Urinary podocyte and TGF-ß1 mRNA as markers for disease activity and progression in anti-glomerular basement membrane nephritis.

BACKGROUND: Podocyte depletion causes glomerulosclerosis, with persistent podocyte loss being a major factor driving disease progression. Urinary podocyte mRNA is potentially useful for monitoring disease progression in both animal models and in humans. To determine whether the same principles apply to crescentic glomerular injury, a rat model of anti-glomerular basement membrane (anti-GBM) nephritis was studied in parallel with a patient with anti-GBM nephritis. METHODS: Podocyte loss was measured by Wilms' Tumor 1-positive podocyte nuclear counting and density, glomerular epithelial protein 1 or synaptopodin-positive podocyte tuft area and urinary podocyte mRNA excretion rate. Glomerulosclerosis was evaluated by Azan staining and urinary transforming growth factor (TGF)-ß1 mRNA excretion rate. RESULTS: In the rat model, sequential kidney biopsies revealed that after a threshold of 30% podocyte loss, the degree of glomerulosclerosis was linearly associated with the degree of podocyte depletion, compatible with podocyte depletion driving the sclerotic process. Urinary podocyte mRNA correlated with the rate of glomerular podocyte loss. In treatment studies, steroids prevented glomerulosclerosis in the anti-GBM model in contrast to angiotensin II inhibition, which lacked a protective effect, and urinary podocyte and TGF-ß1 mRNA markers more accurately reflected both the amount of podocyte depletion and the degree of glomerulosclerosis compared with proteinuria under both scenarios. In a patient successfully treated for anti-GBM nephritis, urinary podocyte and TGB-ß1 mRNA reflected treatment efficacy. CONCLUSION: These results emphasize the role of podocyte depletion in anti-GBM nephritis and suggest that urinary podocyte and TGF-ß1 mRNA could serve as markers of disease progression and treatment efficacy.

Original Research: Potential of urinary nephrin as a biomarker reflecting podocyte dysfunction in various kidney disease models.

Urinary nephrin is a potential non-invasive biomarker of disease. To date, however, most studies of urinary nephrin have been conducted in animal models of diabetic nephropathy, and correlations between urinary nephrin-to-creatinine ratio and other parameters have yet to be evaluated in animal models or patients of kidney disease with podocyte dysfunction. We hypothesized that urinary nephrin-to-creatinine ratio can be up-regulated and is negatively correlated with renal nephrin mRNA levels in animal models of kidney disease, and that increased urinary nephrin-to-creatinine ratio levels are attenuated following administration of glucocorticoids. In the present study, renal nephrin mRNA, urinary nephrin-to-creatinine ratio, urinary protein-to-creatinine ratio, and creatinine clearance ratio were measured in animal models of adriamycin nephropathy, puromycin aminonucleoside nephropathy, anti-glomerular basement membrane glomerulonephritis, and 5/6 nephrectomy. The effects of prednisolone on urinary nephrin-to-creatinine ratio and other parameters in puromycin aminonucleoside (single injection) nephropathy rats were also investigated. In all models tested, urinary nephrin-to-creatinine ratio and urinary protein-to-creatinine ratio increased, while renal nephrin mRNA and creatinine clearance ratio decreased. Urinary nephrin-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA in almost all models, as well as a significant positive correlation with urinary protein-to-creatinine ratio and a significant negative correlation with creatinine clearance ratio. Urinary protein-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA. Following the administration of prednisolone to puromycin aminonucleoside (single injection) nephropathy rats, urinary nephrin-to-creatinine ratio was significantly suppressed and exhibited a significant positive correlation with urinary protein-to-creatinine ratio. In addition, the decrease in number of glomerular Wilms tumor antigen-1-positive cells was attenuated, and urinary nephrin-to-creatinine ratio exhibited a significant negative correlation in these cells. In conclusion, these results suggest that urinary nephrin-to-creatinine ratio level is a useful and reliable biomarker for predicting the amelioration of podocyte dysfunction by candidate drugs in various kidney disease models with podocyte dysfunction. This suggestion will also be validated in a clinical setting in future studies.

