Early Phase Of Diabetic Nephropathy Research Articles

Urinary Podocalyxin Is an Early Marker for Podocyte Injury in Patients with Diabetic Nephropathy as well as Other Glomerular Disease

Abstract Aims/Objective Nephropathy, a major complication of diabetes, is the leading cause of end-stage renal disease. Recent studies have demonstrated that podocyte injury is involved in the onset of and progression to renal insufficiency. Here, we describe a novel, highly sensitive ELISA for detecting urinary podocalyxin, a glycoconjugate on the podocyte apical surface that indicates podocyte injury, particularly in the early phase of diabetic nephropathy. Methods Urine samples from patients with glomerular diseases (n = 40) and diabetic nephropathy (DN) (n = 40) subdivided into 2 groups (DN with macroalbuminuria (n = 20) and DN with microalbuminuria (n = 20)) were used to quantify urinary podocalyxin by ELISA. Urine samples were obtained from 20 healthy controls for whom laboratory data were within normal values. Podocalyxin was detected in urine by ELISA. Results urinary podocalyxin.detective range was 0.156-10 ng/mL, Levels of urinary podocalyxin were elevated in patients with various glomerular diseases and patients with diabetic nephropathy. level of urine podocalyxin was significantly positively correlated regarding LDL cholesterol and urine protein in all patients group, patients with glomerular diseases and patients with DN, while urine podocalyxin was significantly positively correlated regarding diastolic BP, HbA1c in all patients group but neither in patients with glomerular diseases nor patients with DN, while urine podocalyxin was significantly positively correlated regarding CRP in all patients group but in patients with DN it was significantly negatively correlated.while it was not significantly correlated in patients with glomerular diseases. No significant correlation between urine podocalyxin and pathology in renal biopsy. Conclusions/interpretation the level of urinary podocalyxin could provide information of earlier glomerular impairment in patients with Glomerular diseases and diabetic nephropathy and can be used as earlier marker for Glomerular diseases and diabetic nephropathy like microlbuminuria.

Diosgenin protects against podocyte injury in early phase of diabetic nephropathy through regulating SIRT6

BackgroundDiabetic nephropathy (DN) is a serious complication of diabetes mellitus. DN is the main cause of end-stage renal disease (ESRD). SIRT6 becomes the important target of DN. Diosgenin (a monomer from Chinese herbs) is probable to bind to SIRT6. PurposeBased on studies presented in the literature on kidney injuries plus screening for the binding effects of the drug to Sirt6, we aimed to carry out the study to assess the effects of diosgenin involved in improving podocyte damage in the early phase of DN.. MethodsDN model was established in spontaneous diabetic db/db mice. Animal experiment was in two parts. The first part includes four groups consisting of control (Con) group, model (Mod) group, low dose of diosgenin (DL) group and high dose of diosgenin (DH) group. The second part includes four groups consisting of control group, model group, DH+OSS_128167 (OSS, inhibitor of SIRT6) group, MDL800 (agonist of SIRT6) group. MPC5 cell line was selected in cell experiment, which was mainly composed of six groups including Con group, palmitic acid (PA) group, PA+DL group, PA+DH group, PA+DH+OSS group, PA+MDL800 group. Some procedures such as transcriptomics, RT-qPCR and so on were used in the study to explore and verify the mechanism. ResultsThe abnormal changes of mesangial matrix expansion, glomerular basement membrane (GBM) thickness, foot process (FP) width, urine albumin/creatinine (UACR), DESMIN, ADRP, NEPHRIN, PODOCIN, SIRT6 in Mod group were alleviated in DH group rather than DL group in the first part of animal experiment. The effect in DH group could be reversed in DH+OSS group and the same effect was observed in MDL800 group in the second part of animal experiment. The same results were also found in cell experiment. Protein level and mRNA expression of pyruvate dehydrogenase kinase 4 (PDK4) and Angiopoietin-like-4 (ANGPTL4) were increased in PA group, which could be alleviated in DH group, MDL800 group rather than DH+OSS group. ConclusionsDiosgenin could protect against podocyte injury in early phase of diabetic nephropathy by regulating SIRT6.

Mesangial Cell Mammalian Target of Rapamycin Complex 1 Activation Results in Mesangial Expansion.

