Masafumi Kakei

Association of monocyte chemoattractant protein-1 with renal tubular damage in diabetic nephropathy.

Monocyte chemoattractant protein-1 (MCP-1), is a chemokine that mediates renal interstitial inflammation, tubular atrophy, and interstitial fibrosis by recruiting monocytes/macrophages into renal tubulointerstitium. Recent studies have demonstrated that protein overload in renal tubular cells up-regulates MCP-1 gene and its protein expression. Therefore, we hypothesized that increased expression of MCP-1 in renal tubuli, probably triggered by an increase in the leakage of plasma protein from glomerular capillary to tubular fluid, may contribute to renal tubular damage and accelerate the progression of diabetic nephropathy. To test this hypothesis, we examined urinary excretion levels of MCP-1 and N-acetylglucosaminidase (NAG), a sensitive marker of renal tubular damage, in Japanese Type II diabetic patients with normoalbuminuria (n=29), microalbuminuria (n=25), and macroalbuminuria (n=18). The median urinary excretion level of MCP-1 in patients with macroalbuminuria (394.4 ng/g creatinine) was significantly elevated compared to the levels in patients with normoalbuminuria and microalbuminuria (159.6 and 193.9 ng/g creatinine, respectively). Furthermore, the urinary MCP-1 excretion level was positively correlated with urinary excretion levels of albumin (r=.816, P<.001) and NAG (r=.569, P<.001) in all subjects. These results suggest that MCP-1 is produced in renal tubular cells and released into urine in proportion to the degree of proteinuria (albuminuria), and that increased MCP-1 expression in renal tubuli contributes to renal tubular damage. Therefore, we conclude that heavy proteinuria itself may accelerate the progression of diabetic nephropathy by increasing the MCP-1 expression in renal tubuli.

Hepatocyte Nuclear Factor-4α Is Essential for Glucose-stimulated Insulin Secretion by Pancreatic β-Cells

Mutations in the hepatocyte nuclear factor (HNF)-4α gene cause a form of maturity-onset diabetes of the young (MODY1) that is characterized by impairment of glucose-stimulated insulin secretion by pancreatic β-cells. HNF-4α, a transcription factor belonging to the nuclear receptor superfamily, is expressed in pancreatic islets as well as in the liver, kidney, and intestine. However, the role of HNF-4α in pancreatic β-cell is unclear. To clarify the role of HNF-4α in β-cells, we generated β-cell-specific HNF-4α knock-out (βHNF-4αKO) mice using the Cre-LoxP system. The βHNF-4αKO mice exhibited impairment of glucose-stimulated insulin secretion, which is a characteristic of MODY1. Pancreatic islet morphology, β-cell mass, and insulin content were normal in the HNF-4α mutant mice. Insulin secretion by βHNF-4αKO islets and the intracellular calcium response were impaired after stimulation by glucose or sulfonylurea but were normal after stimulation with KCl or arginine. Both NAD(P)H generation and ATP content at high glucose concentrations were normal in the βHNF-4αKO mice. Expression levels of Kir6.2 and SUR1 proteins in the βHNF-4αKO mice were unchanged as compared with control mice. Patch clamp experiments revealed that the current density was significantly increased in βHNF-4αKO mice compared with control mice. These results are suggestive of the dysfunction of KATP channel activity in the pancreatic β-cells of HNF-4α-deficient mice. Because the KATP channel is important for proper insulin secretion in β-cells, altered KATP channel activity could be related to the impaired insulin secretion in the βHNF-4αKO mice.

Glucagon-like peptide-1 evokes action potentials and increases cytosolic Ca2+ in rat nodose ganglion neurons.

