anti-Thy1 Glomerulonephritis Research Articles

P0024TRIPARTITE MOTIF-CONTAINING 55 (TRIM55) PARTICIPATES IN THE IMMUNE RESPONSE IN THE EXPERIMENTAL ANTI-THY1 GLOMERULONEPHRITIS

Abstract Background and Aims Mesangial proliferative glomerulonephritis (MsPGN) is considered as an immune- related disease. In the previous study, tripartite motif family was reported to be closely related to immune regulation. Therefore, we performed a series of experiments in vivo and vitro to investigate the role of tripartite motif-containing 55 (TRIM55), a member of tripartite motif family, in the progression of MsPGN. Method 36 male SD rats were randomly divided into 6 groups to induce anti-Thy1 nephritis. Specimens of the negative control group were collected at 0d after the injection, while those of other groups were collected at 1d, 2d, 3d, 4d, 5d after the injection, respectively. Renal cortex slices were obtained for histological and immunohistochemistry assessmen of CD68. qPCR analysis of glomerulus was performed to examine the expression of TRIM55 . Primary RMCs with overexpression of TRIM55 were obtained through plasmid transfection. In addition, si-RNA tranfection was used to knock down the expression of TRIM55 in primary RMCs. qPCR analysis was conducted to detect the level of cytokines, such as TNF-α, CCL2, CXCL6, CXCL10, and IL-6. Results PAS staining results indicated mesangial dissolution occurred since 1d, followed by inflammatory cell infiltration. CD68 immunohistochemical staining results showed that macrophages infiltration peaked at 1d and then decreased gradually. The expression of TRIM55 mRNA also peaked at 1d and decreased gradually, which was consistent with the trend of macrophages infiltration. In primary RMCs, knockdown of TRIM55 led to down-regulation of the expression of cytokines (TNF-α, CCL2, CXCL6, CXCL10 and IL-6). On the other hand, the expression of those cytokines(TNF-α, CCL2, CXCL6, CXCL10 and IL-6) significantly increased in TRIM55-overexpressed primary RMCs. Conclusion The above results indicate that TRIM55 participates in the immune response in experimental anti-Thy1 glomerulonephritis by regulating the production of cytokines. We duce TRIM55 may be a promising therapeutic intervention to ameliorate leukocyte infiltration in MsPGN.

IL-17 Expression in the Time Course of Acute Anti-Thy1 Glomerulonephritis.

BackgroundInterleukin-17 (IL-17) is a new pro-inflammatory cytokine involved in immune response and inflammatory disease. The main source of IL-17 is a subset of CD4+ T-helper cells, but is also secreted by non-immune cells. The present study analyzes expression of IL-17 in the time course of acute anti-thy1 glomerulonephritis and the role of IL-17 as a potential link between inflammation and fibrosis.MethodsAnti-thy1 glomerulonephritis was induced into male Wistar rats by OX-7 antibody injection. After that, samples were taken on days 1, 5, 10 (matrix expansion phase), 15 and 20 (resolution phase). PBS-injected animals served as controls. Proteinuria and histological matrixes score served as the main markers for disease severity. In in vitro experiments, NRK-52E cells were used. For cytokine expressions, mRNA and protein levels were analyzed by utilizing RT-PCR, in situ hybridization and immunofluorescence.ResultsHighest IL-17 mRNA-expression (6.50-fold vs. con; p<0.05) was found on day 5 after induction of anti-thy1 glomerulonephritis along the maximum levels of proteinuria (113 ± 13 mg/d; p<0.001), histological glomerular-matrix accumulation (82%; p<0.001) and TGF-β1 (2.2-fold; p<0.05), IL-6 mRNA expression (36-fold; p<0.05). IL-17 protein expression co-localized with the endothelial cell marker PECAM in immunofluorescence. In NRK-52E cells, co-administration of TGF-β1 and IL-6 synergistically up-regulated IL-17 mRNA 4986-fold (p<0.001).ConclusionsThe pro-inflammatory cytokine IL-17 is up-regulated in endothelial cells during the time course of acute anti-thy1 glomerulonephritis. In vitro, NRK-52E cells secrete IL-17 under pro-fibrotic and pro-inflammatory conditions.

