Tsukasa Morii

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.

The protective roles of GLP-1R signaling in diabetic nephropathy: possible mechanism and therapeutic potential.

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone that has an antioxidative protective effect on various tissues. Here, we determined whether GLP-1 has a role in the pathogenesis of diabetic nephropathy using nephropathy-resistant C57BL/6-Akita and nephropathy-prone KK/Ta-Akita mice. By in situ hybridization, we found the GLP-1 receptor (GLP-1R) expressed in glomerular capillary and vascular walls, but not in tubuli, in the mouse kidney. Next, we generated C57BL/6-Akita Glp1r knockout mice. These mice exhibited higher urinary albumin levels and more advanced mesangial expansion than wild-type C57BL/6-Akita mice, despite comparable levels of hyperglycemia. Increased glomerular superoxide, upregulated renal NAD(P)H oxidase, and reduced renal cAMP and protein kinase A (PKA) activity were noted in the Glp1r knockout C57BL/6-Akita mice. Treatment with the GLP-1R agonist liraglutide suppressed the progression of nephropathy in KK/Ta-Akita mice, as demonstrated by reduced albuminuria and mesangial expansion, decreased levels of glomerular superoxide and renal NAD(P)H oxidase, and elevated renal cAMP and PKA activity. These effects were abolished by an adenylate cyclase inhibitor SQ22536 and a selective PKA inhibitor H-89. Thus, GLP-1 has a crucial role in protection against increased renal oxidative stress under chronic hyperglycemia, by inhibition of NAD(P)H oxidase, a major source of superoxide, and by cAMP-PKA pathway activation.

Comparisons of the effects of 12‐week administration of miglitol and voglibose on the responses of plasma incretins after a mixed meal in Japanese type 2 diabetic patients

To compare the effects of miglitol [an alpha‐glucosidase inhibitor (AGI) absorbed in the intestine] and voglibose (an AGI not absorbed) on plasma glucagon‐like peptide‐1 (GLP‐1) and gastric inhibitory polypeptide (GIP) levels, 26 and 24 Japanese type 2 diabetic patients were randomly assigned to receive miglitol or voglibose, respectively. After 12‐week administration of both drugs, during 2‐h meal tolerance test, plasma glucose, serum insulin and total GIP were significantly decreased and active GLP‐1 was significantly increased. Miglitol group showed a significantly lower total GIP level than voglibose group. Miglitol, but not voglibose, significantly reduced body weight (BW). In all participants, the relative change in BW was positively correlated with that of insulin significantly and of GIP with a weak tendency, but not of GLP‐1. In conclusion, both drugs can enhance postprandial GLP‐1 responses and reduce GIP responses. The significant BW reduction by miglitol might be attributable to its strong GIP‐reducing efficacy.

Miglitol induces prolonged and enhanced glucagon-like peptide-1 and reduced gastric inhibitory polypeptide responses after ingestion of a mixed meal in Japanese Type 2 diabetic patients.

