Experimental Mesangial Proliferative Glomerulonephritis Research Articles

Activated mesangial cells induce glomerular endothelial cells proliferation in rat anti-Thy-1 nephritis through VEGFA/VEGFR2 and Angpt2/Tie2 pathway.

ObjectivesWe aimed to investigate the underlying mechanism of endothelial cells (ECs) proliferation in anti‐Thy‐1 nephritis.Materials and methodsWe established anti‐Thy‐1 nephritis and co‐culture system to explore the underlying mechanism of ECs proliferation in vivo and in vitro. EdU assay kit was used for measuring cell proliferation. Immunohistochemical staining and immunofluorescence staining were used to detect protein expression. ELISA was used to measure the concentration of protein in serum and medium. RT‐qPCR and Western blot were used to qualify the mRNA and protein expression. siRNA was used to knock down specific protein expression.ResultsIn anti‐Thy‐1 nephritis, ECs proliferation was associated with mesangial cells (MCs)‐derived vascular endothelial growth factor A (VEGFA) and ECs‐derived angiopoietin2 (Angpt2). In vitro co‐culture system activated MCs‐expressed VEGFA to promote vascular endothelial growth factor receptor2 (VEGFR2) activation, Angpt2 expression and ECs proliferation, but inhibit TEK tyrosine kinase (Tie2) phosphorylation. MCs‐derived VEGFA stimulated Angpt2 expression in ECs, which inhibited Tie2 phosphorylation and promoted ECs proliferation. And decline of Tie2 phosphorylation induced ECs proliferation. In anti‐Thy‐1 nephritis, promoting Tie2 phosphorylation could alleviate ECs proliferation.ConclusionsOur study showed that activated MCs promoted ECs proliferation through VEGFA/VEGFR2 and Angpt2/Tie2 pathway in experimental mesangial proliferative glomerulonephritis (MPGN) and in vitro co‐culture system. And enhancing Tie2 phosphorylation could alleviate ECs proliferation, which will provide a new idea for MPGN treatment.

KLF6 Acetylation Promotes Sublytic C5b-9-Induced Production of MCP-1 and RANTES in Experimental Mesangial Proliferative Glomerulonephritis

Rat Thy-1 nephritis (Thy-1N) is an experimental mesangial proliferative glomerulonephritis (MsPGN) for studying human MsPGN. Although sublytic C5b-9 complex formation on glomerular mesangial cells (GMCs) and renal MCP-1 and RANTES production in rats with Thy-1N have been proved, the role and mechanism of MCP-1 or RANTES synthesis in GMCs induced by sublytic C5b-9 are poorly elucidated. In this study, we first found the expression of transcription factor (KLF6), co-activator (KAT7) and chemokines (MCP-1 and RANTES) was all up-regulated both in renal tissue of Thy-1N rats (in vivo) and in sublytic C5b-9-induced GMCs (in vitro). Further in vitro experiments revealed that KLF6 bound to MCP-1 promoter (-297 to -123 nt) and RANTES promoter (-343 to -191 nt), leading to MCP-1 and RANTES gene transcription. Meanwhile, KAT7 also bound to the same region of MCP-1 and RANTES promoter in a KLF6-dependent manner, and KLF6 was acetylated by KAT7 at lysine residue 100, which finally promoted MCP-1 and RANTES expression. Moreover, our in vivo experiments discovered that knockdown of renal KAT7 or KLF6 gene obviously reduced MCP-1 and RANTES production, GMCs proliferation, ECM accumulation, and proteinuria secretion in Thy-1N rats. Collectively, our study indicates that sublytic C5b-9-induced MCP-1 and RANTES synthesis is associated with KAT7-mediated KLF6 acetylation and elevated KLF6 transcriptional activity, which might provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

Metanephric mesenchyme-derived Foxd1+ mesangial precursor cells alleviate mesangial proliferative glomerulonephritis.