We previously reported that the intraportal appearance of glucagon-like peptide-1 (GLP-1) facilitates the afferent activity (the spike discharge firing rate) of the rat hepatic vagus in a dose-dependent fashion. To examine whether GLP-1 directly activates single neurons isolated from the rat nodose ganglion, GLP-1-induced changes of the membrane potential and cytosolic-free Ca2+ concentration ([Ca2+]i) in the cells were measured using whole-cell patch-clamp and microfluorometric techniques, respectively. GLP-1 application (3 x 10(-12) - 3 x 10(-9) M) induced a gradual depolarization from a mean resting membrane potential of - 55.0 +/- 3.1 mV and evoked a burst of action potentials with a time lag of 7.5 +/- 4.5 min after its starting (n = 4). The burst of action potentials continued during the application and even up to 13 min or more after its cessation. GLP-1 at a concentration of 10(-12) - 10(-8) M induced an increase of [Ca2+]i. The GLP-1-induced [Ca2+]i responses were often oscillatory and lasted even up to 10 min or more after the washout of GLP-1. An adenylate cyclase activator, forskolin, mimicked the GLP-1-induced increase in [Ca2+]i. The present results indicate that GLP-1 activates nodose ganglion neurons as manifested by membrane depolarization, a burst of action potentials and [Ca2+]i increase, possibly via the cAMP pathway. Together with our previous observations, the results strongly suggest cellular mechanisms by which the postprandial humoral information, intraportal appearance of GLP-1, is uniquely converted to the neural information in the hepatoportal area.

Effect of metformin on adipose tissue resistin expression in db/db mice

Resistin, a novel adipose-derived protein, has been proposed to cause insulin-resistant states in obesity. To evaluate whether an insulin-sensitizing drug, metformin, regulates adipose tissue resistin expression, murine models of obesity and diabetes, db/db mice, were treated with metformin (metformin group), insulin (insulin group), and vehicle (control group) for 4 weeks, followed by analyzing resistin protein expression in their adipose tissues. Unexpectedly, resistin protein expression was increased by 66% in the metformin group relative to the control group, while it did not differ between the insulin and control groups. Hyperinsulinemia was improved in the metformin group, while the insulin group exhibited severe hyperinsulinemia, similar to the control group. Furthermore, in comparison between obese mice (db/db mice) and age-matched lean controls, resistin protein expression was reduced by 58% in the obese mice with severe hyperinsulinemia. These data collectively suggest that resistin expression may be suppressed by hyperinsulinemia and that metformin may upregulate resistin expression via the improvement of hyperinsulinemia in obesity.

Increased Urinary Excretion of Monocyte Chemoattractant Protein-1 in Proteinuric Renal Diseases

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is produced mainly by tubular epithelial cells in kidney and contributes to renal interstitial inflammation and fibrosis. More recently, we have demonstrated that urinary MCP-1 excretion is increased in proportion to the degree of albuminuria (proteinuria) and positively correlated with urinary N-acetylglucosaminidase (NAG) levels in type 2 diabetic patients. Based on these findings, we have suggested that heavy proteinuria, itself, probably aggravates renal tubular damage and accelerates the disease progression in diabetic nephropathy by increasing the MCP-1 expression in renal tubuli. In the present study, to evaluate whether urinary MCP-1 excretion is increased in the proteinuric states not only in diabetic nephropathy but also in other renal diseases, we examined urinary MCP-1 levels in IgA nephropathy patients with macroalbuminuria (IgAN group; n = 6), and compared the results with the data obtained from type 2 diabetic patients with overt diabetic nephropathy (DN group; n = 23) and those without diabetic nephropathy (non-DN group; n = 27). Urinary MCP-1 excretion levels in non-DN, DN, IgAN groups were 157.2 (52.8–378.5), 346.1 (147.0–1276.7), and 274.4 (162.2–994.5) ng/g creatinine, median (range), respectively. Expectedly, urinary MCP-1 and NAG excretion levels in DN and IgAN groups were significantly elevated as compared with non-DN group. Therefore, we suggest that MCP-1 expression in renal tubuli is enhanced in proteinuric states, irrespective of the types of renal disease, and that increased MCP-1 expression probably contributes to renal tubular damage in proteinuric states.

Enhanced urinary adiponectin excretion in IgA-nephropathy patients with proteinuria.

Adiponectin is secreted specifically by adipose tissue. It was reported that the serum adiponectin level was markedly increased in patients with end-stage renal disease and was positively associated with abnormal renal function in type 2 diabetes. Recently, we found that urinary adiponectin level was significantly increased in type 2 diabetic patients with overt diabetic nephropathy, but not in those without nephropathy. The aim of the present study was to evaluate whether the urinary adiponectin level is increased not only in diabetic patients with macroalbuminuria but also in IgA-nephropathy patients with macroalbuminuria. We measured urinary adiponectin levels in 24 healthy control subjects, 12 IgA-nephropathy patients, and 19 type 2 diabetic nephropathy patients, and they were, in medians, 2.24 µg/g creatinine (ranges of 0.85 ~ 3.70), 59.2 µg/g creatinine (4.95 ~ 186), and 33.1 µg/g creatinine (4.69 ~ 114), respectively. In the two patient groups, urinary adiponectin levels were significantly higher than in control subjects (P < 0.01). Moreover, positive correlations between urinary adiponectin levels and albumin-to-creatinine ratios were observed in IgA-nephropathy (R2 = 0.53, P < 0.01) and diabetic nephropathy patients (R2 = 0.61, P < 0.01), but not in control subjects. Serum adiponectin levels were unchanged in these three groups. These findings suggested that the increase of urinary adiponectin levels partly results from enhanced filtration of circulating adiponectin through the changes of glomerular permselectivity and intraglomerular hydruric pressure. However, clinical implication of urinary adiponectin excretion in healthy control remains to be elucidated.

Increased urinary excretion of N-acetylglucosaminidase in subjects with impaired glucose tolerance.

N-acetylglucosaminidase (NAG) is a lysosomal enzyme produced by renal proximal tubular cells and has been widely used as a marker, which indicates a degree of renal tubular damage. An increase in urinary NAG excretion is thought to result from the renal tubular damage. The aim of this study was to evaluate whether even mild hyperglycemia causes an increase in urinary excretion of NAG, which is a renal tubular protein. We examined urinary NAG excretion in overnight urine in 22 Japanese men with impaired glucose tolerance (IGT) for more than two years (IGT group) and 41 healthy control subjects matched in age, gender, BMI and blood pressure (control group). Urinary NAG excretion levels of IGT group and control group were 2.89 (1.23–7.97) and 2.22 (0.60–4.93) U/g creatinine, median (range), respectively. The IGT group showed significantly higher urinary excretion of NAG compared to the control group (p<0.01). Several studies have indicated that plasma proteins filtered through the glomerular capillary may have intrinsic renal toxicity. Recently, we have reported that urinary excretion of plasma proteins (ceruloplasmin, IgG4 and IgG) with molecular radii of approximately 45–55 Å is increased in subjects with IGT compared to healthy control subjects with normal glucose tolerance. Considering the present result together with our recent finding, we suggest that increased urinary excretion of NAG in the mildly hyperglycemic subjects may be due to the adverse effects of the plasma proteins highly filtered through the glomerular capillary on tubular cells.

Association of upregulated activity of KATP channels with impaired insulin secretion in UCP1-expressing insulinoma cells

