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Inflammation and fibrosis are pathological mechanisms that are partially regulated by cell signalling through the p38 mitogen-activated protein kinase (MAPK) pathway. Elements of the diabetic milieu such as high glucose and advanced glycation end-products induce activation of this pathway in renal cells. Therefore, we examined whether p38 MAPK signalling is associated with the development of human and experimental diabetic nephropathy.Immunostaining identified phosphorylated (active) p38 MAPK in human biopsies with no abnormality ( n=6) and with Type 2 diabetic nephropathy ( n=12). Changes in kidney levels of phosphorylated p38 were assessed by immunostaining and western blotting in mice with streptozotocin-induced Type 1 diabetes that had been killed after 0.5, 2, 3, 4 and 8 months, and in Type 2 diabetic db/db mice at 2, 4, 6 and 8 months of age.Phosphorylated p38 was detected in some intrinsic cells in normal human kidney, including podocytes, cortical tubules and occasional interstitial cells. Greater numbers of these phosphorylated p38+ cells were observed in diabetic patients, and phosphorylated p38 was identified in accumulating interstitial macrophages and myofibroblasts. A similar pattern of p38 activation was observed in both mouse models of diabetes. In mice, kidney levels of phosphorylated p38 increased (2-6 fold) following the onset of Type 1 and Type 2 diabetes. In both mouse models, interstitial phosphorylated p38+ cells were associated with hyperglycaemia, increased HbA(1)c levels and albuminuria. Further assessment of streptozotocin-induced diabetic nephropathy showed that interstitial phosphorylated p38+ cells correlated with interstitial fibrosis (myofibroblasts, collagen).Increased p38 MAPK signalling is a feature of human and experimental diabetic nephropathy. Time course studies in mouse models suggest that phosphorylation of p38 plays a pathological role, particularly in the development of interstitial fibrosis.

The p38 mitogen-activated protein kinase (MAPK) pathway transduces external stress stimuli and is important in extracellular matrix synthesis in cell types in vitro; however, its role in renal fibrosis is not known. Explored was the role the p38 MAPK pathway in rat unilateral ureteric obstruction (UUO), a model of renal fibrosis induced by a noninflammatory surgical insult. In a time-course study, a marked increase in phosphorylation (activation) of p38 in both interstitial myofibroblasts and tubules was shown. Rats were then treated daily with a specific inhibitor of p38alpha, NPC 31169, from the time of UUO surgery until being killed 7 d later. Compared with vehicle, NPC 31169-treated rats had a significant reduction in renal fibrosis assessed by interstitial volume, collagen IV deposition, and mRNA levels. This was primarily due to a reduction in the accumulation of interstitial myofibroblasts, as shown by a reduction in the area of immunostaining for alpha-smooth muscle actin and heat shock protein 47. The increase in renal TGF-beta1 mRNA and protein levels in UUO was unaltered with NPC 31169 treatment; however, connective tissue growth factor mRNA was reduced. These results demonstrate that p38alpha MAPK plays an important role in renal fibrosis, acting downstream of TGF-beta1. Blockade of p38 MAPK reduces extracellular matrix production and may be considered a potential therapeutic option in the treatment of renal fibrosis.

Examined was extracellular-signal regulated kinase (ERK) activation in normal human kidney ( n = 2) and a cohort of glomerulopathies by immunohistochemistry staining for the dual-phosphorylated form of ERK (p-ERK). Cell proliferation was determined by expression of the proliferating cell nuclear antigen (PCNA). In normal human kidney, p-ERK was largely restricted to the cytoplasm of cells of the collecting duct (CD). In glomerulopathies, glomerular ERK activation was highly variable. However, there was colocalization of cell proliferation and ERK activation in the glomerular tuft and crescents. Tubular ERK activation in the different glomerulopathies was confined to the CD in areas with normal architecture. In contrast, ERK activation was prominent in tubules and interstitial cells in areas of tubulointerstitial damage. ERK activation was observed in glomerular and interstitial α-smooth muscle actin–positive myofibroblasts, but few macrophages or T cells showed ERK activation. There was a significant correlation between glomerular p-ERK+ and PCNA+ cells and between tubular p-ERK+ and PCNA+ cells. Glomerular p-ERK+ cells correlated with glomerular cellularity and the percentage of glomeruli with segmental lesions. Tubular p-ERK+ cells correlated with renal dysfunction and interstitial fibrosis and tubular atrophy. In conclusion, activation of the ERK pathway in human glomerulopathies correlates with cell proliferation, histologic lesions, and renal dysfunction. ERK activation may promote renal repair through tubular proliferation while promoting renal fibrosis via proliferation of glomerular and interstitial myofibroblasts.

