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.

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.

SOD1, but not SOD3, deficiency accelerates diabetic renal injury in C57BL/6-Ins2(Akita) diabetic mice.

Superoxide dismutase (SOD) is a major defender against excessive superoxide generated under hyperglycemia. We have recently reported that renal SOD1 (cytosolic CuZn-SOD) and SOD3 (extracellular CuZn-SOD) isoenzymes are remarkably down-regulated in KK/Ta-Ins2(Akita) diabetic mice, which exhibit progressive diabetic nephropathy (DN), but not in DN-resistant C57BL/6- Ins2(Akita) (C57BL/6-Akita) diabetic mice. To determine the role of SOD1 and SOD3 in DN, we generated C57BL/6-Akita diabetic mice with deficiency of SOD1 and/or SOD3 and investigated their renal phenotype at the age of 20 weeks. Increased glomerular superoxide levels were observed in SOD1(-/-)SOD3(+/+) and SOD1(-/-)SOD3(-/-) C57BL/6-Akita mice but not in SOD1(+/+)SOD3(-/-) C57BL/6-Akita mice. The SOD1(-/-)SOD3(+/+) and SOD1(-/-)SOD3(-/-) C57BL/6-Akita mice exhibited higher glomerular filtration rate, increased urinary albumin levels, and advanced mesangial expansion as compared with SOD1(+/+)SOD3(+/+) C57BL/6-Akita mice, yet the severity of DN did not differ between the SOD1(-/-)SOD3(+/+) and SOD1(-/-)SOD3(-/-) C57BL/6-Akita groups. Increased renal mRNA expression of transforming growth factor-ß1 (TGF-ß1) and connective tissue growth factor (CTGF), reduced glomerular nitric oxide (NO), and increased renal prostaglandin E2 (PGE2) production were noted in the SOD1(-/-)SOD3(+/+) and SOD1(-/-)SOD3(-/-) C57BL/6-Akita mice. This finding indicates that such renal changes in fibrogenic cytokines, NO, and PGE2, possibly caused by superoxide excess, would contribute to the development of overt albuminuria by promoting mesangial expansion, endothelial dysfunction, and glomerular hyperfiltration. The present results demonstrate that deficiency of SOD1, but not SOD3, increases renal superoxide in the setting of diabetes and causes overt renal injury in nephropathy-resistant diabetic mice, and that SOD3 deficiency does not provide additive effects on the severity of DN in SOD1-deficient C57BL/6-Akita mice.

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-Ins2(Akita) (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.

Reduction of circulating superoxide dismutase activity in type 2 diabetic patients with microalbuminuria and its modulation by telmisartan therapy.

Growing evidence indicates that oxidative stress induced by excessive superoxide has a central role in the pathogenesis of diabetic nephropathy (DN). Telmisartan, one of the currently available angiotensin II type 1 receptor blockers (ARBs), has been shown to exert a more powerful proteinuria (albuminuria) reduction in patients with DN, but whether the prominent renoprotective effect of telmisartan is mediated through enhancing antioxidant defense capacity and reducing oxidative stress has not been fully elucidated. The present study first revealed that the serum activity of superoxide dismutase (SOD) responsible for superoxide removal is reduced in the DN stage of microalbuminuria, but not in normoalbuminuria in type 2 diabetic patients. We next examined the alteration of SOD and oxidative stress following an 8-week treatment with telmisartan (40 mg per day) in 12 type 2 diabetic patients with microalbuminuria. Interestingly, the telmisartan treatment not only reduced the circulating levels of two oxidative stress markers, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine (NT), but also enhanced serum SOD activity. Notably, a significant correlation was observed between the increase in serum SOD activity and the reduction in albuminuria. We further compared the anti-oxidative effect of telmisartan with that of losartan, another member of the ARB class, by implementing an 8-week interval crossover treatment with these ARBs in another 12 microalbuminuric type 2 diabetic patients. The patients showed higher serum SOD activity, and lower circulating levels of 8-OHdG and NT, during treatment with telmisartan than with losartan. These results suggest that telmisartan has a more potent antioxidative effect through its ability to enhance SOD activity in type 2 diabetic patients with microalbuminuria.

Reduction of renal superoxide dismutase in progressive diabetic nephropathy.

Superoxide excess plays a central role in tissue damage that results from diabetes, but the mechanisms of superoxide overproduction in diabetic nephropathy (DN) are incompletely understood. In the present study, we investigated the enzyme superoxide dismutase (SOD), a major defender against superoxide, in the kidneys during the development of murine DN. We assessed SOD activity and the expression of SOD isoforms in the kidneys of two diabetic mouse models (C57BL/6-Akita and KK/Ta-Akita) that exhibit comparable levels of hyperglycemia but different susceptibility to DN. We observed down-regulation of cytosolic CuZn-SOD (SOD1) and extracellular CuZn-SOD (SOD3), but not mitochondrial Mn-SOD (SOD2), in the kidney of KK/Ta-Akita mice which exhibit progressive DN. In contrast, we did not detect a change in renal SOD expression in DN-resistant C57BL/6-Akita mice. Consistent with these findings, there was a significant reduction in total SOD activity in the kidney of KK/Ta-Akita mice compared with C57BL/6-Akita mice. Finally, treatment of KK/Ta-Akita mice with a SOD mimetic, tempol, ameliorated the nephropathic changes in KK/Ta-Akita mice without altering the level of hyperglycemia. Collectively, these results indicate that down-regulation of renal SOD1 and SOD3 may play a key role in the pathogenesis of DN.

