Hitomi Usui

Methotrexate prevents renal injury in experimental diabetic rats via anti-inflammatory actions.

Recent studies suggested the involvement of inflammatory processes in the pathogenesis of diabetic nephropathy. Methotrexate (MTX), a folic acid antagonist, is widely used for the treatment of inflammatory diseases. Recently, it has been shown that treatment with low-dose MTX reduces the cardiovascular mortality in patients with rheumatoid arthritis, suggesting that MTX has anti-atherosclerotic effects via its anti-inflammatory actions. This study was designed to determine the anti-inflammatory effects of this agent on diabetic nephropathy. Diabetes was induced in Sprague-Dawley rats with streptozotocin, and MTX (0.5 or 1.0 mg/kg) was administered once a week for 8 wk. Treatment with MTX reduced urinary albumin excretion, mesangial matrix expansion, macrophage infiltration, expression of TGF-beta and type IV collagen, and intercellular adhesion molecule-1 in glomeruli. MTX also reduced the high glucose-induced NF-kappaB activation in vitro and in vivo. The results indicate that intermittent administration of MTX prevented renal injuries without changes in blood glucose level and BP in experimental diabetic rats. The protective effects of MTX are suggested to be mediated by its anti-inflammatory actions through inhibition of NF-kappaB activation and consequent reduction of intercellular adhesion molecule-1 expression and macrophage infiltration. The results suggest that anti-inflammatory agents might be beneficial for the treatment of diabetic nephropathy.

Macrophage Scavenger Receptor-A–Deficient Mice Are Resistant Against Diabetic Nephropathy Through Amelioration of Microinflammation

Microinflammation is a common major mechanism in the pathogenesis of diabetic vascular complications, including diabetic nephropathy. Macrophage scavenger receptor-A (SR-A) is a multifunctional receptor expressed on macrophages. This study aimed to determine the role of SR-A in diabetic nephropathy using SR-A–deficient (SR-A−/−) mice. Diabetes was induced in SR-A−/− and wild-type (SR-A+/+) mice by streptozotocin injection. Diabetic SR-A+/+ mice presented characteristic features of diabetic nephropathy: albuminuria, glomerular hypertrophy, mesangial matrix expansion, and overexpression of transforming growth factor-β at 6 months after induction of diabetes. These changes were markedly diminished in diabetic SR-A−/− mice, without differences in blood glucose and blood pressure levels. Interestingly, macrophage infiltration in the kidneys was dramatically decreased in diabetic SR-A−/− mice compared with diabetic SR-A+/+ mice. DNA microarray revealed that proinflammatory genes were overexpressed in renal cortex of diabetic SR-A+/+ mice and suppressed in diabetic SR-A−/− mice. Moreover, anti–SR-A antibody blocked the attachment of monocytes to type IV collagen substratum but not to endothelial cells. Our results suggest that SR-A promotes macrophage migration into diabetic kidneys by accelerating the attachment to renal extracellular matrices. SR-A may be a key molecule for the inflammatory process in pathogenesis of diabetic nephropathy and a novel therapeutic target for diabetic vascular complications.

Dipeptidyl peptidase-4 inhibitor ameliorates early renal injury through its anti-inflammatory action in a rat model of type 1 diabetes

INTRODUCTION Dipeptidyl peptidase-4 (DPP-4) inhibitors are incretin-based drugs in patients with type 2 diabetes. In our previous study, we showed that glucagon-like peptide-1 (GLP-1) receptor agonist has reno-protective effects through anti-inflammatory action. The mechanism of action of DPP-4 inhibitor is different from that of GLP-1 receptor agonists. It is not obvious whether DPP-4 inhibitor prevents the exacerbation of diabetic nephropathy through anti-inflammatory effects besides lowering blood glucose or not. The purpose of this study is to clarify the reno-protective effects of DPP-4 inhibitor through anti-inflammatory actions in the early diabetic nephropathy. MATERIALS AND METHODS Five-week-old male Sprague-Dawley (SD) rats were divided into three groups; non-diabetes, diabetes and diabetes treated with DPP-4 inhibitor (PKF275-055; 3 mg/kg/day). PKF275-055 was administered orally for 8 weeks. RESULTS PKF275-055 increased the serum active GLP-1 concentration and the production of urinary cyclic AMP. PKF275-055 decreased urinary albumin excretion and ameliorated histological change of diabetic nephropathy. Macrophage infiltration was inhibited, and inflammatory molecules were down-regulated by PKF275-055 in the glomeruli. In addition, nuclear factor-κB (NF-κB) activity was suppressed in the kidney. CONCLUSIONS These results indicate that DPP-4 inhibitor, PKF275-055, have reno-protective effects through anti-inflammatory action in the early stage of diabetic nephropathy. The endogenous biological active GLP-1 might be beneficial on diabetic nephropathy besides lowering blood glucose.

Cerebroside sulfotransferase deficiency ameliorates L-selectin-dependent monocyte infiltration in the kidney after ureteral obstruction.