Mesangial proliferative glomerulonephritis (MsPGN) is a glomerular disease characterized by the proliferation of mesangial cells and the accumulation of mesangial matrix. No effective treatment is currently able to stop or reverse the disease process. Here, we isolated metanephric mesenchyme-derived Foxd1+ mesangial precursor cells from E13.5 Foxd1Cre; DTRflox double transgenic embryo mice. The Foxd1+ cells showed the cell-specific expression of high levels of Foxd1 without Six2 and manifested specific cell surface markers of mesenchymal stem cells while retaining their differentiation potential. Next, an anti-Thy1 MsPGN rat model was established, and the Foxd1+ cells were injected into the tail veins 24h later. We found that the Foxd1+ cells could improve the pathological changes to the kidney and significantly reduce proteinuria by inhibiting the sonic hedgehog pathway. Moreover, the Foxd1+ cells could inhibit PDGF-BB-induced activation of mesangial cells by secreting multiple cytokines, including hepatocyte growth factor. Our study uncovered the novel function of Foxd1+ mesangial precursor cells in repairing the damaged mesangium, stabilizing the structure and function of mesangial cells, and restoring their therapeutic effect on the MsPGN. KEY MESSAGES: It is viable to isolate highly purified metanephric mesenchyme-derived Foxd1+ mesangial precursor cells using transgenic mice. Foxd1+ cells could alleviate experimental mesangial proliferative glomerulonephritis and PDGF-induced mesangial cell proliferation through a variety of mechanisms including inhibiting the sonic hedgehog pathway and secreting multiple cytokines. As the progenitor cells of mesangial cells, Foxd1+ cells could stabilize the structure and function of mesangial cells that had undergone pathological changes.

Epidermal growth factor receptor inhibition with erlotinib ameliorates anti-Thy 1.1-induced experimental glomerulonephritis.

Mesangial proliferative glomerulonephritis is a common glomerular disorder that may lead to end-stage renal disease. Epidermal growth factor (EGF) plays an important role in the regulation of cell growth, proliferation, and differentiation and in the pathology of various renal diseases. Erlotinib is a novel, oral, highly selective tyrosine kinase inhibitor of the EGF receptor. It is clinically used to treat non-small cell lung and pancreatic cancers. Here, we investigated the effect of erlotinib on the progression of mesangioproliferative glomerulonephritis in an experimental model. Mesangial glomerulonephritis was induced with anti-rat Thy-1.1 antibody in male Wistar rats weighing 150-160g. Rats were treated with erlotinib (10mg/kg/day p.o.) or vehicle only (polyethylene glycol). Native Wistar rat kidneys were used as histological controls. Serum creatinine levels were measured at day 7. Kidneys were harvested 7days after antibody administration for histology. Native controls showed no histological signs of glomerular pathology. In the vehicle group, intense glomerular inflammation developed after 7days and prominent mesangial cell proliferation and glomerular matrix accumulation was seen. Erlotinib was well tolerated and there were no adverse effects during the follow-up period. Erlotinib significantly prevented progression of the glomerular inflammatory response and glomerular mesangial cell proliferation as well as matrix accumulation when compared with the vehicle group. Erlotinib also preserved renal function. These results indicate that erlotinib prevents the early events of experimental mesangial proliferative glomerulonephritis. Therefore, inhibition of the EGF receptor with erlotinib could prevent the progression of glomerulonephritis also in clinical nephrology.

Protein kinase C ß inhibition ameliorates experimental mesangial proliferative glomerulonephritis

Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic nephropathy where therapy targeting the β isoform of this enzyme has been examined. However, PKC-β is also increased in various forms of human glomerulonephritis, including IgA nephropathy. Accordingly, we sought to examine the effects of PKC-β inhibition in the Thy1.1 model of mesangial proliferative glomerulonephritis. Following administration of monoclonal OX-7, anti-rat Thy-1.1 antibody, Male Wistar rats were randomized to receive either the PKC-β inhibitor, ruboxistaurin (10 mg/kg per day in chow) or vehicle. Animals were then examined 6 days later. PKC-β inhibition was associated with reductions in mesangial cellularity and extracellular matrix deposition. Proteinuria was, however, unaffected. In vitro, PKC-β inhibition showed modest, dose-dependent reductions in mesangial cell (3) H-thymidine and (3) H-proline incorporations, indices of cell proliferation and collagen synthesis, respectively. The amelioration of the pathological findings of experimental mesangial proliferative glomerulonephritis by PKC-β inhibition suggests the potential clinical utility of this approach as a therapeutic strategy in non-diabetic glomerular disease.

