Aims/hypothesis Tissue macrophage accumulation is thought to induce insulin resistance during obesity and stimulate the progression of diabetic nephropathy. Monocyte chemoattractant protein-1 (MCP-1) is a potent stimulator of macrophage recruitment. It is increased in adipose tissue during obesity and in diabetic kidneys, suggesting that inflammation of these tissues may be MCP-1-dependent. Based on these findings, the aim of this study was to examine whether a deficiency in MCP-1 would alter the development of type 2 diabetes and its renal complications. Materials and methods The role of MCP-1 in the progression of type 2 diabetes and its associated renal injury was assessed in obese db/db mice that were deficient in the gene encoding MCP-1 (Ccl2). Results The incidence and development of type 2 diabetes were similar in Ccl2 +/+ and Ccl2 −/− db/db mice between 8 and 32 weeks of age. Body mass, hyperglycaemia, hyperinsulinaemia, glucose and insulin tolerance, plasma triacylglycerol and serum NEFA were not different between these strains. Pathological changes in epididymal adipose tissue, including increases in macrophage accumulation and Tnfa mRNA and reductions in Adipoq mRNA, were unaffected by the absence of MCP-1. In contrast, kidney macrophage accumulation and the progression of diabetic renal injury (albuminuria, histopathology, renal fibrosis) were substantially reduced in Ccl2 −/− compared with Ccl2 +/+ db/db mice with equivalent diabetes. Conclusions/interpretation Our study demonstrates that MCP-1 promotes type 2 diabetic renal injury but does not influence the development of obesity, insulin resistance or type 2 diabetes in db/db mice. MCP-1 plays a critical role in inflammation of the kidney, but not adipose tissue, during the progression of type 2 diabetes.

Renal fibrosis and tubular apoptosis are common mechanisms of progressive kidney disease. In vitro studies have implicated the c-Jun amino-terminal kinase (JNK) pathway in these processes. Both of the major JNK isoforms, JNK1 and JNK2, are expressed in the kidney, but their relative contribution to JNK signaling is unknown. This study investigated the role of JNK signaling in renal fibrosis and tubular apoptosis in the unilateral ureteral obstruction model using two different approaches: (1) Mice that were deficient in either JNK1 or JNK2 and (2) a specific inhibitor of all JNK isoforms, CC-401. Western blotting and immunostaining identified a marked increase in JNK signaling in the obstructed kidney, with substantial redundancy between JNK1 and JNK2 isoforms. Administration of CC-401 blocked JNK signaling in the rat obstructed kidney and significantly inhibited renal fibrosis in terms of interstitial myofibroblast accumulation and collagen IV deposition. This effect was attributed to suppression of gene transcription for the profibrotic molecules TGF-␤1 and connective tissue growth factor. CC-401 treatment also significantly reduced tubular apoptosis in the obstructed kidney. Genetic deletion of JNK1 or JNK2 did not protect mice from renal fibrosis in the unilateral ureteral obstruction model, but JNK1 deletion did result in a significant reduction in tubular cell apoptosis. In conclusion, this is the first study to demonstrate that JNK signaling plays a pathogenic role in renal fibrosis and tubular apoptosis. Furthermore, JNK1 plays a nonredundant role in tubular cell apoptosis. These studies identify the JNK pathway as a potential therapeutic target in progressive kidney disease.

Stress-activated kinases p38 MAPK and JNK promote renal fibrosis; however, how the pathways by which these kinases are activated in kidney disease remain poorly defined. Apoptosis signal-regulating kinase 1 (ASK1/MAPKKK5) is a member of the MAPKKK family that can induce activation of p38 and JNK. The present study examined whether ASK1 induces p38/JNK activation and renal fibrosis in unilateral ureteric obstruction (UUO) using wild-type (WT) and Ask1-deficient (Ask1(-/-)) mice. Basal p38 and JNK activation in WT kidneys was increased three- to fivefold in day 7 UUO mice in association with renal fibrosis. In contrast, there was no increase in p38 activation in Ask1(-/-) UUO mice, whereas JNK activation was only partially increased. The progressive increase in kidney collagen (hydroxyproline) content seen on days 7 and 12 of UUO in WT mice was significantly reduced in Ask1(-/-) UUO mice in association with reduced α-smooth muscle actin-positive myofibroblast accumulation. However, cultured WT and Ask1(-/-) renal fibroblasts showed equivalent proliferation and matrix production, indicating that ASK1 acts indirectly on fibroblasts. Tubular epithelial cells are the main site of p38 activation in the obstructed kidney. Angiotensin II and H₂O₂, but not IL-1 or lipopolysaccharide, induced p38 activation and upregulation of transforming growth factor-β₁, platelet-derived growth factor-B, and monocyte chemoattractant protein-1 production was suppressed in Ask1(-/-) tubular epithelial cells. In addition, macrophage accumulation was significantly inhibited in Ask1(-/-) UUO mice. In conclusion, ASK1 is an important upstream activator of p38 and JNK signaling in the obstructed kidney, and ASK1 is a potential therapeutic target in renal fibrosis.

