiogenic markers, and structural lesions. MG accumulation specifically impaired several of the renal disease markers progressively observed in diabetic rats, and thus, it contributes to the progression of diabetic nephropathy.     

Interleukin-18 and diabetic nephropathy: A review

The inflammatory response has an important role in the pathophysiology of diabetic nephropathy that is contributed to by inflammatory mediators such as interleukin-1 (IL-1), IL-6, IL-18, tumor necrosis factor-α, and macrophage chemotactic protein-1; however, the role of IL-18 seems to be more specific than other cytokines in the inflammatory process. IL-18 is expressed in renal tissue and is upregulated by several stimuli including hyperglycemia. The expression/urinary level of IL-18 is positively correlated with the progression of diabetic nephropathy and the urinary albumin excretion rate. In this review, we have focused on the molecular pathways modulating the relationship between IL-18 and diabetic nephropathy.     

Long Term Metabolic Syndrome Induced by a High Fat High Fructose Diet Leads to Minimal Renal Injury in C57BL/6 Mice

Metabolic syndrome can induce chronic kidney disease in humans. Genetically engineered mice on a C57BL/6 background are highly used for mechanistic studies. Although it has been shown that metabolic syndrome induces cardiovascular lesions in C57BL/6 mice, in depth renal phenotyping has never been performed. Therefore in this study we characterized renal function and injury in C57BL/6 mice with long-term metabolic syndrome induced by a high fat and fructose diet (HFFD). C57BL/6 mice received an 8 months HFFD diet enriched with fat (45% energy from fat) and drinking water enriched with fructose (30%). Body weight, food/water consumption, energy intake, fat/lean mass ratio, plasma glucose, HDL, LDL, triglycerides and cholesterol levels were monitored. At 3, 6 and 8 months, renal function was determined by inulin clearance and measure of albuminuria. At sacrifice, kidneys and liver were collected. Metabolic syndrome in C57BL/6 mice fed a HFFD was observed as early 4 weeks with development of type 2 diabetes at 8 weeks after initiation of diet. However, detailed analysis of kidney structure and function showed only minimal renal injury after 8 months of HFFD. HFFD induced moderate glomerular hyperfiltration (436,4 µL/min vs 289,8 µL/min; p-value=0.0418) together with a 2-fold increase in albuminuria only after 8 months of HFFD. This was accompanied by a 2-fold increase in renal inflammation (p-value=0.0217) but without renal fibrosis or mesangial matrix expansion. In addition, electron microscopy did not show alterations in glomeruli such as basal membrane thickening and foot process effacement. Finally, comparison of the urinary peptidome of these mice with the urinary peptidome from humans with diabetic nephropathy also suggested absence of diabetic nephropathy in this model. This study provides evidence that the HFFD C57BL/6 model is not the optimal model to study the effects of metabolic syndrome on the development of diabetic kidney disease.