Role of Macrophage Stimulating Protein 1 (MSP1) in the Development of Glomerulopathy in Sickle Cell Disease

BackgroundMore than 50% patients with sickle cell disease (SCD) develop renal glomerular disease. Its pathophysiology is likely to be multifactorial, being affected by hyperfiltration, glomerular hypertension, ischemia-reperfusion injury, oxidative stress, and endothelial dysfunction. Ischemia-reperfusion is associated with significant recruitment of glomerular and interstitial macrophages. Recently, Macrophage Stimulating Protein 1 (MSP1) was shown to be involved in the development of anti-Thy1 glomerulonephritis in rat model that also associated with renal macrophages infiltration. MSP1 is produced by the liver and secreted into the circulation as a non-active protein. It is cleaved and activated locally by a macrophage membrane-associated proteinase. We hypothesesize that infiltrating macrophages in SCD activate MSP1 protein that accumulates in the renal glomeruli and induces endothelial cell and podocytes injury.ObjectiveWe analyzed MSP1 expression in vivo in SCD mouse model and the effect of recombinant MSP1 on cultured human podocytes and endothelial cells.MethodsTransgenic SCD mice were obtained from Jackson Laboratory (B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow/Hbbtm3(HBG1,HBB)Tow/J Townes strain). Townes mice produce approximately 94% human sickle (HbS), 6% human fetal hemoglobin (HbF), and no murine hemoglobin. Control animals are carrying two copies of the transgene encoding human α1-globin gene and two copies of the Hbbtm3(HBG1,HBB)Tow mutation (human hemoglobin gamma (Aγ) gene and the human wildtype hemoglobin beta (βA) gene). Townes mice spontaneously develop renal glomerular lesions. Kidneys were collected from 5 months old mice and immunohistochemistry (IHC) was carried to detect macrophages, endothelial cells (CD34), podocytes (WT-1) and MSP1. Human glomerular endothelial cell line (HGEC) and two human podocyte lines (PD1 and PD2) were treated with recombinant MCP1 and the cellular motility, permeability, growth and capillary formation were analyzed.ResultsSCD mice developed focal segmental glomerulosclerosis. It was associated with glomerular capillary aneurisms, loss of podocytes and increased macrophages infiltration. IHC demonstrated accumulation of MSP1 in the capillaries of affected glomeruli. In vitro, MSP1 treatment increased motility of both endothelial cells and podocytes. In addition, MSP1 significantly reduced podocytes growth and viability. MSP1 also inhibited capillary formation by endothelial cells on Matrigel.ConclusionActivated MSP1 is likely to be involved in the development of glomerular lesions in SCD which could be due to the modulation of endothelial cell and podocytes function. Further analysis is needed to elucidate the role of MSP1 in the development of glomerular disease in humans.AcknowledgmentsThis work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005 and 5G12MD007597). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. DisclosuresNo relevant conflicts of interest to declare.

Pentoxifylline Attenuates Proteinuria in Anti-Thy1 Glomerulonephritis via Downregulation of Nuclear Factor-κB and Smad2/3 Signaling.

Anti-Thy1 glomerulonephritis is a rat nephritis model closely simulating human mesangial proliferative glomerulonephritis. It affects primarily the mesangium, yet displays substantial proteinuria during the course. This study investigated the molecular signals underlying proteinuria in this disease and the modulation of which by the known antiproteinuric agent, pentoxifylline. Male Wistar rats were randomly divided into a control group and nephritic groups with or without treatment with IMD-0354 (an IκB kinase inhibitor), SB431542 (an activin receptor-like kinase inhibitor) or pentoxifylline. Kidney sections were prepared for histological examinations. Glomeruli were isolated for mRNA and protein analysis. Urine samples were collected for protein and nephrin quantitation. One day after nephritis induction, proteinuria developed together with ultrastructural changes of the podocyte and downregulation of podocyte mRNA and protein expression. These were associated with upregulation of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β/activins mRNAs and activation of nuclear factor (NF)-κB p65 and Smad2/3. IMD-0354 attenuated proteinuria on d 1, whereas SB431542 decreased proteinuria on d 3 and 5, in association with partial restoration of downregulated podocyte mRNA and protein expression. Pentoxifylline attenuated proteinuria and nephrinuria through the course, plus inhibition of p-NF-κB p65 (d 1) and p-Smad2/3 (d 5) and partial reversal of downregulated podocyte mRNA and protein. Our data show that the pathogenesis of proteinuria in anti-Thy1 glomerulonephritis involves TNF-α and TGF-β/activin pathways, and the evolution of this process can be attenuated by pentoxifylline via downregulation of NF-κB and Smad signals and restoration of the podocyte component of the glomerular filtration barrier.