Aims/hypothesis Obesity and diabetes are associated with increased intracellular p38 mitogen-activated protein kinase (MAPK) signalling, which may promote tissue inflammation and injury. Activation of p38 MAPK can be induced by either of the immediate upstream kinases, MAP kinase kinase (MKK)3 or MKK6, and recent evidence suggests that MKK3 has non-redundant roles in the pathology attributed to p38 MAPK activation. Therefore, this study examined whether MKK3 signalling influences the development of obesity, type 2 diabetes and diabetic nephropathy. Methods Wild-type and Mkk3 (also known as Map2k3) gene-deficient db/db mice were assessed for the development of obesity, type 2 diabetes and renal injury from 8 to 32 weeks of age. Results Mkk3+/+ db/db and Mkk3 −/− db/db mice developed comparable obesity and were similar in terms of incidence and severity of type 2 diabetes. At 32 weeks, diabetic Mkk3 +/+ db/db mice had increased kidney levels of phospho-p38 and MKK3 protein. In comparison, kidney levels of phospho-p38 in diabetic Mkk3 −/− db/db mice remained normal, despite a fourfold compensatory increase in MKK6 protein levels. The reduced levels of p38 MAPK signalling in the diabetic kidneys of Mkk3 −/− db/ db mice was associated with protection against the following: declining renal function, increasing albuminuria, renal hypertrophy, podocyte loss, mesangial cell activation and glomerular fibrosis. Diabetic Mkk3 −/− db/db mice were also significantly protected from tubular injury and interstitial fibrosis, which was associated with reduced Ccl2 mRNA expression and interstitial macrophage accumulation. Conclusions/interpretation MKK3-p38 MAPK signalling is not required for the development of obesity or type 2 diabetes, but plays a distinct pathogenic role in the progression of diabetic nephropathy in db/db mice.

Activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway is involved in the immune response; however, little is known of its role in immune-induced renal injury. In this study, we examine JNK signaling in the rat anti-glomerular basement membrane (GBM) disease model using CC-401, a specific JNK inhibitor. Animals were given CC-401, vehicle alone or no treatment starting before anti-GBM serum injection and continued treatment until killing. In acute disease, CC-401 blocked JNK signaling and reduced proteinuria in the first 24 h. The transient neutrophil influx seen at 3 h of disease was not affected, however. Continued CC-401 treatment suppressed glomerular and tubulointerstitial damage usually seen at 14 days. The protective effect may be due to modulation of macrophage activation, as CC-401 had no effect upon glomerular macrophage infiltration at day 14 despite the suppression of glomerular lesions and a marked reduction in renal tumor necrosis factor-alpha and inducible nitric oxide synthase messenger RNA levels. Treatment with CC-401 had no apparent effect on T cell or humoral immune responses. These studies suggest that JNK signaling promotes renal injury in acute and progressive rat anti-GBM disease. JNK inhibitors may be a novel therapeutic approach for the treatment of human glomerulonephritis.

These results show that lymphocytes contribute to the development of diabetic albuminuria, which may partly arise from increasing glomerular macrophage activation and podocyte damage. In contrast, lymphocytes do not appear to promote tubular injury, increased matrix deposition or decline in renal function in a mouse model of type 1 diabetes. Our findings suggest that innate immunity rather than adaptive immune responses are the major inflammatory contributor to the progression of diabetic renal injury.

p38 mitogen-activated protein kinase (MAPK) signaling promotes diabetic kidney injury. Apoptosis signal-regulating kinase (ASK)1 is one of the upstream kinases in the p38 MAPK-signaling pathway, which is activated by inflammation and oxidative stress, suggesting a possible role for ASK1 in diabetic nephropathy. In this study, we examined whether a selective ASK1 inhibitor can prevent the induction and progression of diabetic nephropathy in mice. Diabetes was induced in hypertensive endothelial nitric oxide synthase (Nos3)-deficient mice by five low-dose streptozotocin (STZ) injections. Groups of diabetic Nos3−/− mice received ASK1 inhibitor (GS-444217 delivered in chow) as an early intervention (2–8 weeks after STZ) or late intervention (weeks 8–15 after STZ). Control diabetic and nondiabetic Nos3−/− mice received normal chow. Treatment with GS-444217 abrogated p38 MAPK activation in diabetic kidneys but had no effect upon hypertension in Nos3−/− mice. Early intervention with GS-444217 significantly inhibited diabetic glomerulosclerosis and reduced renal dysfunction but had no effect on the development of albuminuria. Late intervention with GS-444217 improved renal function and halted the progression of glomerulosclerosis, renal inflammation, and tubular injury despite having no effect on established albuminuria. In conclusion, this study identifies ASK1 as a new therapeutic target in diabetic nephropathy to reduce renal inflammation and fibrosis independent of blood pressure control.

The ability of macrophages to cause acute inflammatory glomerular injury is well-established; however, the role of macrophages in the fibrotic phase of chronic kidney disease remains poorly understood. This study examined the role of macrophages in the fibrotic phase (days 14 to 35) of established crescentic glomerulonephritis. Nephrotoxic serum nephritis (NTN) was induced in groups of eight Wistar-Kyoto rats that were given a selective c-fms kinase inhibitor, fms-I, or vehicle alone from day 14 until being killed on day 35. Rats killed on day 14 NTN had pronounced macrophage infiltration with glomerular damage, fibrocellular crescents in 50% of glomeruli, tubulointerstitial damage, heavy proteinuria, and renal dysfunction. Glomerulosclerosis was more severe by day 35 in vehicle-treated rats, as was periglomerular and interstitial fibrosis, while proteinuria and renal dysfunction continued unabated and some parameters of tubular damage worsened. During the day 14-to-35 period, glomerular and interstitial macrophage infiltration decreased with an apparent change from a proinflammatory M1 phenotype to an alternatively activated M2 phenotype. Treatment with fms-I over days 14 to 35 selectively reduced blood monocyte numbers and abrogated glomerular and interstitial macrophage infiltration. This resulted in improved renal function, significantly reduced glomerular and interstitial fibrosis, and protection against further peritubular capillary loss. However, sustained proteinuria, tubular damage, and interstitial T cell infiltration and activation were unaffected. In conclusion, this study demonstrates that macrophages contribute to renal dysfunction and tissue damage in established crescentic glomerulonephritis as it progresses from the acute inflammatory to a chronic fibrotic phase.