Human glomerular diseases can be caused by several different diseases, many of which include mesangial expansion and/or proliferation followed by glomerulosclerosis. However, molecular mechanisms underlying the pathologic mesangial changes remain poorly understood. Here, we investigated the role of the mammalian target of rapamycin complex 1 (mTORC1)-S6 kinase pathway in mesangial expansion and/or proliferation by ablating an upstream negative regulator, tuberous sclerosis complex 1 (TSC1), using tamoxifen-induced Foxd1-Cre mice [Foxd1ER(+) TSC1 mice]. Foxd1ER(+) TSC1 mice showed mesangial expansion with increased production of collagen IV, collagen I, and α-smooth muscle actin in glomeruli, but did not exhibit significant mesangial proliferation or albuminuria. Furthermore, rapamycin treatment of Foxd1ER(+) TSC1 mice suppressed mesangial expansion. Among biopsy specimens from patients with glomerular diseases, analysis of phosphorylated ribosomal protein S6 revealed mesangial cell mTORC1 activation in IgA nephropathy and in lupus mesangial proliferative nephritis but not in the early phase of diabetic nephropathy. In summary, mesangial cell mTORC1 activation can cause mesangial expansion and has clinical relevance for human glomerular diseases. This report also confirms that the tamoxifen-induced mesangium-specific Cre-loxP system is useful for studies designed to clarify the role of the mesangium in glomerular diseases in adults.

Assessment of the diagnostic value of different biomarkers in relation to various stages of diabetic nephropathy in type 2 diabetic patients

Albuminuria is widely used to indicate early phases of diabetic nephropathy although it is limited by the fact that structural damage might precede albumin excretion. This necessitates identifying better biomarkers that diagnose or predict diabetic nephropathy. This is a cross-sectional hospital based study recruiting type 2 diabetic patients cohort aged 35–75 years with diabetes duration of ≥10 years. Out of total eligible 467 patients, 200 patients were with normal albumin excretion, 184 patients with microalbuminuria and 83 patients with macroalbuminuria. All the patients were tested for the 22 selected biomarkers including serum, plasma and urinary markers. Sensitivity, specificity, and area under the curve (AUC) were calculated as measures of diagnostic accuracy. Out of the tested biomarkers, urinary transferrin, urinary Retinol binding protein (RBP) and serum osteopontin had the best diagnostic value for diabetic nephropathy presence based on the AUC value. The rest of the biomarkers had comparatively less or even no discriminative power. The urinary transferrin and RBP and serum osteopontin, had the best diagnostic value in type 2 diabetic patients at different stages of diabetic nephropathy. Further longitudinal prospective studies are needed to evaluate the predictive power of those markers for detecting diabetic nephropathy before any structural damage occurs.

Endocan and albuminuria in type 2 diabetes mellitus

Background: Endocan is a newly identified proteoglycan released from endothelium, stimulating angiogenesis and when increased, indicates endothelial activation (inflammation). Our aim was to examine the association between serum endocan levels and urine albumin–creatinine ratio (UACR).Method: One hundred and thirty-seven patients with type 2 diabetes mellitus and normal serum creatinine who had no co-morbidities other than hypertension, diabetic nephropathy, retinopathy, or neuropathy were divided into normoalbuminuria (G1), microalbuminuria (G2), and macroalbuminuria (G3) groups and compared cross-sectionally regarding serum endocan levels.Result: There were 55, 47, and 35 patients in G1, G2, and G3, respectively. The groups were comparable in terms of gender, age, duration of diabetes, diabetic neuropathy/retinopathy, fasting glucose, HbA1c, serum creatinine level, and eGFR. Patients in G3 had significantly higher blood pressure but lower serum albumin and endocan levels. UACR showed a negative bivariate correlation with serum endocan levels (r = −.282, p = .001). There was bivariate positive correlation between endocan and systolic blood pressure (r=.185, p = .030). In linear regression analysis, UACR was negatively correlated with endocan while positively correlated with systolic blood pressure, duration of diabetes, and platelet distribution width.Conclusion: Patients with macroalbuminuria had lower endocan levels, and increasing UACR was associated with decreasing serum endocan levels. Despite the occurrence of angiogenesis and glomerular hypertrophy in the early phase of diabetic nephropathy, ensuing significant renal injury over time may reduce the expression of endocan. Serum endocan levels may represent a novel marker for nephropathy progression.