Mononuclear cells infiltrating the interstitium are involved in renal tubulointerstitial injury. The unilateral ureteral obstruction (UUO) is an established experimental model of renal interstitial inflammation. In our previous study, we postulated that L-selectin on monocytes is involved in their infiltration into the interstitium by UUO and that a sulfated glycolipid, sulfatide, is the physiological L-selectin ligand in the kidney. Here we tested the above hypothesis using sulfatide- and L-selectin-deficient mice. Sulfatide-deficient mice were generated by gene targeting of the cerebroside sulfotransferase (Cst) gene. Although the L-selectin-IgG chimera protein specifically bound to sulfatide fraction in acidic lipids from wild-type kidney, it did not show such binding in fractions of Cst-/- mice kidney, indicating that sulfatide is the major L-selectin-binding glycolipid in the kidney. The distribution of L-selectin ligand in wild-type mice changed after UUO; sulfatide was relocated from the distal tubules to the peritubular capillaries where monocytes infiltrate, suggesting that sulfatide relocated to the endothelium after UUO interacted with L-selectin on monocytes. In contrast, L-selectin ligand was not detected in Cst-/- mice irrespective of UUO treatment. Compared with wild-type mice, Cst-/- mice showed a considerable reduction in the number of monocytes/macrophages that infiltrated the interstitium after UUO. The number of monocytes/macrophages was also reduced to a similar extent in L-selectin-/- mice. Our results suggest that sulfatide is a major L-selectin-binding molecule in the kidney and that the interaction between L-selectin and sulfatide plays a critical role in monocyte infiltration into the kidney interstitium.

Pathological Roles of Advanced Glycation End Product Receptors SR‐A and CD36

Abstract: The pathological significance of advanced glycation end product (AGE)‐modified proteins deposited in several lesions is generally accounted for by their cellular interaction via the AGE receptors and subsequent acceleration of the inflammatory process. In this study, we focused on two AGE receptors—specifically, the role of SR‐A in pathogenesis of diabetic nephropathy and the role of CD36 in AGE‐induced downregulation of leptin by adipocytes. In terms of SR‐A, diabetic wild‐type mice exhibited increased urinary albumin excretion, glomerular hypertrophy, and mesangial matrix expansion, whereas SR‐A‐knockout mice showed reduced glomerular size and mesangial matrix area. In these diabetic SR‐A‐knockout mice, the number of macrophages that infiltrated into glomeruli was remarkably reduced (P < 0.05), suggesting that SR‐A‐dependent glomerular migration of macrophages plays an important role in the pathogenesis of diabetic nephropathy. In terms of CD36, incubation of glycolaldehyde‐modified bovine serum albumin (GA‐BSA) with 3T3‐L1 adipocytes reduced leptin secretion by these cells. The binding of GA‐BSA to these cells and subsequent endocytic degradation were effectively inhibited by a neutralizing anti‐CD36 antibody. AGE‐induced downregulation of leptin was protected by N‐acetyl‐cysteine, an antioxidant. These results indicate that the interaction of AGE ligands with 3T3‐L1 adipocytes via CD36 induces oxidative stress and leads to inhibition of leptin expression by these cells, suggesting a potential link of this phenomenon to exacerbation of the insulin sensitivity in metabolic syndrome.

Changes of gene expression profiles in macrophages stimulated by angiotensin II--angiotensin II induces MCP-2 through AT1-receptor.

Introduction. Macrophages play critical roles in the development of atherosclerosis and diabetic nephropathy as well as many inflammatory diseases. Angiotensin II type 1 receptor antagonists (AIIA) are beneficial for the prevention of atherosclerosis and diabetic nephropathy suggesting that angiotensin II (Ang II) promotes the development of these diseases. It has recently been reported that Ang II exerts proinflammatory actions in vivo and in vitro. This study was aimed to clarify the direct effects of Ang II on monocytes/macrophages. Materials and methods. PMA-treated THP-1 cells, a human monocytic leukaemia cell line, were treated with Ang II (10-6 mol/L) for 24 hours with or without AIIA (CV11974). We evaluated gene expression profiles of these cells using DNA microarray system and quantified them by real-time RT-PCR. Results. DNA microarray revealed that in total 19 genes, including monocyte chemoattractant protein (MCP)-2, were up-regulated by Ang II and down-regulated by AIIA. Real-tim D e RT-PCR showed that up-regulation of MCP-2 with Ang II is blocked by the AIIA (CV11974) but not by an AT2-receptor antagonist. Conclusions. These results suggest that Ang II directly stimulates MCP-2 expression through AT1-receptors in activated macrophages.Ang II may contribute to the persistence or amplification of microinflammation in vessel walls, heart and kidney.Vasculoprotective or renoprotective effects of AIIA might partly depend on direct antiinflammatory effects on macrophages.

