Renal epithelial cells retain primary cilia during human acute renal allograft rejection injury.

OBJECTIVES: Primary cilia are sensory organelles which co-ordinate several developmental/repair pathways including hedgehog signalling. Studies of human renal allografts suffering acute tubular necrosis have shown that length of primary cilia borne by epithelial cells doubles throughout the nephron and collecting duct, and then normalises as renal function returns. Conversely the loss of primary cilia has been reported in chronic allograft rejection and linked to defective hedgehog signalling. We investigated the fate of primary cilia in renal allografts suffering acute rejection. RESULTS: Here we observed that in renal allografts undergoing acute rejection, primary cilia were retained, with their length increasing 1 week after transplantation and remaining elevated. We used a mouse model of acute renal injury to demonstrate that elongated renal primary cilia in the injured renal tubule show evidence of smoothened accumulation, a biomarker for activation of hedgehog signalling. We conclude that primary cilium-mediated activation of hedgehog signalling is still possible during the acute phase of renal allograft rejection.

Renal primary cilia lengthen after acute tubular necrosis.

Renal primary cilia are sensory antennas required for the maintenance of normal epithelial differentiation and proliferation in the kidney, but they also have a potential role in epithelial differentiation during renal injury and repair. In mice, tubular damage causes an increase in the length of renal cilia, which may modify their sensory sensitivity during repair. Here, we investigated whether the alteration of renal cilium length during renal injury is clinically relevant. Using biopsies of human renal transplants that suffered acute tubular necrosis during transplantation, we compared the length of renal primary cilia with renal function. Serial biopsies showed that acute tubular necrosis resulted in more than a doubling of cilium length throughout the nephron and collecting duct approximately 1 wk after injury. Allografts displayed a trend toward normalization of cilium length in later biopsies, and this correlated with functional recovery. A mouse model of renal ischemia-reperfusion confirmed the increase and subsequent regression of cilium length during renal repair, displaying complete normalization of cilium length within 6 wk of injury. These findings demonstrate that the length of renal cilia is a clinically relevant indicator of renal injury and repair.

Reversal of dialysis-dependent renal failure in light-chain deposition disease by autologous peripheral blood stem cell transplantation.

Specific treatment of light-chain deposition disease has been reported as ineffective in altering the course of the severe or end-stage renal failure it causes. The authors describe a case of biopsy-proven primary light-chain deposition disease of the kidney, severe renal failure, and incipient dialysis dependency, treated by autologous peripheral blood stem cell transplantation, that led to reversal of dialysis dependency and sustained improvement in renal function.

Activation of the extracellular-signal regulated protein kinase pathway in human glomerulopathies.

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 alpha-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.

p38 Mitogen-activated protein kinase activation and cell localization in human glomerulonephritis: correlation with renal injury.

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.

Activation and cellular localization of the p38 and JNK MAPK pathways in rat crescentic glomerulonephritis.

BACKGROUND: The p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) are intracellular signal transduction pathways involved in the production of inflammatory mediators. Little, however, is known about the contribution of these pathways to renal inflammation, nor the cell types in which these pathways are activated within normal and inflamed kidneys. The aim of this study was therefore to delineate the pattern and cellular localization of p38 and JNK activation in normal rat kidney and rat acute and chronic inflammatory renal disease. METHODS: Normal male Sprague-Dawley rats and groups of rats given accelerated anti-glomerular basement membrane (GBM) disease were killed at 3 hours, day 1, day 7, or day 28 and examined for p38 and JNK pathway activation by Western blotting and immunolocalization of the phosphorylated p38 (p-p38) and JNK (p-JNK) kinases. RESULTS: In terms of glomerular MAPK activation, Western blotting identified the presence of both p-p38 and p-JNK in normal glomeruli, localized by immunohistochemistry to podocytes and epithelial cells of Bowman's capsule. In anti-GBM disease, Western blotting showed that p38 activation peaked at 3 hours and remained elevated above normal throughout the disease time course. JNK activation (via the 54 kD isoform) likewise increased at 3 hours of anti-GBM disease and remained elevated throughout disease. At 3 hours, p-p38, but not p-JNK, was localized to neutrophils and glomerular endothelial cells. p-JNK was localized to glomerular endothelial cells at day 7. Macrophages, lymphocytes, activated podocytes, and myofibroblasts were positive for both p-p38 and p-JNK. In terms of tubular MAPK activation, Western blotting identified p38 and JNK activation in tubules of normal kidney. Immunostaining showed that most cortical tubules contained some p-p38 and p-JNK stained cells. There was a significant increase in tubular p38 activation at 3 hours of anti-GBM disease, followed by increased JNK activation of the 54 kD isoform from day 7 onward, and the 46 kD isoform at day 28. Immunostaining of diseased tissue localized p-p38 and p-JNK to virtually all cortical tubular cells. CONCLUSION: The p38 and JNK MAPK pathways are activated in glomeruli and tubules of normal kidney. In acute anti-GBM disease, there was an increase in p38 activation within glomerular endothelial cells and within infiltrating neutrophils, suggesting an important role for p38 MAPK in acute inflammation. In progressive anti-GBM disease, p38 and JNK activation in podocytes, glomerular endothelial cells, infiltrating macrophages, T cells, and myofibroblasts suggests that both the p38 and JNK MAPK pathways are important in chronic inflammation and fibrosis. Blockade of these pathways may therefore be potentially therapeutic in the treatment of acute and chronic renal inflammation.

Activation of the ERK pathway precedes tubular proliferation in the obstructed rat kidney.

BACKGROUND: In vitro studies suggest that activation of the extracellular signal-regulated kinase (ERK) pathway plays a critical role in the proliferation of tubular epithelial and myofibroblast-like cells. However, little is known of ERK activation in individual cell types in normal or diseased kidney. The aims of this study were to (1) localize ERK activation within the kidney, and (2) examine the relationship between ERK activation and cell proliferation in the injured kidney. METHODS: Unilateral ureteric obstruction (UUO) was induced in groups of six Wistar rats, which were killed at 30 minutes, 6 hours, and 1, 4, or 7 days after obstruction. Activation of ERK was identified using antibodies specific for the phosphorylated form of ERK (pERK) in Western blots and immunostaining. Proliferating cells were detected using bromodeoxyuridine (BrdU). RESULTS: Western blotting showed abundant expression of the two ERK isoforms, ERK-1 and ERK-2, in normal rat kidney. Low levels of activated ERK (pERK-2> pERK-1) were detected in normal rat kidney by Western blotting. Immunostaining showed that ERK activation in normal kidney was largely restricted to collecting ducts in the outer medulla. Within 30 minutes of ureter obstruction, Western blotting showed a sixfold increase in ERK activation followed by a second peak (14-fold increase) on days 4 and 7. The initial peak of ERK activation was localized to medullary collecting ducts and the thick ascending limb of Henle (TALH), whereas the second peak corresponded to a progressive increase in ERK activation in dilated collecting ducts and in interstitial cells in the cortex. Proliferation of tubular epithelial cells closely followed the pattern of ERK activation, being evident first in medullary collecting ducts and TALH on day 1, and then in cortical collecting ducts from day 4. CONCLUSION: This study has identified a discrete pattern of ERK activation in normal rat kidney and an increase in ERK activation following obstruction. The temporal and spatial relationship in which ERK activation preceded tubular cell proliferation suggest that ERK signaling plays a key role in tubular epithelial cell proliferation in the injured kidney.

Blockade of p38alpha MAPK ameliorates acute inflammatory renal injury in rat anti-GBM glomerulonephritis.

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 (p38alpha, 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.

Interferon-gamma induces macrophage migration inhibitory factor synthesis and secretion by tubular epithelial cells.

Macrophage migration inhibitory factor (MIF) promotes macrophage accumulation and leucocyte activation during inflammation. Macrophage migration inhibitory factor is upregulated in intrinsic renal cells in many types of kidney diseases, and has a pathogenic role in rat crescentic nephritis. However, little is known about the factors that regulate the production and secretion of MIF in kidney cells. In this study, we evaluated whether interferon-gamma (IFN-gamma), a cytokine implicated in the development of kidney disease and a potent inducer of MIF production in macrophages, could promote MIF synthesis and secretion from renal tubular epithelial cells. Northern blot analysis detected constitutive expression of MIF mRNA in rat tubular epithelial cells (NRK52E), which increased twofold after a 6-h stimulation with IFN-gamma. Macrophage migration inhibitory factor protein was found only in the cytoplasm of NRK52E cells. Following IFN-gamma stimulation, intracellular MIF in NRK52E cells was rapidly secreted with a maximal reduction of 50% after 20 min, which returned to normal levels after 2-4 h. Rapid secretion of MIF in response to IFN-gamma was also seen in rat mesangial cells. These findings indicate that IFN-gamma induces rapid secretion of MIF by tubular epithelial cells, and suggest that this may be an important mechanism leading to inflammatory cell accumulation and activation during kidney disease.