Effect of selective cyclooxygenase-2 (COX-2) inhibitor treatment on glucose-stimulated insulin secretion in C57BL/6 mice.

Previous studies have shown that Prostaglandin E(2) (PGE(2)) inhibits glucose-stimulated insulin secretion. However, the role of cyclooxygenase (COX)-1 vs. COX-2 derived PGE(2) production in glucose-stimulated insulin secretion remains poorly understood. Here we investigated the expression of COX-1 and COX-2 in pancreatic islets and the effect of selective inhibition of COX-1 and COX-2 on glucose-stimulated insulin secretion using C57BL/6 (B6) mice. Although immunofluorescence histochemistry showed the constitutive expression of both COX-1 and COX-2 in B6 mouse pancreatic islets, insulin secretion and hyperglycemia after glucose loading were ameliorated in B6 mice treated with selective COX-2 inhibitor (SC58236) for 18 weeks. Interestingly, incubation with selective COX-2 inhibitor for 24h led to a reduction in PGE(2) production in pancreatic islets isolated from B6 mice. In addition, selective COX-2 inhibition enhanced insulin secretion from the isolated islets. These results collectively suggest that selective inhibition of COX-2 enhances glucose-stimulated insulin secretion through a reduction in PGE(2) production in pancreatic islets.

Effects of antidiabetic treatment with metformin and insulin on serum and adipose tissue adiponectin levels in db/db mice.

Decreased circulating levels of adiponectin, a novel adipose-derived adipocytokine, in obesity possibly contribute to the development of insulin resistance which is a major factor in the pathogenesis of type 2 diabetes. The present study was conducted to examine whether circulating and adipose tissue adiponectin levels are modulated by chronic treatment with metformin and intensive treatment with insulin in murine models of obesity and type 2 diabetes, db/db mice with a C57BL/KsJ genetic background. Nine-week-old male db/db mice were treated with metformin, insulin, and vehicle for 4 weeks. Expectedly, metformin treatment led to inhibition of weight gain and improvement of hyperinsulinemia. Insulin treatment lowered fasting blood glucose levels to normal values, although it sustained hyperinsulinemic state. However, after 4 weeks of treatment, serum adiponectin levels were not significantly elevated in either metformin-treated or insulin-treated db/db mouse group (14.2 +/- 0.7 and 16.7 +/- 1.0 microg/ml, respectively) compared to vehicle-treated group (14.9 +/- 0.6 microg/ml). Similarly, adipose tissue adiponectin levels determined by Western blot analysis were not increased in either metformin-treated or insulin-treated group relative to vehicle-treated group. Recent studies have shown that adiponectin possibly has the same physiological effects on lipid and glucose metabolism that metformin has. Therefore, an elevation in blood concentration of metformin following the treatment might lead to suppression in adiponectin synthesis in adipose tissue, independent of inhibition in weight gain and improvement in hyperinsulinemia by metformin treatment. The present results indicate that adiponectin is not involved in the mechanism by which metformin treatment enhances insulin sensitivity. Moreover, our results suggest that adiponectin synthesis in adipose tissue may be suppressed under hyperinsulinemic state sustained by insulin treatment, even though hyperglycemia is markedly reduced. We conclude that antidiabetic treatment with metformin and insulin does not affect circulating and adipose tissue adiponectin levels.

Urinary adiponectin excretion is increased in patients with overt diabetic nephropathy.

Adiponectin, a novel adipose-derived adipocytokine, has beneficial effects not only on improvement of insulin sensitivity but also on mitigation of vascular damage. To evaluate whether adiponectin is implicated in the pathogenesis of diabetic nephropathy characterized by microvascular damage, we examined urinary and serum adiponectin levels in type 2 diabetic patients with different stages of nephropathy. We first confirmed adiponectin is excreted into urine through Western blot analysis, followed by measurements of urinary and serum adiponectin levels by radioimmunoassay. Interestingly, urinary adiponectin excretion levels were markedly increased in patient group with overt nephropathy relative to the groups without nephropathy and with incipient nephropathy. Surprisingly, serum adiponectin levels were also elevated in patient group with overt nephropathy. Increased urinary adiponectin excretion may result from elevations in circulating adiponectin levels and enhanced filtration of circulating adiponectin through the damaged kidney. Furthermore, adiponectin synthesis in adipose tissue and its secretion into circulating blood may be enhanced to mitigate microvascular damage in the advanced stage of diabetic nephropathy.

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.

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.

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.