Activation of Src Mediates PDGF-Induced Smad1 Phosphorylation and Contributes to the Progression of Glomerulosclerosis in Glomerulonephritis

Platelet-derived growth factor (PDGF) plays critical roles in mesangial cell (MC) proliferation in mesangial proliferative glomerulonephritis. We showed previously that Smad1 contributes to PDGF-dependent proliferation of MCs, but the mechanism by which Smad1 is activated by PDGF is not precisely known. Here we examined the role of c-Src tyrosine kinase in the proliferative change of MCs. Experimental mesangial proliferative glomerulonephritis (Thy1 GN) was induced by a single intravenous injection of anti-rat Thy-1.1 monoclonal antibody. In Thy1 GN, MC proliferation and type IV collagen (Col4) expression peaked on day 6. Immunohistochemical staining for the expression of phospho-Src (pSrc), phospho-Smad1 (pSmad1), Col4, and smooth muscle α-actin (SMA) revealed that the activation of c-Src and Smad1 signals in glomeruli peaked on day 6, consistent with the peak of mesangial proliferation. When treated with PP2, a Src inhibitor, both mesangial proliferation and sclerosis were significantly reduced. PP2 administration also significantly reduced pSmad1, Col4, and SMA expression. PDGF induced Col4 synthesis in association with increased expression of pSrc and pSmad1 in cultured MCs. In addition, PP2 reduced Col4 synthesis along with decreased pSrc and pSmad1 protein expression in vitro. Moreover, the addition of siRNA against c-Src significantly reduced the phosphorylation of Smad1 and the overproduction of Col4. These results provide new evidence that the activation of Src/Smad1 signaling pathway plays a key role in the development of glomerulosclerosis in experimental glomerulonephritis.

Effects of TJN-598, a new selective phosphodiesterase type IV inhibitor on anti-Thy1 nephritis in rats

Phosphodiesterase type IV (PDEIV) plays an important role in the immune response and inflammation. However, it is well known that classical PDEIV inhibitors have systemic side effects, so the clinical and chronic use of these agents as therapy for glomerulonephritis is difficult. This study was performed to elucidate the anti-nephritic effects of TJN-598, a new chemical compound derived from herbal components, on experimental mesangial proliferative glomerulonephritis. We first examined the effects of TJN-598 and captopril on mesangial expansion induced by anti-Thy1 serum in rats. Second, to investigate the effects of TJN-598 and rolipram, which are typical PDEIV inhibitors, on the production of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1, glomeruli were isolated from rats with anti-Thy1 nephritis and incubated with the test drugs in vitro for 48 h. Treatment with TJN-598 prevented an increase in the mesangial area/total glomerular area, in the number of cells in the glomerular cross section and matrix index. TJN-598 also inhibited the increases in the expression of α-smooth muscle actin, the TGF-β1-positive area, in the number of ED-1 positive cells and proliferating cell nuclear antigen-positive cells in the glomeruli. Furthermore, administration of TJN-598 inhibited increases in the levels of TGF-β1 protein derived from glomeruli with anti-Thy-1 nephritis. The addition of both TJN-598 and rolipram to the culture supernatant inhibited both increased expression of TGF-β1 and increases in levels of TNF-α in glomeruli isolated from rats with anti-Thy1 nephritis in a dose-dependent manner. These results suggest that TJN-598, a PDEIV inhibitor, is effective against expansion of mesangial cells, via the suppression of secretion of TGF-β1 and TNF-α from inflamed glomeruli.

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.

Transactivation of RON receptor tyrosine kinase by interaction with PDGF receptor β during steady-state growth of human mesangial cells