R-268712, an orally active transforming growth factor-β type I receptor inhibitor, prevents glomerular sclerosis in a Thy1 nephritis model

R-268712 is a novel and specific inhibitor of activin receptor-like kinase 5 (ALK5), a transforming growth factor β (TGF-β) type I receptor. Evaluation of in vitro inhibition indicated that R-268712 is a potent and selective inhibitor of ALK5 with an IC50 of 2.5nM, an approximately 5000-fold more selectivity for ALK5 than p38 mitogen-activated protein kinase (MAPK). Oral administration of R-268712 at doses of 1, 3 and 10mg/kg also inhibited the development of renal fibrosis in a dose-dependent manner in a unilateral ureteral obstruction (UUO) model. Additionally, we evaluated the efficacy of R-268712 in a heminephrectomized anti-Thy1 glomerulonephritis model at doses of 0.3 and 1mg/kg. R-268712 reduced proteinuria and glomerulosclerosis significantly with improvement of renal function. Collectively, these results suggested that R-268712 and other ALK5 inhibitors could suppress glomerulonephritis as well as glomerulosclerosis by an inhibitory mechanism that involves suppression of TGF-β signaling.

ACE Inhibition in Anti-Thy1 Glomerulonephritis Limits Proteinuria but Does Not Improve Renal Function and Structural Remodeling

Background/Aims: ACE inhibitor (ACE-I) treatment effectively inhibits proteinuria and ameliorates the course of various renal diseases. In experimental glomerulonephritis, however, angiotensin II (AngII) infusion has also been shown to be renoprotective. We evaluated the long-term (28 days) course of anti-Thy1 glomerulonephritis in animals with suppressed AngII formation by ACE-I treatment. Methods: Brown Norway rats received perindopril (2.8 mg/kg/day, n = 12), dihydropyridine calcium-antagonist amlodipine (Ca-A; 13 mg/kg/day, n = 6) or were left untreated (n = 14). All animals were monitored for blood pressure, proteinuria, and creatinine clearance after anti-Thy1 injection. Renal histology was assessed at day 7 and 28. Results: Systolic blood pressure was equally reduced by ACE-I and Ca-A treatment. AngII suppression prevented development of proteinuria, but did not protect against glomerular microaneurysm formation or reduction in creatinine clearance. After resolution of the microaneurysms, animals with suppressed AngII production showed a modest increase in glomerulosclerosis and vasculopathic thickening of intrarenal vessels. Conclusions: In anti-Thy1 glomerulonephritis, suppression of AngII formation does not protect against the induction of glomerular damage and is associated with mild aggravation of adverse renal fibrotic remodeling. Proteinuria, however, is effectively prevented by ACE-I treatment. Ca-A treatment did not affect the course of glomerulonephritis, indicating that ACE-I effects are blood pressure independent.

Change of MAX Interactor 1 Expression in an Anti-Thy1 Nephritis Model and its Effect on Mesangial Cell Proliferation

Background/Aims: During the disease process of mesangial proliferative glomerulonephritis, the expression of various factors that influence mesangial proliferation is altered. MAX interactor 1 (Mxi1) antagonizes the transcription factor Myc and is believed to be a tumor suppressor. However, no studies have investigated its effect on mesangial cell proliferation. Methods: To investigate the effect of Mxi1 on renal mesangial cell proliferation, we established a classic rat anti-Thy1 mesangial proliferative glomerulonephritis model. Mesangial proliferation was estimated by immunohistochemical analysis of Ki67. Mxi1 expression at each time point was assessed by real-time RT-PCR and Western blot analyses. Furthermore, we altered the expression level of Mxi1 by a plasmid and siRNA to detect its effect on rat mesangial cell proliferation in vitro. Results: Mxi1 expression decreased significantly during the proliferative period of anti-Thy1 nephritis model and then gradually increased as proliferation declined, indicating that Mxi1 may be linked to mesangial cell proliferation. Upregulation of Mxi1 expression via plasmid transfection in vitro reduced the expression of the positive-acting cell cycle regulatory proteins cyclin B1, cyclin D1, cyclin E, CDC2 and CDK2; significantly reduced mesangial cell proliferation; reduced the percentage of S phase cells; and increased the percentage of G2/M phase cells. Inhibition of Mxi1 expression by siRNA in vitro produced the opposite effects: increased expression of cyclin B1, cyclin D1, cyclin E, CDC2 and CDK2; markedly increased cell proliferation; higher percentage of S phase cells; and dramatically lower percentage of G2/M phase cells. Transcription factor c-myc protein expression showed no obvious difference after Mxi1 plasmid and siRNA transfection. The expressions of cell cycle regulatory proteins mentioned above were negative correlated with Mxi1 expression in anti-Thy1 nephritis model. Conclusion: These results suggest that Mxi1 expression levels were inversely correlated with proliferation in anti-Thy1 nephritis rats and it may influence cell cycle progression and thus the rate of mesangial cell proliferation by regulating the expression of c-myc target cell cycle regulatory proteins.