The p38 mitogen-activated protein kinase (MAPK) pathway is a pro-inflammatory signal transduction pathway. The aim of this study was to examine the role of this pathway in acute renal inflammation. Immunostaining localized components of the p38 MAPK pathway (p38α, p-p38, p-ATF-2) in normal glomeruli, to podocytes, and occasional endothelial cells. This study identified an eightfold increase in glomerular activation of p38 MAPK (phosphorylated p38, p-p38) within 3 h of the induction of rat anti–glomerular basement membrane (GBM) glomerulonephritis and localized p-p38 and p-ATF-2 to infiltrating neutrophils, with increased staining of podocytes and endothelial cells. The relevance of these findings to human acute inflammatory renal disease was determined by examination of biopsy specimens. In patients with post-infectious glomerulonephritis, there was an increased number of positive p-p38 glomerular cells, including p-p38 staining of infiltrating neutrophils, compared with normal human kidney. In rats, administration of a specific p38 MAPK inhibitor, NPC 31145, before induction of anti-GBM disease prevented a loss of renal function and substantially reduced proteinuria. The reduction in renal injury was attributed to a 55% reduction in glomerular neutrophil infiltration and a 68% reduction in platelet accumulation. This was associated with an abrogation of glomerular P-selectin immunostaining and inhibition of glomerular P-selectin gene expression. In summary, this study has localized the components of the p38 MAPK pathway to cells in normal and diseased rat and human kidney and identified a number of important mechanisms by which signaling through the p38 MAPK pathway induces inflammatory renal disease. Blockade of the p38 pathway may be a novel therapeutic strategy for the treatment of acute renal inflammation. E-mail: cosimo.stambe@med.monash.edu.au

Tubules are the major site of M-CSF production in experimental kidney disease: Correlation with local macrophage proliferation . Background Local proliferation of macrophages occurs within both the glomerulus and the interstitium in severe forms of human and experimental glomerulonephritis and plays an important role in amplifying renal injury. Macrophage colony-stimulating factor (M-CSF) is thought to be the growth factor driving this local macrophage proliferation. Previous studies have found that glomeruli are the predominant source of M-CSF production. However, this is difficult to reconcile with the prominent macrophage accumulation and proliferation seen in the interstitial compartment in glomerulonephritis. To address this issue, we localized M-CSF expression in rat models of glomerular versus tubulointerstitial injury and examined its relationship to local macrophage proliferation. Methods M-CSF expression (Northern blotting, in situ hybridization, immunostaining, Western blotting) and local macrophage proliferation (double immunostaining) was examined in normal rat kidney on days 1 and 14 of rat anti-glomerular basement membrane (anti-GBM) glomerulonephritis and on day 5 following unilateral ureteric obstruction. Results M-CSF mRNA and protein expression were identified in small numbers of glomerular podocytes, approximately 25% of cortical tubules, and most medullary tubules in normal rat kidney. Northern blotting showed a significant increase in whole kidney M-CSF mRNA in rat anti-GBM glomerulonephritis. Up-regulation of glomerular and, most prominently, tubular M-CSF production was confirmed by three independent methods: in situ hybridization, immunostaining, and Western blotting. The increase in M-CSF expression colocalized with local macrophage proliferation (ED1+PCNA+ cells) in both the glomerulus and tubulointerstitium. On day 5 after ureter ligation, there was a significant increase in tubular M-CSF mRNA and protein expression in the obstructed kidney, with no change in glomerular M-CSF. In parallel with M-CSF expression, macrophage accumulation and proliferation was prominent in the interstitium, but was absent from glomeruli. Conclusions The tubular epithelial cell is the major site of M-CSF production within the injured kidney. Indeed, substantial macrophage accumulation and local proliferation can occur in the tubulointerstitium in the absence of glomerular inflammation. These results suggest that M-CSF production within the kidney, particularly by tubular epithelial cells, plays an important role in regulating local macrophage proliferation in experimental kidney disease.

Activation of the p38 mitogen-activated protein kinase (MAPK) signal transduction pathway plays an important role in the inflammatory response. It was postulated that p38 MAPK is important in the pathogenesis of human glomerulonephritis and contributes to the development of renal injury. p38 MAPK activation was examined by immunodetection for dual phosphorylated p38 (p-p38) in normal human kidney and 77 renal biopsy specimens encompassing a wide spectrum of glomerulonephritides. In normal kidney, p-p38 immunostaining was restricted to the nuclei of a small number of podocytes, parietal epithelial cells, and tubular cells. There was a dramatic increase in the number of p-p38-positive cells in glomeruli and tubules in nonproliferative and proliferative glomerulonephritis and a substantial increase in the number of interstitial p-p38-positive cells in proliferative glomerulonephritis. Double immunostaining identified p38 activation in intrinsic renal cells (podocytes and endothelial and tubular cells), infiltrating macrophage and neutrophils, and myofibroblasts. Renal failure correlated with the number of p-p38-positive glomerular, tubular, and interstitial cells. Proteinuria correlated with the number of p-p38-positive tubular and interstitial cells and the number of p-p38-positive podocytes in nonproliferative glomerulonephritis. Furthermore, glomerular p38 activation correlated with segmental proliferative and necrotic lesions, and interstitial p38 activation correlated with the degree of interstitial inflammation. In conclusion, activation of p38 MAPK in intrinsic renal cells and infiltrating leukocytes correlated with renal dysfunction and histopathology, suggesting an important pathogenic role for p38 MAPK activation in human glomerulonephritis.