GAS6 intron 8 c.834 + 7G > A gene polymorphism in diabetic nephropathy

Background - Aim: In animal experiments, growth arrest-specific 6 (Gas6) protein plays a key role in the development of mesangial cell and glomerular hypertrophy in the early phase of diabetic nephropathy, and diabetic nephropathy is prevented by warfarin-induced inhibition of GAS6 protein. It was shown that GAS6 intron 8 c.834 + 7G > A polymorphism is protective against type 2 diabetes mellitus, and AA genotype is associated with higher blood levels of GAS6 protein. Our aim is to investigate whether this polymorphism is a risk factor for diabetic nephropathy in type 2 diabetes mellitus. Method: Eighty-seven patients with diabetic nephropathy were compared with 66 non-diabetic controls in terms of GAS6 intron 8 c.834 + 7G > A polymorphism. Patients with history of stroke, ischemic heart disease were excluded. Each patient was examined by the ophthalmologist to determine diabetic retinopathy. Results: Frequency of GG, GA and AA genotypes are similar in diabetic nephropathy and control groups according to GAS6 intron 8 c.834 + 7G > A polymorphism (p = 0.837). Rate of diabetic retinopathy was 54.02%. In the subgroup analysis, GA genotype was significantly more frequent than GG genotype in patients with diabetic retinopathy when compared to without diabetic retinopathy (p = 0.010). Conclusion: In our study, GAS6 intron 8 c.834 + 7G > A polymorphism was not associated with diabetic nephropathy in type 2 diabetes mellitus. However, heterozygous state of this polymorphism may be a risk factor for diabetic retinopathy in patients with diabetic nephropathy.

Effect of Astragalus membranaceus extract on diabetic nephropathy.

SummaryDiabetic nephropathy, a microvascular complication of diabetes, is a progressive kidney disease caused by angiopathy of the capillaries in the kidney glomeruli. Herein, we report a case of a 62-year-old patient with a 30 year history of diabetes, who showed a substantial improvement in diabetic nephropathy on administration of 30 g of Astragalus membranaceus extract per day. After 1 month, estimated glomerular filtration rate increased from 47 to 72 ml/min per 1.73 m2 and was subsequently maintained at the 1-month follow-up. Urinary protein levels also decreased following treatment. Herein, we present and discuss the evidence and mechanism of A. membranaceus on diabetic nephropathy in this patient.Learning points Diabetic nephropathy is a progressive kidney disease.Angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) are currently used to prevent and delay the progression of diabetic nephropathy. However, their effects are not sufficient to prevent a decline in kidney function.Furthermore, combination therapy with an ACE inhibitor and an ARB can produce adverse effects without additional benefits.In the early phase of diabetic nephropathy, administration of Astragalus membranaceus can be a therapeutic option.

Alterations in renal hemodynamics are associated with increased renal MMP‐2 expression during the early phase of diabetic nephropathy

The aim of the present study was to compare the early changes in proteinuria, renal hemodynamics and matrix metalloproteinase‐2 (MMP‐2, marker of renal injury) expression in type‐1 diabetic SS rats that are susceptible to renal injury versus diabetic Sprague Dawley (SD) rats that are resistant to renal disease. Rats were fed a low salt diet (0.3% NaCl) to minimize the development of hypertension and injected with streptozotocin (STZ, 50 mg/kg, i.p.) to induce diabetes. Blood glucose levels were similar in both strains 3 weeks after diabetes (487±53 mg/dL). Proteinuria increased to 227±19 mg/day in diabetic SS rats while it only increased to 28±5 mg/day in diabetic SD rats. Mean arterial pressure was significantly increased in diabetic SS rats compared to diabetic SD rats (124±4 vs. 106±7 mmHg, respectively). Three weeks of diabetes caused a 2‐fold rise in glomerular filtration rate in both strains; however, renal blood flow was significantly higher in diabetic SS rats compared to values observed in diabetic SD rats. The expression of the active form of MMP‐2 protein was markedly increased in the renal cortex of SS rats compared to SD rats when treated with STZ for 3 weeks. These data suggest that the upregulation in renal MMP‐2 expression may be due to the hyperglycemia‐mediated elevation in hemodynamic stress to the glomeruli leading to the development of severe proteinuria in the SS strain compared to the SD strain.

Experimental diabetic nephropathy is accelerated in matrix metalloproteinase-2 knockout mice

Matrix metalloproteinase-2 (MMP-2) is responsible for the degradation of various types of extracellular matrix (ECM) proteins such as type IV collagen. Decreased MMP-2 expression and activity has been generally thought to contribute to increased accumulation of ECM at the advanced stage of diabetic nephropathy. However, the kinetics and role of MMP-2 in the early phase of diabetic nephropathy remain unclear. To address this issue, we examined whether streptozotocin (STZ)-induced early diabetic nephropathy was accelerated in MMP-2 knockout (KO) mice. Diabetes was induced by the injection of STZ in 6-week-old control and MMP-2 KO mice. Animals were killed after 16 weeks of diabetes of after observation alone. Compared with non-diabetic control mice, renal MMP-2 expression and activity were increased in 16-week old diabetic mice. Serum levels of blood urea nitrogen and creatinine and urinary excretion levels of albumin and N-acetyl-β-D-glucosaminidase were significantly elevated in diabetic MMP-2 KO mice when compared with wild-type diabetic littermates. Further, accumulation of ECM in the glomeruli and atrophy and fibrosis in the tubulointerstitium were exacerbated, and renal α-smooth muscle actin expression was enhanced in diabetic MMP-2 KO mice. Our present study suggests that renal expression and activity of MMP-2 are increased as a compensatory mechanism in the early phase of diabetic nephropathy. Since MMP-2 could play a protective role against the progression of diabetic nephropathy, further enhancement of MMP-2 expression and/or activity in the kidney may be a therapeutic target for the treatment of early diabetic nephropathy.

Reduction in urinary albumin excretion with a moderate low-carbohydrate diet in patients with type 2 diabetes: a 12-month intervention

IntroductionUrinary albumin excretion (UAE) is a marker of the early phase of diabetic nephropathy. Although a low-carbohydrate diet (LCD) has been shown to effectively improve glycemic control in patients with type 2 diabetes (T2DM), its effects on UAE remain unknown.Patients and methodsA total of 124 patients (mean age ± standard deviation, 61.6 ± 9.2 years) with T2DM were instructed to consume a moderate LCD (1734 ± 416 kcal/d; % carbohydrate:fat:protein = 38:37:19) for 12 months. We measured the levels of UAE, hemoglobin A1c, fasting plasma glucose, fasting serum insulin (IRI), and the serum lipid profiles in the patients and recorded their dosages of antidiabetic drugs during this 12-month period.ResultsOf the 124 patients, 68 were normoalbuminuric, 50 were microalbuminuric, and six were macroalbuminuric at baseline. The patients had relatively good compliance with the moderate LCD diet. After 12 months, the mean levels of hemoglobin A1c, fasting plasma glucose, IRI, homeostasis model assessment-estimated insulin resistance, and the body mass index of all participants significantly decreased (P = 0.003 for IRI and P < 0.001 for the other parameters). Among the microalbuminuric patients (n = 50), remission to normoalbuminuria was frequently achieved (52%) and the geometric mean UAE significantly decreased by 53% (95% confidence interval: 43, 62) over 12 months (P < 0.001). After patients taking an angiotensin 2 receptor blocker were excluded, the UAE significantly decreased by 41% (n = 26, 95% confidence interval: 25, 54; P < 0.001). Moreover, the reduction in UAE was significantly and positively correlated with a reduction in homeostasis model assessment-estimated insulin resistance levels (rs = 0.308; P = 0.031).ConclusionA moderate LCD (38% carbohydrate diet) achieved a remarkable reduction in UAE over 12 months in microalbuminuric patients with T2DM.

Roles of Coagulation Pathway and Factor Xa in Chronic Kidney Disease (CKD)

Considering that fibrin deposition is observed in glomerulonephritis as well as in diabetic nephropathy, we performed studies to clarify the roles of the coagulation pathway and the active type of coagulation factor X (factor Xa) in the development of chronic kidney disease (CKD) using animal models. Factor Xa activates various cell types through protease-activated receptor 2 (PAR2). Several in vitro studies have demonstrated that PAR2 can mediate factor Xa signaling, but not thrombin signaling. Coagulation processes proceed together with the extracellular matrix (ECM) accumulation through factor V expression in rat Thy-1 nephritis. DX-9065a, a factor Xa inhibitor, suppresses this type of glomerulonephritis. The factor Xa inhibitor danaparoid ameliorated proteinuria, cellular proliferation, and fibrin deposition in lipopolysaccharide (LPS)-triggered activation of High IgA (HIGA) strain of ddY mice. Another factor Xa inhibitor, fondaparinux, suppressed urinary protein, glomerular hypertrophy, and connective tissue growth factor (CTGF), and ECM protein deposition together with angiogenesis in diabetic db/db mice. Finally, in the model of peritoneal fibrosis, fondaparinux treatment decreased the thickness of submesothelial fibrotic tissue and angiogenesis. In consideration of the results to potential human therapy, factor Xa regulation may be promising for the treatment of the aggravation in glomerulonephritis and of the early phase of diabetic nephropathy. In the near future, novel factor Xa inhibitors with the characteristics of oral administration and biliary elimination may appear in the clinical use for treatment of cardiovascular diseases.

Albuminuria in non-primary renal disease: risk marker rather than risk factor

The impact of an increased urinary albumin excretion in predicting future renal function loss in subjects with diabetes was emphasized already in the 1980s–1990s [1,2]. During these years, the terminology of ‘early diabetic nephropathy’ was introduced to indicate diabetic subjects with microalbuminuria. In this early phase of diabetic nephropathy, the glomerular filtration rate (GFR) is still generally well preserved. Early nephropathy contrasts with the later phase of overt nephropathy in which albuminuria increases into the macroalbuminuria range and the GFR falls below 60 ml/min to finally progress to the level of endstage renal disease (ESRD). The documentation of the early phase of kidney damage with microalbuminuria but still normal GFR helped to better understand the impact of albuminuria in the loss of renal function in diabetes. Moreover, it led to the demonstration that early intervention by interfering in the renin–angiotensin–aldosterone system (RAAS) [3,4] slows the progression of nephropathy in diabetic patients. Data on the impact of albuminuria on the progressive decline of renal function in non-diabetic renal disease received attention in the 1990s. The findings from large clinical trials, showing the beneficial effect of lowering albuminuria by using agents that interfere with the RAAS to (partially) prevent progression to ESRD [5,6], gave a boost to the concept of focussing the treatment in renal patients on albuminuria and not only on blood pressure [7]. These studies often focused on subjects with macroalbuminuria and a GFR in the range of Stage 3 or 4 chronic kidney disease (CKD) which was mostly due to glomerular disorders. Thus far, no trials have shown that the lowering of albuminuria in the early phase (which is the case in microalbuminuria with a GFR >60 ml/min, thus in Stage 1 or 2 CKD) slows the progressive decline of renal function. There is, however, some evidence that ACE inhibition in subjects with non-diabetic microalbuminuria may prevent future cardiovascular events [8]. In this issue of the journal, Lorenzo et al. nicely show the parallel impact of albuminuria on progressive loss of renal function in patients with diabetic and non-diabetic CKD [9]. In an observational cohort study, they followed the loss of GFR in diabetic and non-diabetic patients. Progression was faster in the diabetic compared to the nondiabetic group. The more rapid progression was, however, fully related to the higher albuminuria in the diabetic group: after adjustment for albuminuria, the progression in the diabetic group was comparable to that in the nondiabetic group. This led the authors to conclude that it is not the diabetes itself, but the albuminuria related to the underlying disease that best predicts progressive decline of renal function. Of particular interest is the underlying diagnosis of the CKD in non-diabetic subjects. These were mostly patients with ischaemic or vascular nephropathy. Interestingly, these causes of ESRD currently form the great majority of new ESRD cases and outweigh the number of subjects with ESRD due to the classical glomerular or interstitial renal diseases [10].

