Tomohito Gohda

Circulating TNF Receptors 1 and 2 Predict ESRD in Type 2 Diabetes

Levels of proinflammatory cytokines associate with risk for developing type 2 diabetes but whether chronic inflammation contributes to the development of diabetic complications, such as ESRD, is unknown. In the 1990s, we recruited 410 patients with type 2 diabetes for studies of diabetic nephropathy and recorded their characteristics at enrollment. During 12 years of follow-up, 59 patients developed ESRD (17 per 1000 patient-years) and 84 patients died without ESRD (24 per 1000 patient-years). Plasma markers of systemic inflammation, endothelial dysfunction, and the TNF pathway were measured in the study entry samples. Of the examined markers, only TNF receptors 1 and 2 (TNFR1 and TNFR2) associated with risk for ESRD. These two markers were highly correlated, but ESRD associated more strongly with TNFR1. The cumulative incidence of ESRD for patients in the highest TNFR1 quartile was 54% after 12 years but only 3% for the other quartiles (P<0.001). In Cox proportional hazard analyses, TNFR1 predicted risk for ESRD even after adjustment for clinical covariates such as urinary albumin excretion. Plasma concentration of TNFR1 outperformed all tested clinical variables with regard to predicting ESRD. Concentrations of TNFRs moderately associated with death unrelated to ESRD. In conclusion, elevated concentrations of circulating TNFRs in patients with type 2 diabetes at baseline are very strong predictors of the subsequent progression to ESRD in subjects with and without proteinuria.

Circulating TNF Receptors 1 and 2 Predict Stage 3 CKD in Type 1 Diabetes

Elevated plasma concentrations of TNF receptors 1 and 2 (TNFR1 and TNFR2) predict development of ESRD in patients with type 2 diabetes without proteinuria, suggesting these markers may contribute to the pathogenesis of renal decline. We investigated whether circulating markers of the TNF pathway determine GFR loss among patients with type 1 diabetes. We followed two cohorts comprising 628 patients with type 1 diabetes, normal renal function, and no proteinuria. Over 12 years, 69 patients developed estimated GFR less than 60 mL/min per 1.73 m(2) (16 per 1000 person-years). Concentrations of TNFR1 and TNFR2 were strongly associated with risk for early renal decline. Renal decline was associated only modestly with total TNFα concentration and appeared unrelated to free TNFα. The cumulative incidence of estimated GFR less than 60 mL/min per 1.73 m(2) for patients in the highest TNFR2 quartile was 60% after 12 years compared with 5%-19% in the remaining quartiles. In Cox proportional hazards analysis, patients with TNFR2 values in the highest quartile were threefold more likely to experience renal decline than patients in the other quartiles (hazard ratio, 3.0; 95% confidence interval, 1.7-5.5). The risk associated with high TNFR1 values was slightly less than that associated with high TNFR2 values. TNFR levels were unrelated to baseline free TNFα level and remained stable over long periods within an individual. In conclusion, early GFR loss in patients with type 1 diabetes without proteinuria is strongly associated with circulating TNF receptor levels but not TNFα levels (free or total).

The early decline in renal function in patients with type 1 diabetes and proteinuria predicts the risk of end-stage renal disease

The risk of end-stage renal disease (ESRD) remains high in patients with type 1diabetes and proteinuria; however, little is known about the rate of decline in their renal function. To help determine this we enrolled patients with 1 diabetes and proteinuria whose estimated glomerular filtration rate (eGFR) was normal (equal to or above 60 ml/min/1.73m2). Using a minimum of 5 serial measurements of serum creatinine for 161 patients, we determined individual trajectories of eGFR change and the occurrence of ESRD during 5–18 years of follow-up. The rates were linear for 110 patients, for 24 the non-linear rate was mild enough to satisfy a linear model, and the rates were clearly non-linear for only 27 patients. Overall, in more than one third of patients, the eGFR decline was less than 3.5 ml/min/1.73m2 per year and the lifetime risk of ESRD could be considered negligible. In the remainder of patients, eGFR declined with widely different slopes and ESRD developed within 2 to 18 years. Based on up to five years observation when renal function was within the normal range, the estimates of early eGFR slope predicted the risk of ESRD during subsequent follow-up better than the baseline clinical characteristics of glycated hemoglobin, blood pressure, or the albumin to creatinine ratio. Thus, the early slope of eGFR decline in patients with type 1diabetes and proteinuria can be used to predict the risk of ESRD.

Attenuating effect of angiotensin-(1–7) on angiotensin II-mediated NAD(P)H oxidase activation in type 2 diabetic nephropathy of KK-Ay/Ta mice

ANG-(1-7) is associated with vasodilation and nitric oxide synthase stimulation. However, the role of ANG-(1-7) in type 2 diabetes mellitus is unknown. In this study, we examined the hypothesis that ANG-(1-7) attenuates ANG II-induced reactive oxygen species stress (ROS)-mediated injury in type 2 diabetic nephropathy of KK-A(y)/Ta mice. KK-A(y)/Ta mice were divided into four groups: 1) a control group; 2) ANG II infusion group; 3) ANG II+ANG-(1-7) coinfusion group; and 4) ANG II+ANG-(1-7)+d-Ala(7)-ANG-(1-7) (A779) coinfusion group. In addition, primary mesangial cells were cultured and then stimulated with 25 mM glucose with or without ANG II, ANG-(1-7), and A779. The ANG II+ANG-(1-7) coinfusion group showed a lower urinary albumin/creatinine ratio increase than the ANG II group. ANG-(1-7) attenuated ANG II-mediated NAD(P)H oxidase activation and ROS production in diabetic glomeruli and mesangial cells. ANG II-induced NF-κB and MAPK signaling activation was also attenuated by ANG-(1-7) in the mesangial cells. These findings were related to improved mesangial expansion and to fibronectin and transforming growth factor-β1 production in response to ANG II and suggest that ANG-(1-7) may attenuate ANG II-stimulated ROS-mediated injury in type 2 diabetic nephropathy. The ACE2-ANG-(1-7)-Mas receptor axis should be investigated as a novel target for treatment of type 2 diabetic nephropathy.

Relationship between polymorphism in the angiotensinogen, angiotensin-converting enzyme or angiotensin II receptor and renal progression in Japanese NIDDM patients.

We determined the relationship between the gene polymorphism of angiotensinogen (AGT), angiotensin-converting enzyme (ACE), or angiotensin II receptor (AT1R) and the progression of diabetic nephropathy in a multicenter trial of ethnically homogeneous Japanese patients with non-insulin-dependent diabetes (NIDDM). Gene polymorphism of ACE I/D, AGT M235T and AT1R A1166C was determined by polymerase chain reaction amplification using allele-specific primers. Japanese NIDDM patients (n = 1,152) were selected from several diabetic clinics. All patients were divided into three groups as follows: (1) group I (n = 407): normoalbuminuric patients; (2) group II (n = 327): microalbuminuric patients, and (3) group III (n = 418): overt albuminuric patients. Clinical factors for investigation in all patients were the date of birth, gender, levels of urinary albumin excretion, findings of the ocular fundus, duration of diabetes, hemoglobin A1c and blood pressure. It appears that genetic polymorphisms in the renin-angiotensin systems, i.e. ACE or AT1R, may affect the progression to renal failure of patients (especially females) with NIDDM.

Glomerular changes in the KK‐Ay/Ta mouse: A possible model for human type 2 diabetic nephropathy

Background:  In type 2 diabetic nephropathy, there is no animal model which has been completely matched with humans. Advanced glycation end products (AGE) and transforming growth factor‐beta (TGF‐β) are closely related to hyperglycaemia and their pathobiochemistry could explain diabetic nephropathy. The objective of the present study was to evaluate the KK‐Ay/Ta mouse as a suitable model for type 2 diabetic nephropathy including pathological changes and immunohistochemical analyses of AGE and TGF‐β, compared with the non‐diabetic BALB/cA mouse.

Pioglitazone attenuates TGF-β1-induction of fibronectin synthesis and its splicing variant in human mesangial cells via activation of peroxisome proliferator-activated receptor (PPAR)γ

The peroxisome proliferator‐activated receptor (PPAR)γ is expressed not only in adipose tissue but also in macrophages/monocytes and plays important roles in acute/chronic inflammation. Transforming growth factor (TGF)‐β is a common pathogenic indicator of sclerosis because it induces the accumulation of extracellular matrix (ECM) in the glomerular mesangium of the kidney. Among components of the ECM, fibronectin (FN) is an acute reactant in inflammation, and isoforms of it produced by splicing of gene variants appear during abnormal conditions such as wound healing. In this study, we examined the effects of pioglitazone, a PPARγ agonist, on TGF‐β1‐induced FN synthesis in cultured mesangial cells using RT‐PCR and Western blot analysis. We also analyzed its splicing variant, extra domain (ED) A, containing FN (EDA+FN). TGF‐β1 enhanced the production of both FN and EDA+ FN and down‐regulated PPARγ expression. Pioglitazone reversed both these effects of TGF‐β1. These findings suggest that PPARγ activation by pioglitazone may affect the TGF‐β1‐induced FN accumulation observed in the glomerular mesangium in cases of glomerulosclerosis, although further in vivo experiments are needed to evaluate this inference.

Increased serum endogenous secretory receptor for advanced glycation end-product (esRAGE) levels in type 2 diabetic patients with decreased renal function.

