Prudence A. Hill

Macrophage accumulation in human progressive diabetic nephropathy.

Background:  Diabetic nephropathy is a major global health problem. Progression to renal failure is common; however, the mechanisms are unknown. Experimental models suggest a role for macrophages. Therefore, macrophage accumulation and its relationship to the subsequent clinical course were studied.

Blockade of p38α 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.

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, w 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.

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.

Ascorbic acid protects against the nephrotoxicity and apoptosis caused by colistin and affects its pharmacokinetics

OBJECTIVES The use of colistin in the treatment of life-threatening Gram-negative infections is associated with a high rate of nephrotoxicity that is dose limiting. This study aimed to examine the nephroprotective effect of ascorbic acid against colistin-induced nephrotoxicity. METHODS Rats were treated intravenously twice daily with saline, colistin (cumulative dose of 36.5 mg/kg), a combination of ascorbic acid (50 or 200 mg/kg) and colistin, or ascorbic acid (200 mg/kg) over 7 days. Colistin-induced apoptosis was examined in rats over 5 days and in vitro using rat renal proximal tubular cells NRK-52E over 24 h with and without ascorbic acid. The effect of co-administered ascorbic acid on colistin pharmacokinetics was investigated. RESULTS The 24 h urinary excretion of N-acetyl-β-D-glucosaminidase, a sensitive marker for tubular damage, was significantly lower (P < 0.0001) in the colistin/ascorbic acid 200 mg/kg group. Significant histological abnormalities (P < 0.01) were detected only in the kidneys of the colistin group, which also had the highest percentage (30.6 ± 7.8%) of apoptotic cells (P < 0.005). In the cell culture studies, the percentage of apoptotic cells was significantly higher in the presence of 0.1 mM colistin alone (51.8 ± 2.0%; P < 0.0001) than in the presence of ascorbic acid, which decreased the apoptotic effect in a concentration-dependent manner. Ascorbic acid (200 mg/kg) altered colistin pharmacokinetics, as the total body clearance decreased from 3.78 ± 0.36 mL/min/kg (colistin group) to 2.46 ± 0.57 mL/min/kg (P = 0.0024). CONCLUSIONS This is the first study demonstrating the protective effect of ascorbic acid against colistin-induced nephrotoxicity and tubular apoptosis. Co-administration of ascorbic acid has the potential to increase the therapeutic index of colistin.

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 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.

CD44 and hyaluronan expression in the development of experimental crescentic glomerulonephritis

CD44 is a widely expressed cell surface glycoprotein which is involved in both cell–matrix and cell–cell interactions which regulate a variety of processes, including leucocyte migration and activation. Therefore, we examined the expression of CD44, and its major ligand hyaluronan, during the induction and progression of experimental glomerulonephritis. Antibody staining of normal rat kidney showed constitutive CD44 expression by resident glomerular macrophages, parietal epithelial cells, medullary and occasional cortical tubules. There was a marked increase in CD44 expression over days 1, 7 and 21 of rat crescentic anti‐glomerular basement membrane (GBM) glomerulonephritis. Infiltrating monocytes and lymphocytes were CD44+, with ultrastructural studies showing high levels of CD44 expressed on the surface of lymphocytes adherent to activated endothelium. Marked hyaluronan deposition was seen in areas of fibrosis on days 7 and 21, such as glomerular crescents and the periglomerular area. Hyaluronan deposition was accompanied by the presence of many CD44+ cells. Double immunohistochemistry showed that both CD44+ED1+ macrophages and CD44+ myofibroblasts (identified by expression of α‐smooth muscle actin) were present in areas of fibrosis. There was also a dramatic increase in cortical tubular CD44 expression, which was most evident in areas of tubular damage. Although tubular epithelial cells expressed CD44 upon both the basolateral and luminal surface, CD44 expression was most prominent within tight junctions, suggesting a role for CD44–CD44 interactions in cell–cell adhesion within the tubule. Analysis of CD44 isoforms by reverse transcriptase‐polymerase chain reaction (RT‐PCR) showed that the standard form of CD44 predominated in both normal and diseased kidney. However, a series of alternatively spliced CD44 isoforms was also detected, whose expression was markedly increased during disease. At least seven isoforms containing the v6 domain were identified, with the smallest form representing activated T cells. In conclusion, CD44 is constitutively expressed in normal kidney and is dramatically up‐regulated in rat anti‐GBM disease, suggesting possible roles for the CD44–hyaluronan interaction in leucocyte recruitment, renal fibrosis and tubular cell–matrix and cell–cell interactions during the induction and progression of crescentic glomerulonephritis.

High mammographic density is associated with an increase in stromal collagen and immune cells within the mammary epithelium

IntroductionMammographic density (MD), after adjustment for a women’s age and body mass index, is a strong and independent risk factor for breast cancer (BC). Although the BC risk attributable to increased MD is significant in healthy women, the biological basis of high mammographic density (HMD) causation and how it raises BC risk remain elusive. We assessed the histological and immunohistochemical differences between matched HMD and low mammographic density (LMD) breast tissues from healthy women to define which cell features may mediate the increased MD and MD-associated BC risk.MethodsTissues were obtained between 2008 and 2013 from 41 women undergoing prophylactic mastectomy because of their high BC risk profile. Tissue slices resected from the mastectomy specimens were X-rayed, then HMD and LMD regions were dissected based on radiological appearance. The histological composition, aromatase immunoreactivity, hormone receptor status and proliferation status were assessed, as were collagen amount and orientation, epithelial subsets and immune cell status.ResultsHMD tissue had a significantly greater proportion of stroma, collagen and epithelium, as well as less fat, than LMD tissue did. Second harmonic generation imaging demonstrated more organised stromal collagen in HMD tissues than in LMD tissues. There was significantly more aromatase immunoreactivity in both the stromal and glandular regions of HMD tissues than in those regions of LMD tissues, although no significant differences in levels of oestrogen receptor, progesterone receptor or Ki-67 expression were detected. The number of macrophages within the epithelium or stroma did not change; however, HMD stroma exhibited less CD206+ alternatively activated macrophages. Epithelial cell maturation was not altered in HMD samples, and no evidence of epithelial–mesenchymal transition was seen; however, there was a significant increase in vimentin+/CD45+ immune cells within the epithelial layer in HMD tissues.ConclusionsWe confirmed increased proportions of stroma and epithelium, increased aromatase activity and no changes in hormone receptor or Ki-67 marker status in HMD tissue. The HMD region showed increased collagen deposition and organisation as well as decreased alternatively activated macrophages in the stroma. The HMD epithelium may be a site for local inflammation, as we observed a significant increase in CD45+/vimentin+ immune cells in this area.

Chronic intestinal pseudo-obstruction in systemic lupus erythematosus due to intestinal smooth muscle myopathy.

We report the case of a woman with systemic lupus erythematosus initially manifesting with fever, rash and arthritis, and two years later with Class IV lupus nephritis. Following treatment with cyclophosphamide she developed symptoms and signs of chronic intestinal pseudo-obstruction (CIPO) that was initially thought to be due to a neutropenic enterocolitis. However, persistence of symptoms resulted in segmental resection of the ileum which showed widespread myocyte necrosis and active inflammation within the muscularis propria. A subsequent, more extensive ileocolic resection showed severe diffuse atrophy and fibrosis of the muscularis propria throughout the resected bowel. The absence of mesenteric vasculitis and the clinical response of the CIPO to the immunosupressive regimen of prednisolone and cyclosporin A suggest that the bowel muscle coat changes reflect an intestinal myopathy secondary to systemic lupus erythematosus, and may have an auto-immune etiology.