Decreased renal corin expression contributes to sodium retention in proteinuric kidney diseases

Patients with proteinuric kidney diseases often have symptoms of salt and water retention. It has been hypothesized that dysregulated sodium absorption is due to increased proteolytic cleavage of epithelial sodium channels (ENaCs) and increased Na,K-ATPase expression. Microarray analysis identified a reduction in kidney corin mRNA expression in rat models of puromycin aminonucleoside-induced nephrotic syndrome and acute anti-Thy1 glomerulonephritis (GN). As atrial natriuretic peptide (ANP) resistance is a mechanism accounting for volume retention, we analyzed the renal expression and function of corin; a type II transmembrane serine protease that converts pro-ANP to active ANP. Immunohistochemical analysis found that corin colocalized with ANP. The nephrotic and glomerulonephritic models exhibited concomitant increased pro-ANP and decreased ANP protein levels in the kidney consistent with low amounts of corin. Importantly, kidneys from corin knockout mice had increased amounts of renal β-ENaC and its activators, phosphodiesterase (PDE) 5 and protein kinase G II, when compared to wild-type mice. A similar expression profile was also found in cell culture suggesting the increase in PDE5 and kinase G II could account for the increase in β-ENaC seen in nephrotic syndrome and GN. Thus, we suggest that corin might be involved in the salt retention seen in glomerular diseases.

Effects of Adrenomedullin on the Glomerular Adrenomedullin System in a Rat Model of Anti-Thy1 Glomerulonephritis

Background: Adrenomedullin (ADM) has antiproliferative effects on glomerular mesangial cells. The study was performed to determine changes in glomerular gene expression of the ADM system by ADM treatment in anti-Thy1 glomerulonephritis (GN). Methods: GN in rats was induced by injecting anti-Thy-1 antibody. To show the effect of ADM treatment, rats received ADM from day 3 to day 6 of GN. Supplemental rats were sacrificed on day 3, 7 and 14 of GN to show the expression pattern of adrenomedullin and its receptors. Glomeruli were prepared by sieving or laser-assisted microdissection. Expression of ADM, calcitonin receptor-like receptor (CLR), receptor activity-modifying proteins (RAMP) 1–3, CD34, Thy1 and nephrin was analyzed using real-time PCR. Results: During GN a reduction of CLR and RAMP 2 + 3 expressions was detected on days 3, 7 and 14, while RAMP 1 rose. ADM mRNA decreased on days 3 and 7. Thy1 expression as a surrogate of mesangial cell number was downregulated during GN. A significant reduction of CD34 expression, as a surrogate for endothelial cell number, was detected on day 7. A tendency towards reduction of nephrin gene expression, as a surrogate for number of podocytes, was seen. The administration of ADM during GN did not change the expression on Thy1, CD34 or nephrin. The results were similar for microdissected and sieved glomeruli. In ADM-treated GN animals ADM gene expression rose compared to untreated GN animals on day 6. These effects were detected both in sieved and microdissected glomeruli. ADM administration did not change the expression of the receptors. Conclusion: The downregulation of adrenomedullin during GN at the gene level can be improved by ADM application.

Thrombospondin-2 therapy ameliorates experimental glomerulonephritis via inhibition of cell proliferation, inflammation, and TGF-β activation

We recently identified thrombospondin-2 (TSP-2) as an endogenous regulator of matrix remodelling and inflammation in experimental kidney disease by studying TSP-2-deficient mice. In this study, we asked whether systemic TSP-2 overexpression via thigh muscle transfection is able to ameliorate the time course of the anti-Thy1 glomerulonephritis model. After induction of anti-Thy1 nephritis, rats were transfected either with an overexpression plasmid for TSP-2 or lacZ as a control. Biopsies, urine, and blood samples were taken on days 1, 3, and 6 after disease induction. Muscular overexpression of TSP-2 reduced glomerular transforming growth factor (TGF)-beta activation and glomerular extracellular matrix formation as determined by collagen IV and fibronectin. In addition, activation of mesangial cells to the myofibroblast-like phenotype was also significantly decreased in TSP-2-overexpressing animals. TSP-2 overexpression inhibited both glomerular endothelial and mesangial cell proliferation, resulting in a reduced glomerular cell number and glomerular tuft area. The inflammatory response, as monitored by T cells and antigen-presenting cells, was reduced significantly by TSP-2 overexpression, but influx of macrophages was unchanged. These data demonstrate TSP-2 as a potential therapeutic agent to inhibit the glomerular proliferative and inflammatory response as well as TGF-beta activation and extracellular matrix accumulation in experimental mesangial proliferative glomerulonephritis.

NO Signaling Through cGMP in Renal Tissue Fibrosis and Beyond: Key Pathway and Novel Therapeutic Target

Nitric oxide (NO) produced by endothelial NO synthase (NOS) in low concentrations is a unique messenger molecule with key homeostatic functions concerning the prevention of pathological vascular and tissue changes such as increases in blood pressure, platelet degranulation, mononuclear cell infiltration, cell proliferation and extracellular matrix protein accumulation. This is in contrast to high levels of NO derived from inducible NOS which act as detrimental effector molecules and free radicals in immune response. Deficiency in NO's protective signaling actions is a major characteristic in numerous experimental and human disease situations. The main function of the NO signaling pathway is activation of the soluble guanylate cyclase (sGC) enzyme with subsequent generation of cyclic guanosine monophosphate (cGMP) as a second messenger and downstream mediator. In the past, attempts to overcome deficiency in endothelial NO effects were focused primarily on increasing the supply with the NO precursor L-arginine or on the use of directly NO-releasing compounds. The clinical impact of these strategies, however, was rather limited. Recent state-of-the-art studies have revealed that NO signaling is highly regulated at the transcriptional level and that deficiency in NO signaling correlates closely with pathological changes. In parallel efforts, novel pharmacological compounds which specifically enhance NO/cGMP signaling have been developed and have demonstrated remarkable efficacy in experimental disease settings. In this review, we summarize the current state of knowledge on the impairment of NO/cGMP signaling and about its pharmacological stimulation. In the first part, experimental renal fibrosis, i.e. the tandem rat model of acute anti-thy1 glomerulonephritis and progressive anti-thy1 renal fibrosis will serve as a paradigm for introducing this new and exciting field. In the second part, we will address the most recent findings on NO signaling in non-renal diseases. Together, these results point out that deficiency in NO/cGMP is a common key pathway as well as a novel therapeutic target in a number of diseases.

Activation of the nitric oxide-cGMP signaling pathway precedes resolution in acute anti-thy1 glomerulonephritis

Activation of the nitric oxide-cGMP signaling pathway precedes resolution in acute anti-thy1 glomerulonephritis

Stimulation of soluble guanylate cyclase slows progression in anti-thy1-induced chronic glomerulosclerosis

A critical role of soluble guanylate cyclase and nitric oxide-dependent cyclic 3',5'-guanosine monophosphate (cGMP) production for glomerular matrix expansion has recently been documented in a rat model of acute anti-thy1 glomerulonephritis. The present study analyzes the renal activity of the nitric oxide-cGMP signaling cascade in and the effect of the specific soluble guanylate cyclase stimulator Bay 41-2272 on a progressive model of anti-thy1-induced chronic glomerulosclerosis. Anti-thy1 glomerulosclerosis was induced by injection of anti-thy1 antibody into uninephrectomized rats. One week after disease induction, animals were randomly assigned to chronic glomerulosclerosis, chronic glomerulosclerosis plus Bay 41-2272 (10 mg/kg body weight/day) or chronic glomerulosclerosis plus hydralazine (15 mg/kg body weight/day). In week 16, analysis included effects on systolic blood pressure, proteinuria, kidney function, glomerular and tubulointerstitial matrix protein accumulation, expression of transforming growth factor-beta1 (TGF-beta1), fibronectin and plasminogen activator inhibitor type 1 (PAI-1), macrophage infiltration, cell proliferation, basal and nitric oxide-stimulated cGMP production as well as tubulointerstitial mRNA expression of alpha 1 and beta 1 soluble guanylate cyclase. The moderately elevated systolic blood pressure seen in the chronic glomerulosclerosis group was comparably decreased by both treatments. Compared to normal controls, soluble guanylate cyclase mRNA expression and nitric oxide-stimulated cGMP production were up-regulated in the tubulointerstitium of the untreated chronic glomerulosclerosis animals, while its activity was decreased in glomeruli. Bay 41-2272 treatment enhanced glomerular and tubulointerstitial nitric oxide-cGMP signaling significantly. This went along with markedly reduced glomerular and tubulointerstitial macrophage infiltration, number of proliferating cells, matrix expression and accumulation, as well as improved kidney function. In contrast, hydralazine therapy did not significantly affect renal nitric oxide-cGMP signaling, macrophage number, cell proliferation, matrix protein expression and accumulation. Glomerular and tubulointerstitial soluble guanylate cyclase activity are discordantly altered in anti-thy1-induced chronic glomerulosclerosis. Stimulation of soluble guanylate cyclase signaling by Bay 41-2272 limits the progressive course of this model toward tubulointerstitial fibrosis and impaired renal function at least in part in a blood pressure-independent manner. The results suggest that soluble guanylate cyclase activation counteracts fibrosis and progression in chronic renal disease.

Role of soluble guanylate cyclase in progressive renal fibrosis

Pathological expansion of extracellular matrix proteins is a hallmark of acute and chronic kidney diseases. In both, matrix accumulation results from an orchestrated overexpression of the key profibrotic cytokine transforming growth factor(TGF)-β and subsequent increases in matrix protein production and decreases in matrix protein degradation. A critical role of soluble guanylate cyclase (sGC) and nitric oxide(NO)-dependent cGMP production for glomerular matrix expansion has recently been documented in acute anti-thy1 glomerulonephritis, a rat model of short-term acute, reversible glomerular TGF-β overexpression and matrix protein accumulation [1]. Subsequently, we have analyzed the renal activity of the NOcGMP signaling cascade in and the effect of the specific sGC stimulator Bay 41-2272 on anti-thy1-induced chronic glomerulosclerosis (cGS), a long-term rat model with progressive fibrosis of the whole organ and progressive renal insufficiency [2].

Glomerular activin A overexpression is linked to fibrosis in anti-Thy1 glomerulonephritis

Activin A, a member of the transforming growth factor-beta (TGF-beta) superfamily of proteins, shares many biological features with the pro-fibrotic cytokine TGF-beta1, which is primarily responsible for the accumulation of extracellular matrix proteins in renal disease. This study was designed to identify regulators of activin A production in glomerular mesangial cells and test if activin A acts as a pro-fibrotic cytokine in mesangial cells. The effect of inflammatory cytokines on activin A production and the effect of exogenous activin A on mediators of fibrosis were analysed in cultured rat mesangial cells. Expression of activin A and of established mediators of fibrosis was analysed in a rat model of glomerular fibrosis (anti-Thy1 glomerulonephritis). In cultured mesangial cells, interleukin-1 and basic fibroblast growth factor, both mediators of glomerular inflammatory injury, dose-dependently increased activin A expression. Incubation with activin A significantly stimulated TGF-beta1, PAI-1 and connective tissue growth factor RNA expression and increased production of extracellular matrix proteins in mesangial cells. In rats with anti-Thy1 glomerulonephritis, expression of glomerular activin A mRNA and protein paralled the expression of TGF-beta and other indices of fibrosis, showing little change from normal on day 1, a marked, 70-fold increase of activin protein production on day 6, and a subsequent decrease at day 12. Antifibrotic therapy with the angiotensin-converting enzyme inhibitor enalapril significantly reduced glomerular activin A production. Taken together, the results of this study link overexpression of activin A to glomerular matrix protein expansion in vivo and in vitro, suggesting that activin A acts as pro-fibrotic cytokine in renal disease.

Expression and activity of soluble guanylate cyclase in injury and repair of anti-thy1 glomerulonephritis

Activation of soluble guanylate cyclase and generation of cyclic 3',5'-guanosine monophosphate (cGMP) is the main signal transducing event of the L-arginine-nitric oxide pathway. The present study analyzes the expression and activity of the nitric oxide-cGMP signaling cascade in and the effect of the specific soluble guanylate cyclase stimulator Bay 41-2272 on the early injury and subsequent repair phase of acute anti-thy1 glomerulonephritis. Anti-thy1 glomerulonephritis was induced by OX-7 antibody injection in rats. In protocol 1 (injury), Bay 41-2272 was given starting 6 days before antibody injection. One day after disease induction, parameters of mesangial cell injury (glomerular cell number and inducible nitric oxide synthesis) were analyzed. In protocol 2 (repair), Bay 41-2272 treatment was started one day after antibody injection. On day 7, parameters of glomerular repair [glomerular matrix score, expression of transforming growth factor (TGF)-beta1, fibronectin, and plasminogen-activator-inhibitor (PAI)-1, infiltration with macrophages and fibrinogen deposition (indicating platelet localization)] were determined. In both protocols, tail bleeding time, systolic blood pressure, plasma cGMP levels, glomerular mRNA expression of endothelial nitric oxide synthase (eNOS), alpha1 and beta1 soluble guanylate cyclase, and basal and nitric oxide-stimulated glomerular cGMP production were analyzed. Bay 41-2272 prolonged bleeding time, reduced blood pressure, and increased plasma cGMP levels in both protocols. In the injury experiment, disease induction increased inducible nitric oxide synthesis and reduced glomerular cell number, while expression and activity of soluble guanylate cyclase was almost completely diminished. Bay 41-2272 did not affect parameters of mesangial cell injury and glomerular soluble guanylate cyclase expression and activity. In the repair protocol, expression and activity of soluble guanylate cyclase was markedly increased by disease. Bay 41-2272 further enhanced soluble guanylate cyclase expression and activity. This went along with significant reductions in proteinuria, glomerular matrix accumulation, expression of TGF-beta1, fibronectin, and PAI-1, macrophage infiltration and fibrinogen deposition as compared to the untreated anti-thy1 animals. Glomerular nitric oxide signaling via cGMP is markedly impaired during injury of anti-thy1 glomerulonephritis, while it is highly up-regulated during subsequent repair. Further pharmacologic soluble guanylate cyclase stimulation limits glomerular TGF-beta overexpression and matrix expansion, suggesting that the soluble guanylate cyclase enzyme represents an important antifibrotic pathway in glomerular disease.

CCAAT/Enhancer-binding protein delta contributes to myofibroblast transdifferentiation and renal disease progression.

Myofibroblasts are pivotal participants in pathologic processes in a wide variety of organs, such as lung, liver, and kidney, by producing several inflammatory cytokines and extracellular matrices. The mechanism by which transdifferentiation from original cell to myofibroblast occurs, however, is still unclear. The expression of smooth muscle alpha-actin (SMalphaA) is the most characteristic feature of myofibroblasts; therefore, it was speculated that any factors that promote SMalphaA expression might be the key to transdifferentiation to myofibroblasts and disease exacerbation. A transcription factor CCAAT/enhancer-binding protein delta (C/EBPdelta) was identified and demonstrated to bind to sequences including the CArG motif from SMalphaA intron 1 and to increase transcriptional activity of this promoter. Expression of SMalphaA and C/EBPdelta in the glomerular area was upregulated in rat anti-Thy1 glomerulonephritis and mouse Habu-venom glomerulonephritis, both of which are models of mesangioproliferative glomerulonephritis. In the latter model, C/EBPdelta knockout mice demonstrated significantly less SMalphaA expression in the glomerular area on day 8 and less renal functional deterioration on day 14, compared with wild-type mice. These data suggest an important role for C/EBPdelta in myofibroblast transdifferentiation and glomerulonephritis exacerbation.

Platelet inhibition limits TGF-β overexpression and matrix expansion after induction of anti-thy1 glomerulonephritis

Although a role of platelets is well established in atherosclerosis, only little is known about their contribution to pathologic renal matrix expansion. The present study analyzes the effect of the platelet inhibitor clopidogrel on the early injury and subsequent repair phase of experimental anti-thy1 glomerulonephritis. In male Sprague-Dawley rats, acute anti-thy1 glomerulonephritis was induced by intravenous injection of OX-7 antibody. In protocol 1 (injury), clopidogrel was given starting 5 days before antibody injection. One day after disease induction, parameters of mesangial cell injury (glomerular cell number, inducible NO synthesis, and macrophage infiltration) were analyzed. In protocol 2 (repair), clopidogrel treatment was started one day after antibody injection. On day 6, parameters of glomerular repair [glomerular matrix score, expression of transforming growth factor (TGF)-beta 1, fibronectin, and plasminogen activator inhibitor (PAI)-1] and thrombosis (aneurysm formation and fibrinogen deposition) were determined. In both protocols, an additional group of rats was treated with the angiotensin-converting enzyme (ACE) inhibitor enalapril. In the injury protocol, platelet inhibition did not affect mesangial cell lysis, glomerular NO production, and macrophage infiltration, while ACE inhibition was protective. In the repair protocol, clopidogrel significantly limited aneurysm formation and fibrinogen deposition, as well as glomerular matrix expansion, TGF-beta 1, fibronectin, and PAI-1 expression. In comparison, enalapril was less effective in preventing glomerular thrombosis, but was significantly superior to clopidogrel in limiting matrix protein expression and accumulation. The present study shows that platelets play a significant role in the sequence from mesangial cell injury to renal matrix expansion in anti-thy1 glomerulonephritis. The results, directly comparing renin-angiotensin-system and platelet inhibition, suggest that platelets contribute less than angiotensin II to TGF-beta overexpression and matrix accumulation in this model of acute glomerular wound repair.