Angiotensin-Receptor Blockers in the Prevention or Treatment of Microalbuminuria

Editorials7 July 2009Angiotensin-Receptor Blockers in the Prevention or Treatment of MicroalbuminuriaPatrick S. Parfrey, MDPatrick S. Parfrey, MDFrom Health Sciences Centre, Memorial University, St. John's, Newfoundland A1B 3V6, Canada.Search for more papers by this authorAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/0003-4819-151-1-200907070-00011 SectionsAboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail Type 1 and type 2 diabetes mellitus are the most frequent causes of end-stage renal disease in the United States (1). The natural history of diabetic nephropathy starts with microalbuminuria, which may progress to macroalbuminuria, gradual deterioration in glomerular filtration rate (GFR), and eventually end-stage kidney disease. Microalbuminuria in diabetes strongly predicts end-stage renal disease and adverse cardiovascular events (2–4). Although inhibitors of the renin–angiotensin system (RAS) have become the mainstays of treating established diabetic nephropathy (5), the role of these agents in the early phases of diabetic nephropathy is unclear. Kidney damage may occur in diabetics who do not ...

Non enzymatic glycosylation of IgG and their urinary excretion in patients with diabetic nephropathy.

Diabetic nephropathy is a major cause of end stage renal disease. Increased excretion of albumin has widely been recognized as an early manifestation of diabetic nephropathy particularly in subjects with diabetes mellitus. However, certain other proteins besides albumin may be excreted in high amount during early phase of diabetic nephropathy. The serum and urinary IgG, Glycosylated hemoglobin, fructosamine and glycosylated IgG were evaluated in the present study. Thirty-two patients of Type 2 Diabetes without any complications, thirty-one patients of Type 2 Diabetes with nephropathy, twenty-six patients of non-diabetic nephropathy and forty normal healthy individuals were enrolled in this study. Subjects were grouped based on their serum creatinine level. Serum IgG, glycosylation of IgG and urinary IgG excretion were increased significantly in diabetic patients compared to healthy controls, which were further increased significantly in chronic renal failure patients with respect to the clinical stage of nephropathy. A positive correlation was observed between glycosylation of IgG and IgG excretion (R(2)=0.5995, 0.7114 respectively) in diabetic patients without any complications and diabetic nephropathy patients only, suggesting a significant role of IgG glycosylation in the vascular clearances of IgG during diabetic nephropathy.

Involvement of asymmetric dimethylarginine (ADMA) in tubulointerstitial ischaemia in the early phase of diabetic nephropathy

Decreased peritubular capillary (PTC) flow due to impaired endothelial function elicits tubulointerstitial ischaemia, thereby enhancing renal damage in chronic kidney disease, including diabetic nephropathy. Since nitric oxide (NO) is a vasodilator and known to play an important role in the maintenance of PTC flow, it is conceivable that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, may cause tubulointerstitial ischaemia, thus being involved in the progression of diabetic nephropathy. In this study, we investigated whether overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that degrades ADMA, could improve tubulointerstitial ischaemia in streptozotocin (STZ)-induced diabetic rats. Recombinant adenovirus vector encoding DDAH-I (Adv-DDAH) or control vector expressing bacterial beta-galactosidase (Adv-LZ) was intravenously administrated to diabetic rats. Three days after the treatment, effects of DDAH overexpression on plasma or urinary levels of ADMA or NO metabolites (NOx), tubulointerstitial ischaemia and renal expression of transforming growth factor-beta (TGF-beta) were evaluated. Renal DDAH expression and activity were reduced in diabetic rats. Urinary levels of ADMA and TGF-beta were increased, while NOx levels were decreased in diabetic rats. Compared with control rats, pimonidazole-detected hypoxic areas were larger in the kidney of diabetic rats, although the number of capillaries in tubulointerstitial regions was not different between the two groups. In addition, renal expression levels of hypoxia-inducible factor-1alpha (HIF-1alpha) and TGF-beta were also increased in diabetic rats. DDAH overexpression significantly inhibited the increase of ADMA and the decrease of NOx and subsequently decreased urinary albumin excretion levels and ameliorated tubulointerstitial hypoxia and HIF-1alpha as well as TGF-beta expression in diabetic rats. The present study demonstrated for the first time that the suppression of ADMA by DDAH overexpression could improve tubulointerstitial ischaemia and subsequent renal damage in experimental diabetic nephropathy. Substitution of DDAH protein or enhancement of its activity may become a novel therapeutic strategy for the treatment of early diabetic nephropathy.