Although it is well known that platelet-derived growth factor (PDGF) causes mesangial cell proliferation (presumably contributing to progression of glomerular disease), targeted inhibition of the PDGF receptor system has shown only limited efficacy against glomerular diseases. To examine whether this discrepancy is due to the involvement of other pathways, we used phosphorylated receptor tyrosine kinase arrays and found that RON (recepteur d'origine nantais) was phosphorylated while the PDGF receptor was dephosphorylated (thus inactive) in human mesangial cells (HMCs) at the time of cell cycle entry. Further, RON remained active during steady-state growth. Activation of RON was independent of its canonical ligand, macrophage-stimulating protein, but was mediated by transactivation from the PDGF-engaged PDGF receptor. Following stimulation with PDGF we found that the two receptors physically interacted. Knockdown of RON by siRNA increased the number of apoptotic cells without affecting the rate of DNA synthesis, suggesting that RON has anti-apoptotic functions. Immunohistochemical analysis found phosphorylated RON in glomerular lesions of patients with IgA nephropathy but not those with minimal change nephrotic syndrome, a disease not associated with mesangial proliferation. These results suggest that RON is involved in mesangial cell proliferation under both physiological and pathological conditions, and may be a relevant target for therapeutic intervention.

C3G overexpression in glomerular epithelial cells during anti-GBM-induced glomerulonephritis

The guanine nucleotide exchange factor C3G, along with the CrkII adaptor protein, mediates GTP activation of the small GTPase proteins Rap1 and R-Ras, facilitating their activation of downstream signaling pathways, which had been found to be important in the pathogenesis of glomerulonephritis. We found that expression of C3G protein was upregulated in glomerular epithelial cells in an experimental model of accelerated anti-GBM antibody-induced glomerulonephritis expression. To determine the consequence of its increased expression, we transfected C3G (using adenoviral constructs) into cultured glomerular epithelial cells and measured the activated forms (i.e., GTP-bound) forms of Rap1 and R-Ras. Activation of Rap1 was not affected by C3G; however, the basal level of GTP-bound R-Ras was decreased. Further, C3G over-expression enhanced the activation of R-Ras in response to endothelin. Overexpression of C3G also led to a significant reduction in glomerular epithelial cell spreading and decreased the cells' E-cadherin expression and augmented their migration. We found that C3G was overexpressed in accelerated anti-GBM antibody-induced glomerulonephritis and suggest that this modulates glomerular epithelial cell morphology and behavior.

Tranilast Ameliorates Experimental Mesangial Proliferative Glomerulonephritis

Background: Immunoglobulin A nephropathy and its related animal model Thy1.1 nephritis are characterized by mesangial hypercellularity, extracellular matrix expansion and overexpression of the proproliferative and profibrotic growth factors, platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β). Tranilast [n-(3,4-dimethoxycinnamoyl) anthranilic acid] has been shown to block the actions of PDGF and TGF-β. Methods: Experimental mesangial proliferative glomerulonephritis was induced in male Wistar rats with a monoclonal anti-rat Thy-1.1 antibody (OX-7) with rats randomized to receive either tranilast 400 mg/kg/day or vehicle control. Collagen synthesis and proliferation of cultured mesangial cells following incubation with PDGF (50 ng/ml) and tranilast (10–100 µM) was determined by ³H-proline and ³H-thymidine incorporation, respectively. Results: Tranilast treatment resulted in a significant reduction in mesangial cell proliferation, macrophage infiltration, activated (α-smooth muscle actin positive) mesangial cells, glomerular type IV collagen deposition and proteinuria compared to control rats. Also, PDGF stimulation of mesangial cell ³H-thymidine and ³H-proline incorporation was reduced by tranilast in a dose-dependent manner. Conclusion: These in vitro data and the amelioration of the pathological findings of experimental mesangial proliferative glomerulonephritis by tranilast suggest the potential clinical utility of this approach as a therapeutic strategy in mesangial proliferative conditions such as immunoglobulin A nephropathy.

The mTOR Inhibitor Everolimus Induces Proteinuria and Renal Deterioration in the Remnant Kidney Model in the Rat

Under certain circumstances the nonnephrotoxic, antiproliferative, immunosuppressive mammalian target of rapamycin (mTOR) inhibitors can cause renal deterioration and proteinuria after conversion from calcineurin inhibitors, especially in long-term renal transplant patients with low glomerular filtration rates. The mTOR inhibitors also show an impaired glomerular healing reaction during acute renal injury in experimental mesangial proliferative glomerulonephritis. In this study, everolimus treatment was investigated in a low nephron number model, the remnant kidney model in rats. The remnant kidney model was induced by uninephrectomy and infarction of 2/3 of the remaining kidney in 31 male Sprague-Dawley rats. