Kazuhiko Funabiki

Serum Cystatin C Is a More Sensitive Marker of Glomerular Function than Serum Creatinine

We determined the relationship between the levels of serum cystatin C or creatinine (s-Cr) and the grade of creatinine clearance (CCr) in patients with various glomerular diseases. Serum samples from 96 patients with glomerular diseases were obtained from our hospital. The levels of serum cystatin C were measured using the Dade Behring Cystatin C assay with the automated Dade Behring Nephelometer II (BNII). CCr levels were classified into six groups according to the Guidelines of the Japanese Society of Nephrology as follows: grade 1 (normal renal function); grade 2 (slight decrease of renal function); grade 3 (moderate decrease of renal function); grade 4 (severe decrease of renal function); grade 5 (renal failure), and grade 6 (uremia). The mean levels of serum cystatin C in grade 3 patients were significantly higher than those in grade 1. The mean levels of serum cystatin C in grades 4, 5 and 6 patients were also significantly higher than those in grade 1. However, the mean levels of serum Cr in grade 3 patients were not significantly higher than those in grade 1. The levels of s-Cr in grades 4, 5 or 6 patients were significantly higher than those in grade 1. In this study, an increase of serum cystatin C levels occurred earlier than that of s-Cr in various glomerular diseases. It appears that the levels of serum cystatin C may provide early prognostic marker of patients with various glomerular diseases rather than the levels of s-Cr.

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.

Susceptibility and negative epistatic loci contributing to type 2 diabetes and related phenotypes in a KK/Ta mouse model

The KK/Ta mouse strain serves as a suitable polygenic model for human type 2 diabetes. Using 93 microsatellite markers in 208 KK/Ta x (BALB/c x KK/Ta)F1 male backcross mice, we carried out a genome-wide linkage analysis of KK/Ta alleles contributing to type 2 diabetes and related phenotypes, such as obesity and dyslipidemia. We identified three major chromosomal intervals significantly contributing to impaired glucose metabolism: one quantitative trait locus for impaired glucose tolerance on chromosome 6 and two loci for fasting blood glucose levels on chromosomes 12 and 15. The latter two loci appeared to act in a complementary fashion. Two intervals showed significant linkages for serum triglyceride levels, one on chromosome 4 and the other on chromosome 8. The KK allele on chromosome 8 acts to promote serum triglyceride levels, whereas the KK allele on chromosome 4 acts to suppress this effect in a recessive fashion. In addition, it is suggested that the chromosome 4 locus also acts to downregulate body weight and that the chromosome 8 locus acts to upregulate serum insulin levels. Our data clearly showed that each disease phenotype of type 2 diabetes and related disorders in KK/Ta mice is under the control of separate genetic mechanisms. However, there appear to be common genes contributing to different disease phenotypes. There are potentially important candidate genes that may be relevant to the disease.

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.

Podocyte Injury Predicts Prognosis in Patients with IgA Nephropathy Using a Small Amount of Renal Biopsy Tissue

To predict the progression in patients with IgA nephropathy, we analyzed glomerular lesions except for sclerosis, adhesion and/or crescents in 34 patients with this disease by morphometric analysis. Levels of urinary protein excretion (UP), creatinine clearance (Ccr), serum creatinine (sCr) and mean blood pressure (MBP) at the time of renal biopsy were used as the clinical parameters. The slope of 1/sCr was also used as a prognostic parameter. Renal specimens were obtained by echo-guided biopsy. In PAS-stained light microscopic renal sections, three midsections of open glomeruli were selected and photographed. Stereologic estimation was performed as follows: absolute values of glomerular volume (V_G), glomerular surface area (S_G), podocyte and nonpodocyte cell number per glomerulus (N_G(pod) and N_G(Non-pod)), glomerular surface area covered by one podocyte S_G/N_G(pod)) and glomerular volume occupied by one nonpodocyte cell (V_G/N_G(Non-pod)). There was a significant correlation between the levels of UP and the change of podocyte injury parameters (N_G(pod) and S_G/N_G(pod)) or N_G(Non-pod). N_G(pod) was negatively but S_G/N_G(pod) and N_G(Non-pod) were positively correlated with UP. S_G/N_G(pod) or N_G(Non-pod) was correlated with MBP. N_G(pod), S_G/N_G(pod), N_G(Non-pod), UP or MBP was significantly correlated with the slope of 1/sCr. High specificity was observed for N_G(pod), S_G/N_G(pod) and MBP. High sensitivity was also observed for N_G(Non-pod) and UP. It appears that podocyte injury might provide additional prognostic information in patients with IgA nephropathy.

Urinary levels of interleukin-6 and disease activity in patients with IgA nephropathy.