At present, strategies of enhancing glucagon-like peptide-1 (GLP-1) action have received attention as new therapies in Type 2 diabetic patients (T2DM). In experimental studies in high-fat-fed animals, gastric inhibitory polypeptide (GIP) signals are reduced, obesity prevented and glycaemic control improved through reduction of insulin resistance [1]. Administration of α -glucosidase inhibitors (AGIs) simultaneously with nutrient intake leads to reduction of GIP responses and prolonged elevation of GLP-1 in healthy control subjects. These findings can be explained by the fact that AGIs move the absorption site of carbohydrates from the GIP-producing upper portions of intestine to the GLP-1 producing lower intestine. However, in T2DM, only a few studies in obese patients after a mixed meal have reported conflicting effects of AGIs on GLP-1 secretion [2–4] and a consistent finding of reduced GIP secretion [2,4]. Therefore, we measured plasma glucose (PG), insulin, GLP-1 (active and total forms) and GIP periodically for 3 h after ingestion of a mixed meal (56.5 g of carbohydrates, 18 g of protein, 18 g of fat) before and after administration of 50 mg miglitol three times per day for 14 days in nine Japanese T2DM subjects without severe obesity [glycated haemoglobin (HbA 1c ) 6.6 ± 0.16%; body mass index (BMI) 24.1 ± 1.0 kg/m 2 , (mean ± SE )] treated with diet therapy alone ( n = 5) or with oral glucose-lowering agents ( n = 4; glimepiride 1 or 2 mg/day in three cases; pioglitazone 30 mg/day in one case). Active forms of GLP-1 and total GIP levels, and total GLP-1 levels were measured with commercially available ELISA and RIA kits (Linco Research, St Charles, MO, USA), respectively. As seen in Fig. 1, in response to administration of miglitol, PG, insulin and GIP were unchanged at time 0 min, but during the early phase were significantly decreased. However, both active and total GLP-1 levels were significantly higher during the late phase. Our present study and that of Lee et al . [4] have demonstrated consistent enhancing effects of miglitol on GLP-1 responses to a mixed meal in T2DM. This is in contrast to results after treatment with acarbose, where the GLP-1 response to a mixed meal is unchanged [2,3]. Miglitol is absorbed in the upper portion of the intestine, where AGIs have their main effects [5]. In contrast, acarbose cannot be absorbed from the intestine. Accordingly, the relatively higher amount of carbohydrate absorption in the lower intestine with miglitol than with acarbose may explain the strong enhancement of GLP-1 secretion with miglitol treatment in T2DM subjects, although the percentages of miglitol and carbohydrates actually remaining in the lower part of the intestine are unknown. Additionally, the fact that our patients were not severely obese might partially explain the significant enhancement of GLP-1 response after miglitol; the GLP-1 response to carbohydrate is attenuated in obese individuals [6]. It is possible that the enhancement of postprandial active GLP-1 in response to miglitol might be as a result of dipeptidyl peptidase-4 (DPP-4) inhibition. However, enhancement of both active and total GLP-1 in our present study does not support this implication as DPP-4 inhibitors enhance active GLP-1 and reduce total GLP-1 levels [7]. In this 14-day study, we did not observe any change in body weight or determine directly a change in pancreatic B-cell function. A long-term follow-up study to examine the effects of miglitol on these parameters in relation to enhanced GLP-1 and reduced GIP secretion is warranted.

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.

Modulation of renal superoxide dismutase by telmisartan therapy in C57BL/6-Ins2(Akita) diabetic mice.

Renal superoxide excess, which is induced by an imbalance of the superoxide-producing enzyme NAD(P)H oxidase and the superoxide-scavenging enzyme superoxide dismutase (SOD) under hyperglycemia, increases oxidative stress and contributes to the development of diabetic nephropathy. In this study, we treated non-obese and hypoinsulinemic C57BL/6-Ins2Akita (C57BL/6-Akita) diabetic mice with telmisartan (5 mg kg−1 per day), an angiotensin II type 1 receptor blocker, or amlodipine (5 mg kg−1 per day), a calcium channel blocker, for 4 weeks and compared the effects of these two anti-hypertensive drugs on renal NAD(P)H oxidase, SOD and transcription factor Nrf2 (NF-E2-related factor 2), which is known to upregulate several antioxidant enzymes including SOD. Vehicle-treated C57BL/6-Akita mice exhibited higher renal NAD(P)H oxidase and lower renal SOD activity with increased levels of renal superoxide than the C57BL/6-wild-type non-diabetic mice. Interestingly, telmisartan treatment not only reduced NAD(P)H oxidase activity but also enhanced SOD activity in C57BL/6-Akita mouse kidneys, leading to a reduction of renal superoxide levels. Furthermore, telmisartan-treated C57BL/6-Akita mice increased the renal protein expression of SOD and Nrf2. In parallel with the reduction of renal superoxide levels, a reduction of urinary albumin levels and a normalization of elevated glomerular filtration rate were observed in telmisartan-treated C57BL/6-Akita mice. In contrast, treatment with amlodipine failed to modulate renal NAD(P)H oxidase, SOD and Nrf2. Finally, treatment of C57BL/6-Akita mice with apocynin, an NAD(P)H oxidase inhibitor, also increased the renal protein expression of SOD and Nrf2. Collectively, our data suggest that NAD(P)H oxidase negatively regulates renal SOD, possibly by downregulation of Nrf2, and that telmisartan could upregulate renal SOD by the suppression of NAD(P)H oxidase and subsequent upregulation of Nrf2, leading to the amelioration of renal oxidative stress and diabetic renal changes.

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.

TCF1 links GIPR signaling to the control of beta cell function and survival

The glucagon-like peptide-1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor transduce nutrient-stimulated signals to control beta cell function. Although the GLP-1 receptor (GLP-1R) is a validated drug target for diabetes, the importance of the GIP receptor (GIPR) for the function of beta cells remains uncertain. We demonstrate that mice with selective ablation of GIPR in beta cells (MIP-Cre:GiprFlox/Flox; Gipr−/−βCell) exhibit lower levels of meal-stimulated insulin secretion, decreased expansion of adipose tissue mass and preservation of insulin sensitivity when compared to MIP-Cre controls. Beta cells from Gipr−/−βCell mice display greater sensitivity to apoptosis and markedly lower islet expression of T cell–specific transcription factor-1 (TCF1, encoded by Tcf7), a protein not previously characterized in beta cells. GIP, but not GLP-1, promotes beta cell Tcf7 expression via a cyclic adenosine monophosphate (cAMP)-independent and extracellular signal–regulated kinase (ERK)-dependent pathway. Tcf7 (in mice) or TCF7 (in humans) levels are lower in islets taken from diabetic mice and in humans with type 2 diabetes; knockdown of TCF7 in human and mouse islets impairs the cytoprotective responsiveness to GIP and enhances the magnitude of apoptotic injury, whereas restoring TCF1 levels in beta cells from Gipr−/−βCell mice lowers the number of apoptotic cells compared to that seen in MIP-Cre controls. Tcf7−/− mice show impaired insulin secretion, deterioration of glucose tolerance with either aging and/or high-fat feeding and increased sensitivity to beta cell injury relative to wild-type (WT) controls. Hence the GIPR-TCF1 axis represents a potential therapeutic target for preserving both the function and survival of vulnerable, diabetic beta cells.

Stromal cell–derived factor-1 is upregulated by dipeptidyl peptidase-4 inhibition and has protective roles in progressive diabetic nephropathy

The role of stromal cell-derived factor-1 (SDF-1) in the pathogenesis of diabetic nephropathy and its modification by dipeptidyl peptidase-4 (DPP-4) inhibition are uncertain. Therefore, we studied this independent of glucagon-like peptide-1 receptor (GLP-1R) signaling using two Akita diabetic mouse models, the diabetic-resistant C57BL/6-Akita and diabetic-prone KK/Ta-Akita. Increased SDF-1 expression was found in glomerular podocytes and distal nephrons in the diabetic-prone mice, but not in kidneys from diabetic-resistant mice. The DPP-4 inhibitor linagliptin, but not the GLP-1R agonist liraglutide, further augmented renal SDF-1 expression in both Glp1r(+/+) and Glp1r(-/-) diabetic-prone mice. Along with upregulation of renal SDF-1 expression, the progression of albuminuria, glomerulosclerosis, periglomerular fibrosis, podocyte loss, and renal oxidative stress was suppressed in linagliptin-treated Glp1r(+/+) diabetic-prone mice. Linagliptin treatment increased urinary sodium excretion and attenuated the increase in glomerular filtration rate which reflects glomerular hypertension and hyperfiltration. In contrast, selective SDF-1 receptor blockade with AMD3100 reduced urinary sodium excretion and aggravated glomerular hypertension in the Glp1r(+/+) diabetic-prone mice. Thus, DPP-4 inhibition, independent of GLP-1R signaling, contributes to protection of the diabetic kidney through SDF-1-dependent antioxidative and antifibrotic effects and amelioration of adverse renal hemodynamics.

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.