Urine proteome analysis in murine nephrotoxic serum nephritis.

BACKGROUND: Urine contains serum proteins filtered by the glomerulus or secreted by the renal tubules and proteins produced locally by the urinary tract. Proteomic analysis of urine holds the potential as a noninvasive means of studying or monitoring disease activity. In mice, large concentrations of albumin and lipocalins have complicated the ability to identify urinary biomarkers in disease models. METHODS: Passive nephrotoxic serum nephritis was induced in mice. Urine proteins were identified and quantified by iTRAQ and MALDI-TOF mass spectrometry. Results were compared to Western blotting and multiplex immunoassays. RESULTS: Large concentrations of major urinary proteins dominate the urine proteome of mice even in the context of acute nephritis. Increased proteinuria caused by nephrotoxic serum nephritis is transient and includes increased albumin excretion. There were no alterations in chemokine excretion. Altered hepcidin excretion was identified, most likely reflecting local production and renal retention. CONCLUSION: Proteomic analysis of mouse urine remains challenging due to the abundance of a limited subset of proteins. iTRAQ analysis does not circumvent these challenges, but can provide information on post-translational processing of some proteins. Hepcidin is identified as a potential urinary marker of nephritis and its role in disease pathogenesis warrants further study.

Therapeutic effect of all-trans retinoic acid on rats with anti-GBM antibody glomerulonephritis.

BACKGROUND: All-trans retinoic acid (ATRA) has antiproliferative and anti-inflammatory effects and is currently used in the treatment of leukemia and dermatologic diseases. We tested the therapeutic potential of ATRA on anti-glomerular basement membrane (GBM) glomerulonephritis rats. METHODS: Glomerulonephritis was induced in male Wistar-Kyoto rats on day 0 by an intravenous injection of antirat GBM antibody. On day 14 after the induction of anti-GBM glomerulonephritis, some rats were sacrificed (N = 5). Another 10 rats were divided into two groups: the vehicle group (N = 5) and the ATRA treated group (N = 5). ATRA was orally administrated from day 14 to day 27 after disease induction. Blood pressure, body weight, urinary protein excretion, and blood chemistry was determined on days 1, 14, 21, and 27. Kidney samples were obtained on day 28. The kidneys were examined with periodic acid-Schiff staining (PAS) and immunohistochemistry using antibodies against the proliferative cell nuclear antigen (PCNA), rat monocyte and macrophage (ED-1), and alpha-smooth muscle actin (alpha-SMA). Glomerular RNA was extracted from isolated glomeruli, and reverse transcription (RT) followed by polymerase chain reaction (PCR) was performed. RESULTS: ATRA administration produced a 55% reduction of proteinuria in glomerulonephritis rats. Light microscopic analysis revealed severe necrosis/crescent formation (>50% of the glomerulus) affecting 34% of glomeruli in vehicle rats, whereas ATRA treatment reduced the glomeruli showing severe change to 14%. ATRA also significantly reduced PCNA-positive cells, ED-1-positive cells and alpha-SMA-positive area in the glomeruli. RT-PCR analyses revealed that a wide variety of genes including inflammation related [tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and CCAAT enhancer-binding protein delta (C/EBPdelta)], cell proliferation-related [platelet-derived growth factor (PDGF)] and fibrosis-related [transforming growth factor-beta1 (TGF-beta1), type I collagen, and alpha-SMA) genes were suppressed in the glomeruli of ATRA-treated rats. CONCLUSION: ATRA administration significantly reduced severe necrosis/crescent formation and urinary protein excretion in glomerulonephritis rats. Suppression of a wide variety of gene expression may partly explain the mechanism of ATRA's antiproliferative and anti-inflammatory effects. These data suggest a novel therapeutic application of ATRA toward glomerulonephritis.

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.

Interleukin-10 inhibits macrophage-induced glomerular injury.