BACKGROUND The binding of advanced glycation end-products (AGEs) to their receptor for AGEs (RAGE) may play an important role in the development of diabetic vascular complications. Recently, soluble RAGE (sRAGE) has been identified as an alternative splicing form of RAGE. Furthermore, administration of sRAGE improved atherosclerosis in type 2 diabetic mice. OBJECTIVE The objective of the present study is to investigate the role of endogenous secretory RAGE (esRAGE) as a biological marker for type 2 diabetic nephropathy, and also to determine whether serum esRAGE levels are associated with serum AGEs [including Nepsilon-(carboxymethyl) lysine-protein adducts (CML) and pentosidine] levels. MATERIALS AND METHODS Serum esRAGE levels were examined in 107 type 2 diabetic patients including those on hemodialysis (HD). Diabetic patients were divided into three groups as follows: Group A [patients without nephropathy, i.e. normoalbuminuric stage (AER<30microg/mg creatinine)], Group B [patients with nephropathy (AER>30microg/mg creatinine) but excluding HD patients], and Group C (HD patients). RESULTS Serum esRAGE and AGEs (including CML and pentosidine) levels in Group C were significantly higher than in Group A or B. In single linear univariate correlation, serum esRAGE levels were correlated using body mass index (BMI), duration of diabetes, and serum creatinine, high-density lipoprotein (HDL)-cholesterol and AGEs (including CML and pentosidine) levels. Furthermore, in stepwise multivariate regression analysis, the levels of serum creatinine and duration of diabetes were independently associated with serum esRAGE levels. CONCLUSION Serum esRAGE levels are associated with the severity of renal dysfunction and duration of diabetes in type 2 diabetic patients.

Relationship between Abdominal Fat Accumulation and Insulin Resistance in Hemodialysis Patients

It is well known that obesity and insulin resistance are closely related to the development of type 2 diabetes. However, the exact pathogenic mechanism underlying the insulin resistance in renal disease has not been clarified. The purpose of the present study was to clarify the contribution of abdominal (visceral and subcutaneous) fat accumulation to insulin resistance and various clinical parameters, including C-reactive protein (CRP), in hemodialysis (HD) patients. Visceral and subcutaneous fat areas (VFA and SFA) were evaluated at the umbilical level by CT. Insulin resistance was estimated by the homeostasis model assessment−insulin resistance index (HOMA-IR) in 80 HD patients. Insulin resistance and CRP seemed to be closely correlated with fat-related parameters such as body mass index (BMI), VFA and SFA. HOMA-IR was positively correlated with BMI, VFA, SFA, triglycerides (TG), remnant-like particle (RLP)-cholesterol and CRP in simple regression analysis. In multiple stepwise regression analysis, SFA and RLP-cholesterol were predominant determinants of HOMA-IR in HD patients. Furthermore, CRP was positively correlated with BMI, VFA, SFA, TG, high-density lipoprotein (HDL)-cholesterol, atherosclerosis index (AI), immunoreactive insulin (IRI) and HOMA-IR in simple regression analysis. In multiple stepwise regression analysis, VFA and HDL-cholesterol were predominant determinants of CRP in HD patients. In conclusion, insulin resistance and CRP were related to fat-related parameters such as BMI, VFA and SFA in HD patients. Furthermore, the contribution of SFA to insulin resistance was much higher than that of VFA, while the opposite relation was recognized for CRP. (Hypertens Res 2008; 31: 83−88)

Effect of Combination Therapy with Angiotensin Receptor Blocker and 1,25-Dihydroxyvitamin D3 in Type 2 Diabetic Nephropathy in KK-Ay/Ta Mice

Background: Although angiotensin II type 1 receptor blockers (ARB) have beneficial effects in patients with diabetic nephropathy, they may induce a compensatory increase in renin. Renin exhibits profibrotic actions independent of angiotensin II, which is regulated by extracellular signal-regulated kinase 1 and 2 (ERK1/2). Calcitriol (1,25(OH)2D3) is a negative inhibitor of the renin-angiotensin system and the present study examined the effects of combination therapy with an ARB and 1,25(OH)2D3 on diabetic nephropathy in KK-Ay/Ta mice. Methods: KK-Ay/Ta mice were divided into four groups: ARB group, 1,25(OH)2D3 group, combination group, and control group. The urinary albumin/creatinine ratio (ACR) was measured and the renal expression of renin, p-ERK1/2 and TGF-β1 protein determined. Results: The levels of urinary ACR in the combination group were significantly lower than those in the ARB or control group. Renal expression of renin in the ARB group was significantly increased compared with the control group but was significantly decreased in both the 1,25(OH)2D3 and combination group. Renal expression of p-ERK1/2 in the combination group was significantly decreased compared with the control or ARB group. Expression of TGF-β1 protein in the ARB and combination groups, especially the combination group, was significantly decreased compared with those in the control group. Conclusions: These data suggest that the addition of 1,25(OH)2D3 to therapy with ARB further reduced proteinuria by suppressing the compensatory increase in renin expression in type 2 diabetic nephropathy. These effects might relate to suppression of renin, ERK1/2 and TGF-β1 expression which may or may not depend on angiotensin II.