A case of congenital generalized lipodystrophy with lipoatrophic diabetes developing anti-insulin antibodies

Human amylin or islet amyloid polypeptide (IAPP) is a 37 amino acid polypeptide which is produced in the pancreatic β -cells and may be diabetogenic both through inhibition of insulin secretion and through formation of the major component of the islet amyloid associated with Type 2 diabetes [1–3]. However, the peptide may also be anti-diabetogenic, since it inhibits gastric emptying [4]. To evaluate the possible antidiabetogenic action of amylin, the human amylin analogue, pramlintide, has been developed. In pramlintide, proline was introduced in positions 25, 28 and 29 in the molecule to provide a therapeutic molecule substantially better than human amylin. Pramlintide reduces post-prandial glycaemia as shown in both short-term studies and in studies up to 4 weeks in subjects with Type 1 diabetes [5–10]. The actions of pramlintide (delayed gastric emptying accompanied by reduced circulating glucagon) resemble the effects of glucagon-like peptide (GLP1), the gut incretin hormone which exerts an anti-diabetogenic effect and which currently is explored for treatment of patients with Type 2 diabetes [11–15]. To examine whether the antidiabetogenic effect of pramlintide depends on a release of GLP-1, we explored the effect of pramlintide over a 4-week period in patients with Type 1 diabetes. Basal and meal-induced GLP-1 concentrations were studied. Nine patients with Type 1 diabetes (three males, six females; age 35.4 ± 2.3 years (mean ± SD ); diabetes duration 13.7 ± 2.5 years; HbA 1c 7.2 ± 0.3%; daily insulin dose 53.3 ± 3.2 U; no clinically diagnosed secondary complications) were studied over 4 weeks. The study protocol was approved by the ethics committee of the Karolinska Institute, Stockholm. Written informed consent was obtained from all participants. The subjects were treated for 4 weeks with four daily subcutaneous injections of 30 μ g pramlintide (Amylin Corp., San Diego, CA, USA). During the entire study period, including during the test day, the patients remained on their usual insulin treatment. A mixed meal (500 kcal, 28% protein, 22% fat and 50% carbohydrates) was served to the overnight fasted subjects before the start of pramlintide treatment and after 4 weeks. Before and at regular time points after meal ingestion, blood samples were taken for analysis of glucose (glucose oxidase), GLP-1 (radioimmunoassay analysing amidated C-terminus of GLP-1, i.e. GLP-1 of intestinal origin; [16]), glucagon (radioimmunoassay; Linco Res., St Charles, MO, USA) and pramlintide (double antibody enzymatic immunoassay; [17]). Comparisons of differences between the groups were performed using two-way analysis of variance for multiple comparisons. Baseline glucose, glucagon or GLP-1 did not change during the treatment. However, pramlintide reduced the post-prandial increase in glucose and glucagon levels (Fig. 1). The 240-min post-meal AUC glucose (suprabasal area under the curve, calculated by the trapezoid rule) was reduced from 364.2 ± 276.6 mmol/ l × 240 min to –486.5 ± 235.9 mmol/ l × 240 min by pramlintide ( P = 0.013) and the AUC glucagon over the first 90-min postmeal study period was reduced from 825 ± 201 ng/ l × 90 min to –94 ± 146 ng/ l × 90 min by pramlintide ( P < 0.001). In contrast, the post-prandial GLP-1 was not altered by pramlintide (Fig. 1c); the peak values were 20.7 ± 2.0 pmol/ l before treatment and 19.8 ± 2.5 pmol/ l after treatment and the suprabasal AUC GLP-1 was 1.57 ± 0.03 before treatment vs. 1.67 ± 0.03 nmol/ l × 240 min after 4 weeks of treatment. However, the time point at which the peak was seen was delayed after treatment with pramlintide. Thus, before treatment, GLP-1 levels peaked at 93 ± 12 min, whereas the GLP-1 peak was observed at 206 ± 19 min ( P < 0.001) after 4 weeks of treatment with pramlintide. Figure 1d shows that following subcutaneous administration of pramlintide, its peak level was observed at 15 min. Thereafter, the elimination of pramlintide followed a first order kinetic, as evidenced by a linear relation between the logarithmic values of pramlintide concentrations vs. time after peak ( r = –0.79, P < 0.001). The calculated circulating half-life of pramlintide was 69.5 ± 9.5 min. The study thus confirms previous reports that subcutaneous administration of the amylin analogue, pramlintide, reduces post-prandial glucose excursions in subjects with Type 1 diabetes [5–10]. This pronounced and rapid action is probably executed by inhibiting gastric emptying, which is a well-known action of amylin [3–5], and through prevention of the mixed meal-induced increase in glucagon levels. In contrast, we found no evidence that the anti-diabetogenic action of pramlintide is mediated by release of GLP-1. We measured total GLP-1 immunoreactivity, i.e. intact hormone and its primary metabolite. This analytical approach is required for the study of L-cell secretion because of the intravascular degradation of GLP-1 [16]. There is still a possibility that pramlintide could alter the degradation of GLP-1 and its effect mediated by increased levels of active GLP-1. However, it is unlikely that pramlintide inhibits the activity of proteases. The anti-diabetogenic action of pramlintide is more likely due to its decelerating effect on gastric emptying, which would cause glucose absorption to be effected over a longer time period and which would inhibit glucagon secretion by delaying the absorption of glucagonotropic amino acids.