The ability of interleukin-10 (IL-10) to inhibit macrophage recruitment, activation, and proliferation in vivo was studied in a macrophage-mediated, but T cell-independent, passive anti-glomerular basement membrane antibody-induced model of glomerulonephritis (GN) in rats. Treatment with recombinant murine IL-10 resulted in dose-dependent reductions in proteinuria (high dose: 16 +/- 1 mg/24 h; low dose: 30 +/- 2 mg/24 h; control treatment: 69 +/- 6 mg/24 h; normal: 7 +/- 1 mg/24 h) and glomerular macrophage recruitment (high dose: 1.8 +/- 0.1 macrophages per glomerular cross section [c/gcs]; low dose: 5.5 +/- 0.2 c/gcs; control treatment: 12.1 +/- 0.6 c/gcs). Macrophage and intrinsic glomerular cell proliferation were reduced at both doses of IL-10, as was glomerular expression of P-selectin and monocyte chemoattractant protein-1. IL-10 treatment also resulted in a dose-dependent reduction of macrophage activation as indicated by MHC class II and IL-1beta expression. Glomerular nitrite production by isolated cultured glomeruli was reduced after IL-10 treatment in vivo (high dose: 2.3 +/- 2.3 nmol/10(4) glomeruli per 72 h; low dose: 28 +/- 5 nmol/10(4) glomeruli per 72 h; control treatment: 82 +/- 11 nmol/10(4) glomeruli per 72 h). Tumor necrosis factor-alpha production was abolished by high-dose treatment and reduced by the lower dose (3.8 +/- 3.8 pg/10(4) glomeruli per 72 h; control treatment: 249 +/- 23 pg/10(4) glomeruli per 72 h). These studies demonstrate that IL-10 directly attenuates glomerular macrophage recruitment, activation, and proliferation in vivo and can significantly attenuate macrophage-mediated GN independent of any effects on T cells.

Immunoelectron microscopic study on type I, II and III TGF-beta receptors on visceral glomerular epithelial cells in relation to glomerular basement membrane alterations in proteinuric rats.

BACKGROUND: Transforming growth factor (TGF)-beta is a regulator of extracellular matrix accumulation. Both TGF-beta receptors, type I (TbetaRI) and type II (TbetaRII), may be required for signal transduction in the TGF-beta pathway. The aim of this study was to investigate the relationship between the TGF-beta pathways and glomerular basement membrane (GBM) accumulation in vivo. METHODS: We examined TbetaRI, II, and III protein expression on visceral glomerular epithelial cells (GEP) in relation to GBM alterations in passive Heymann nephritis (PHN), anti-GBM nephritis and anti-thymocyte serum (ATS) nephritis. Renal tissues were examined by pre-embedding immunoelectron microscopy 3, 7 and 14 days after induction of nephritis in rats. RESULTS: In normal control rats TbetaRI was not detected on GEP, TbetaRII expression was very occasionally found on GEP and TbetaRIII was seen in the cytoplasm of the GEP. TbetaRI, TbetaRII, and TbetaRIII were constitutively expressed on glomerular endothelial cells. By day 3 of anti-GBM nephritis and PHN, expression of TbetaRI, TbetaRII, and TbetaRIII was still similar to that of normal control rats, and GBM alterations in both models were not prominent except for deposit formation in PHN. From day 7 onwards, in both models, expression of TbetaRI and TbetaRII on GEP increased in association with GBM thickening. Expression of TbetaRIII in the cytoplasm of the GEP was increased, with occasional positive staining being seen on the urinary surface of the GEP from day 7 onwards. On the other hand, at day 3 of ATS nephritis, increased expression of TbetaRI and TbetaRII on GEP was noted, but from day 7 onwards, expression of TbetaR II on GEP dramatically decreased. Expression of TbetaRIII in the cytoplasm of the GEP also transiently increased at day 3. GBM thickening was not noted in ATS nephritis. CONCLUSIONS: The results suggest that persistent upregulation of expression of TbetaRI, TbetaRII and possibly TbetaRIII on GEP may contribute to GBM matrix accumulation in vivo.