The current clinical problems for early phase of diabetic nephropathy and approach for pathogenesis of diabetic nephropathy

The important clinical problems of diabetic nephropathy are both proteinuria and decrease of renal function. Pathological analysis showed decrease of GFR was correlated to degree of mesangial expansion but not thickening of GBM nor the other findings in human type 1 diabetic nephropathy. From the perspective in renal dysfunction, mesangial matrix expansion was crucial for diabetic nephropathy. However, there was no difference of mesangial expansion between normal and microalbuminuria stage in type 1 and 2 diabetes mellitus (DM). On the other hand, microalbuminuria definitely shows a key related factor for cardiovascular events, but it does not indicate a clear interaction for glomerulosclerosis. We need to search a new clinical marker for renal injury. We have first shown that Smad1 is a transcription factor for α1 and 2 of type 4 collagen (Col4), which is a major component of glomerulosclerosis. We have also identified Smad1 is a critical responsible molecule for developing glomerulosclerosis in rat diabetic nephropathy. We have found the good correlation between glomerulosclerosis and urinary Smad1 but not between glomerulosclerosis and urine albumin. These data suggests that urine Smad1 is a promising clinical marker for underlying glomerular damages in early stage diabetic nephropathy. The study also implicates that angiotensin II (AngII) -Src-Smad1 signaling pathway has played a key role for development of diabetic nephropathy. These suggest that it is necessary to clarify the whole mechanism related to Smad1 to identify the pathogenesis of diabetic nephropathy.

Rosiglitazone Ameliorates Diabetic Nephropathy by Inhibiting Reactive Oxygen Species and Its Downstream-Signaling Pathways

Aim: To study whether rosiglitazone prevents the development of diabetic nephropathy through reduction of reactive oxygen species and its downstream signal transduction pathways.Methods: The rats were intraperitoneally injected with streptozotocin to induce diabetes, meanwhile the rats in the therapeutic groups were given rosiglitazone (5 or 20 mg/kg/day) for 4 weeks by intragastric administration. Blood glucose, serum lipid and creatinine, urinary albumin excretion were measured. Malondialdehyde content, the activities of nuclear factor-ĸB (NF-ĸB), antioxidant enzymes including Cu-Zn SOD and GSH-Px in kidney were also measured. In addition, the mRNA and protein expression of MCP-1 were semiquantitatively determined with PT-PCR and immunohistochemical staining respectively. Results: No significant difference of blood glucose and lipid were found between diabetic rats and rosiglitazone treatment groups. The renal histopathology was improved significantly. The expressions of MCP-1 mRNA and protein, malondialdehyde level and the activity of NF-ĸB were decreased markedly in rats treated with high-dose rosiglitazone, but the activities of renal Cu-Zn SOD and GSH-Px increased significantly. Conclusions: Rosiglitazone treatment prevented glomerular injury in diabetic rats, which was closely related with its roles of reducing reactive oxygen species, NF-ĸB activation and MCP-1 expression in the early phase of diabetic nephropathy.

Angiotensin II-dependent Src and Smad1 signaling pathway is crucial for the development of diabetic nephropathy

Angiotensin II (Ang II) is known to play a pivotal role in the development of diabetic nephropathy. However, the precise mechanism of Ang II-mediated effects on diabetic nephropathy is still unknown. We have reported that Smad1 plays a key role in diabetic mesangial matrix expansion and directly regulates the transcription of type IV collagen (Col4) in vitro and in vivo. Here we examined the effect of Ang II on the expression of Smad1 and mesangial matrix expansion in streptozotocin (STZ)-induced diabetic rats in vivo, using Ang II type 1 receptor blocker, olmesartan. We also examined the signaling mechanism by which Ang II induces mesangial matrix expansion in vitro. Treatment of diabetic rats with low-dose olmesartan for 20 weeks reduced albuminuria and hyperfiltration without affecting blood pressure and inhibited mesangial matrix expansive changes and the expression of Col4 and smooth muscle alpha actin compared with those in untreated rats. Immunohistochemical staining and Western blotting showed that the increased expression of Smad1, phospho-Smad1, and phospho-Src was inhibited by olmesartan. Ang II induced Col4 synthesis and increased expression of phospho-Src and phospho-Smad1 in cultured mesangial cells, which was blocked by olmesartan. PP2, a Src tyrosine kinase inhibitor, and overexpression of dominant negative Src also reduced the phosphorylation of Smad1. Moreover, addition of small-interfering RNA against Src significantly reduced the phosphorylation of Smad1 and synthesis of Col4. Taken together, these results indicate that Ang II can regulate the development of mesangial matrix expansion in the early phase of diabetic nephropathy through Src and Smad1.