Insulin‐secreting MIN6 cells overexpressing uncoupling protein‐1 (UCP1) were studied regarding insulin secretion in response to various secretagogues. Overexpression of UCP1 prevented an increase of cytosolic ATP levels induced by glucose. In contrast, glucose utilization was not affected, nor was glycerol phosphate flux. The UCP1‐expressing cells showed an inability to increase cytosolic Ca2+ concentration ([Ca2+]i) in response to glucose or α ketoisocaproate and this resulted in less insulin secretion, whereas initial reduction in [Ca2+]i occurring upon either nutrient addition was not affected. Moreover, the effectiveness of tolbutamide on [Ca2+]i increase was reduced and the dose‐response relations for insulin secretion induced by the agent was shifted toward the right in the UCP1‐expressing cells. The resting membrane potential of the UCP1‐expressing cells was significantly hyperpolarized by 6.2 mV compared with control cells. In the perforated and conventional whole‐cell patch‐clamp configurations, the conductance density of ATP‐sensitive K+ (KATP) channels of the UCP1‐expressing cells was 6‐fold and 1.7‐fold greater than that of the control cells, respectively. The sensitivity of KATP channels for tolbutamide was not different between two groups, indicating that in intact cells more than 6‐fold higher concentrations of tolbutamide were required to reduce the KATP channel currents of UCP1‐expressing cells to the same levels as of the control cells. The current density of the voltage‐dependent Ca2+ channels was not influenced. In conclusion, UCP1‐expressing cells showed a refractoriness to respond to tolbutamide as well as nutrients. An upregulated activity of KATP channels was associated with unresponsiveness to the agent in the cells with impaired mitochondrial function.

Effects of Chronic Intake of Vegetable Protein Added to Animal or Fish Protein on Renal Hemodynamics

Background/Aims: To examine whether chronic intake of vegetable protein added to animal protein diet affects renal hemodynamics or not, we studied effects of three kinds of diets containing various amounts of animal and vegetable protein with 1-week dietary program in each on renal hemodynamics. Methods: The crossover design of different amounts of vegetable protein added to the constant amount of animal protein was applied to two groups of 7 healthy individuals after the control dietary program. Renal function and 24 hours’ urinary albumin excretion rate (AER) were examined on every 7th day of three consecutive 1-week dietary programs. Results: Glomerular filtration rate (GFR; sodium thiosulphate clearance) and renal plasma flow (RPF) significantly decreased after decreasing the intake of animal protein by one third with keeping the amount of vegetable protein constant. The results when substituting vegetable protein for some of the animal protein in the diet without changing the total amount of protein were identical. The filtration fraction and AER did not change over the study periods regardless of dietary composition. Conclusion: The lack of an effect a 1-week intake of vegetable protein added to animal protein on GFR and RPF suggests that vegetable protein may be excluded from lists of restriction in low protein diet therapy in patients with renal insufficiency.

Aggressive Antihypertensive Treatment and Serum Lipid Lowering Therapy Are Necessary to Prevent Deterioration of the Renal Function Even in Elderly Type 2 Diabetic Patients with Persistent Albuminuria

Background: In elderly patients with diabetic nephropathy, clinical information aiming at preventing a decline of the renal function is limited. Objective: To clarify factors that could be related to changes in renal function in elderly diabetic patients with persistent albuminuria. Methods: We studied the relationships between the declining rate of creatinine clearance (Ccr) and other clinical features – glycemic control, blood pressure (BP), the degree of albuminuria, and other confounding factors – retrospectively in elderly diabetic patients with persistent albuminuria. Results: We analyzed 17 elderly diabetic patients with persistent albuminuria (4 men and 13 women, mean age 68.0 ± 5.7 years at the beginning of the observation period) for 3.7 ± 1.5 (SD) years. We found that the last value of the urinary albumin excretion (UAE) of the observation period and the individual mean values of systolic BP, mean BP, and serum cholesterol closely correlated with the rate of decline in Ccr estimated by Cockcroft’s formula. The last UAE closely correlated with the individual mean values of systolic BP and mean BP. For patients such as the present subjects, adequate retardation of decline in renal function requires that the estimated goal of antihypertensive treatment should be a BP below 130/70 mm Hg. The level of glycemic control and the use of angiotensin-converting enzyme inhibitors did not correlate with the rate of decline in Ccr. Conclusion: Aggressive antihypertensive treatment and serum lipid lowering therapy may be necessary to prevent deterioration of the renal function in elderly type 2 diabetic patients with persistent albuminuria.