We studied, using ELISA, 27 patients with IgA nephropathy to determine if levels of urinary interleukin-6 (IL-6) might reflect the disease activity. The levels of urinary IL-6 in patients with the advanced stage were significantly higher than those in patients with the mild stage of the disease or in healthy adults. The results showed a significant correlation between the levels of urinary IL-6 and the disease activity, i.e., levels of urinary cast and urinary protein. It was thus suggested that the measurement of urinary IL-6 is useful in evaluating the degree of glomerular injuries and/or prognosis in patients with IgA nephropathy.

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.

Urinary levels of interleukin-8 (IL-8) and disease activity in patients with IgA nephropathy.

Using quantitative sandwich ELISA, we studied 27 patients with IgA nephropathy to determine whether the levels of urinary IL‐8 might reflect the disease activity. The levels of urinary IL‐8 in patients with advanced stage IgA nephropathy were significantly higher than those in the patients with the mild stage of this disease, or in the healthy controls. The results showed a positive significant correlation between the levels of IL‐8 and disease activity, i.e., between levels of urinary protein and urinary casts. A significant correlation between levels of urinary IL‐8 and tubular function damage was also found. It was thus suggested that measurement of urinary IL‐8 might be useful in evaluating the degree of renal injuries and/or prognosis in patients with IgA nephropathy. J. Clin. Lab. Anal. 15:30–34, 2001.

Effect of Exercise on Kidney Function, Oxidative Stress, and Inflammation in Type 2 Diabetic KK-Ay Mice

Exercise is recommended for the management of type 2 diabetes, but its effects on diabetic nephropathy (DN) are still unknown. We hypothesized that appropriate exercise improves early DN via attenuation of inflammation and oxidative damage. Type 2 diabetic KK-A y mice, a spontaneous DN model, underwent two different kinds of exercise (i.e., moderate and low intensity). Sedentary mice or those undergoing an exercise regimen causing no significant body weight loss were used. We examined the urinary excretion of albumin, number of podocytes and macrophages, renal expressions of HIF-1α and MCP-1, and biomarkers of oxidative stress such as urinary 8-OHdG and serum SOD. Exercise reduced urinary levels of albumin and also maintained the number of podocytes in the exercised KK-A y mice independently of improvements of overweight and hyperglycemia, although moderate-intensity exercise increased expression of HIF-1α. Sedentary KK-A y mice showed increased expression of MCP-1 and infiltration of macrophage, increased urinary 8-OhdG, and decreased serum SOD levels compared with exercised KK-A y mice. On the whole, low-intensity exercise attenuates progression of early DN without affecting marked renal ischemia. Reduction rates of urinary albumin and maintained podocyte numbers, with parallel improvements in oxidative damage and inflammation, are related to beneficial effects of exercise in diabetic kidney disease.

Combination effects of enalapril and losartan on lipid peroxidation in the kidneys of KK-Ay/Ta mice.

Background: Adenosine monophosphate activated protein kinase (AMPK) has a protective effect on lipid peroxidation. Adiponectin and AMPK might have a role in the pathogenesis of diabetic nephropathy. Blockade of the renin-angiotensin system (RAS) increases adiponectin levels and reduces oxidative stress. The objective of the present study was to examine lipid peroxidation via adiponectin and AMPK activation in the kidneys of KK-Ay/Ta mice by RAS inhibitors, such as enalapril and/or losartan. Methods: KK-Ay/Ta mice were given enalapril (2.5 mg/kg/day) and/or losartan (25 mg/kg/day), or hydralazine (25 mg/kg/day) in the drinking water for 8 weeks starting at 8 weeks of age. They were divided into 5 groups as follows: enalapril 2.5 mg/kg/day treatment group (n = 5), losartan 25 mg/kg/day treatment group (n = 5), enalapril 2.5 mg/kg/day + losartan 25 mg/kg/day combination treatment group (n = 5), hydralazine 25 mg/kg/day treatment group (n = 5) and tap water group as the untreated group (n = 5). The urinary albumin/creatinine ratio (ACR), serum adiponectin and systemic blood pressure were measured as test parameters. Expressions of adiponectin, phospho-AMPKα (p-AMPKα) and phospho-acetyl CoA carboxylaseβ (p-ACCβ) in the kidneys were evaluated by Western blot analyses. Pathological changes of glomeruli were evaluated by light microscopy. Accumulations of Nε-(carboxymethyl) lysine (CML), malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) in glomeruli were evaluated by immunohistochemical analyses. Results: Enalapril and/or losartan improved levels of urinary ACR with activation of adiponectin, p-AMPKα and p-ACCβ in the kidneys. CML, MDA and 4-HNE expressions in glomeruli were significantly suppressed by enalapril and/or losartan, especially in the combination treatment group. Conclusions: It appears that enalapril and/or losartan, especially in combination, inhibited accumulation of CML/MDA/4-HNE in diabetic renal tissues. These effects might be related to lipid peroxidation via tissue-specific activation of adiponectin and AMPK.