Expression of Smad1 is directly associated with mesangial matrix expansion in rat diabetic nephropathy

Diabetic nephropathy is the leading cause of end-stage renal disease, and glomerular mesangial matrix expansion is the hallmark in diabetic nephropathy. However, the precise mechanism for the development of mesangial matrix expansion has remained unknown. The key component involved in mesangial matrix expansion is type IV collagen (Col4). Recently, we have reported that Smad1 transcriptionally regulates expression of Col4 under diabetic conditions in vitro. Here we show that this direct regulator of Col4 also plays a crucial role for mesangial matrix expansion in vivo. Streptozotocin-induced diabetic rats are the model of incipient diabetic nephropathy, and showed various levels of mesangial matrix expansion at 24 weeks. The glomerular expression of Smad1 was significantly increased in diabetic rats with more mesangial matrix expansion by Western blot and immunohistochemical analysis. Furthermore, the glomerular expression of Smad1 was closely correlated with the glomerular expression of Col4 and smooth muscle alpha actin (α-SMA), while albuminuria or glomerular filtration rate was not correlated with mesangial matrix expansion. We also found that urinary excretion of Smad1 was closely associated with the severity of mesangial matrix expansion. In cultured mesangial cells expression of Smad1 upregulated the transcriptional activity of key molecules in mesangial matrix expansion, such as Col4 and α-SMA. These data indicate the critical involvement of Smad1 in mesangial matrix expansion in the early phase of diabetic nephropathy. Our data imply that urinary Smad1 might be a representative diagnostic marker for mesangial matrix expansion in diabetic nephropathy.

The role of adrenomedullin and receptors in glomerular hyperfiltration in streptozotocin-induced diabetic rats

Since adrenomedullin (AM) elicits vasodilatation by binding to specific AM receptors consisted of calcitonin-receptor-like receptor (CRLR)/receptor-activity-modifying protein 2 (RAMP2) or CRLR/receptor-activity-modifying protein 3 (RAMP3) on endothelial cells and stimulating nitric oxide production, AM possibly involves in glomerular capillary dilatation in early phase of diabetic nephropathy. Streptozotocin (STZ)-induced diabetic Sprague-Dawley rats at 4 weeks after the injection were employed for expression studies of AM, RAPM2, and RAMP3. The measurement of AM peptide levels in kidney tissue, plasma, and urine was performed. Human aortic endothelial cells (HAEC) were used to investigate functional link between glucose-induced AM production and nitric oxide release. STZ rats showed glomerular hypertrophy and increased urinary NO2- and NO3- excretion. By Northern blot analyses, AM and RAPM2 mRNAs significantly increased in the kidneys of STZ rats, while RAMP3 mRNA was not altered. In STZ rats, AM peptide was actively secreted into urine (1280 +/- 360 fmol/day vs. control 110 +/- 36 fmol/day). AM peptide was mainly detected on cortical and medullary collecting duct cells in control rat kidneys and AM peptide and mRNA were up-regulated on afferent arterioles and glomeruli of STZ rats. RAMP2 expression was detected on afferent arterioles and not in glomeruli in control rats and it was up-regulated on glomerular endothelial cells in STZ rats. In HAEC culture, d-glucose stimulated AM and nitric oxide production and they were suppressed by addition of AM antisense oligodeoxynucleotides. Up-regulated expression of AM and RAMP2 in afferent arterioles and glomeruli may be related to selective dilatation of glomerular capillary in acute phase of type 1 diabetes.