Macrophage accumulation at a site of renal inflammation is dependent on the M-CSF/c-fms pathway.

Production of macrophage-colony stimulating factor (M-CSF), the major macrophage growth factor, is increased in tissues during inflammation. Therefore, we determined whether M-CSF, acting through its receptor c-fms, contributes to macrophage accumulation at a site of tissue injury. Daily treatment with anti-c-fms or control antibody was given to mice with renal inflammation resulting from unilateral ureteric obstruction (UUO). Following UUO, kidney M-CSF mRNA increased in association with macrophage accumulation (days 1, 5, and 10) and local macrophage proliferation (days 5 and 10). Anti-c-fms treatment caused a minor inhibition of monocyte recruitment at day 1, reduced macrophage accumulation by 75% at day 10, but did not affect blood monocyte counts or the CD4 and CD8 lymphocytic infiltrate. Prevention of macrophage accumulation by anti-c-fms treatment was associated with a 90% reduction in local macrophage proliferation at days 5 and 10 without evidence of increased macrophage apoptosis. Therefore, M-CSF/c-fms signaling plays a key role in macrophage accumulation during tissue injury.

Membranoproliferative glomerulonephritis in association with chronic lymphocytic leukaemia: a report of three cases.

AIMS: The reported association of glomerular disease with chronic lymphocytic leukaemia (CLL) is rare despite the relative frequency of this type of leukaemia. Hence, we have examined the renal biopsies in three patients with CLL and glomerulonephritis. METHODS: Renal biopsies were examined by light microscopy, immunofluorescence microscopy and electron microscopy. RESULTS: One of two patients with mild impairment of renal function and an active urinary sediment had ultrastructural features of idiopathic type I membranoproliferative glomerulonephritis (MPGN), and the other had features of fibrillary/ immunotactoid glomerulonephritis with deposits of IgG and C3. One patient with nephrotic syndrome had characteristic electron microscopic appearances of type III MPGN. In all three there was an association with monoclonal gammopathy. The parameters of glomerular damage improved in association with response to drug treatment of the CLL. CONCLUSION: There is a spectrum of types of MPGN seen in patients with CLL and there appears to be an association with the presence of monoclonal gammopathy. This is the first reported case of type III MPGN in CLL.

Urine macrophage migration inhibitory factor reflects the severity of renal injury in human glomerulonephritis.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays a pathogenic role in experimental crescentic glomerulonephritis (GN). Renal expression of MIF is also upregulated in human GN and correlates with leukocytic infiltration, histologic damage, and renal dysfunction. The study presented here examined whether MIF can be measured in urine and if so, whether the urine MIF concentration reflects the degree of renal injury. Urine and serum MIF was measured by enzyme-linked immunosorbent assay in 10 normal healthy volunteers and in a cohort of 63 patients with GN (2 thin basement membrane disease [TBM], 15 membranous GN, 10 focal segmental glomerular sclerosis, 20 IgA glomerularnephritis, 11 crescentic GN, 10 systemic lupus erythematosis World Health Organization class IV). Renal MIF expression was assessed by immunostaining of biopsy tissue. MIF was detected in urine from normal volunteers (mean +/- SD; 191 +/- 132 pg MIF/micromol creatinine). The urine MIF concentration was unchanged in patients with nonproliferative nephropathies (343 +/- 397 pg MIF/micromol Cr) but was increased 3.4-fold in proliferative nephropathies (645 +/- 527 pg MIF/micromol Cr; P < 0.05 versus normal and nonproliferative). Stratified analysis showed the greatest increase in urine MIF in crescentic GN (4.5-fold). In contrast, serum MIF levels were not different between normal patients and any patient group. Immunostaining demonstrated a significant increase in renal MIF expression in proliferative glomerulonephritides that was associated with macrophage and T cell infiltration. There was a significant correlation between the urine MIF concentration and renal MIF expression, but not with serum MIF, indicating a renal origin for the excreted urine MIF. The urine MIF concentration also correlated with the degree of renal dysfunction, histologic damage, and leukocytic infiltration, but not with the amount of proteinuria. In conclusion, this study shows that the urine MIF concentration is significantly increased in proliferative forms of GN and correlates with the degree of renal injury. Urine MIF levels reflect MIF expression within the kidney and may be a useful noninvasive tool for monitoring patients with crescentic GN, particularly in disease exacerbation.