Gene Expression Profile in Experimental Mesangial Proliferative Glomerulonephritis

We analyzed gene expression in rat anti-Thy1 antibody-induced glomerulonephritis by using the cDNA microarray method. Ninety-seven genes that differed by more than 1.5-fold intensity in comparison with the controls were selected. Cluster analysis showed that the expression of genes associated with inflammation reached maximum levels at 24 h, while genes involved in the development of fibrosis increased at 7 days after injection. Microarray analysis of animal disease models may be a powerful approach for understanding the gene expression programs that underlie these disorders.

Expression of CX3CL1/fractalkine by mesangial cells in vitro and in acute anti-Thy1 glomerulonephritis in rats.

Mesangial cells (MCs) can promote glomerular macrophage accumulation in glomerulonephritis through production of a variety of chemokines. This study investigated the potential of MCs to synthesize CX3CL1/fractalkine, a CX3C chemokine, both in vitro and in acute anti-Thy1 glomerulonephritis in rats. Anti-Thy1 glomerulonephritis was induced in Wistar rats by a single injection of mouse anti-rat Thy1.1 antibody intravenously. Glomerular mRNAs for CX3CL1/fractalkine, CCL2/monocyte chemoattractant protein (MCP)-1, and their cognate receptors, CX3CR1 and CCR2, were determined by northern blot analysis or reverse-transcription polymerase chain reaction. CX3CL1/fractalkine mRNA and protein expression in vivo was localized by in situ hybridization and immunohistochemistry. Monocytes/macrophages and activated MCs were detected by immunohistochemistry. Regulation of CX3CL1/fractalkine expression in cultured MCs was determined by northern and western blot analysis. After induction of anti-Thy1 disease, glomerular CX3CL1/fractalkine mRNA was significantly up-regulated, peaking at 2 h and sustaining into day 5 of the nephritis. A corresponding increase in urinary CX3CL1/fractalkine protein was evident after day 1 of the nephritis, but became more prominent during the MC proliferative phase (days 3-5). Meanwhile, induction of glomerular CCL2/MCP-1 mRNA and urinary CCL2/MCP-1 protein occurred within 24 h, and was barely detectable after day 3 of the nephritis. Urinary CCL2/MCP-1, but not CX3CL1/fractalkine, correlated with glomerular macrophage accumulation (r = 0.936, P<0.01) and glomerular CCR2 mRNA expression (r = 0.965, P<0.01). In contrast, only urinary CX3CL1/fractalkine coincided temporally to glomerular mRNA for CX3CR1 (r = 0.809, P < 0.01). Combined in situ hybridization and immunohistochemistry revealed that activated MCs were a major source for CX3CL1/fractalkine mRNA and protein during days 3-5 of the nephritis. Incubation of cultured MCs with tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, platelet-derived growth factor (PDGF)-AB or basic fibroblast growth factor (bFGF) significantly up-regulated CX3CL1/fractalkine mRNA and protein expression. This cytokine- and growth factor-stimulated CX3CL1/fractalkine expression could be abolished by the nuclear factor-kappaB inhibitors, curcumin and MG132. Our data demonstrate that activated MCs are a source for the augmented glomerular CX3CL1/fractalkine expression during the proliferative phase of acute anti-Thy1 glomerulonephritis. Up-regulation of MC CX3CL1/fractalkine by TNF-alpha, IL-1beta, PDGF-AB and bFGF is mediated, at least in part, via the nuclear factor-kappaB signalling pathway. The differential expression of CCL2/MCP-1 and CX3CL1/fractalkine may sequentially recruit distinct subsets of monocytes to the glomerulus during acute anti-Thy1 glomerulonephritis.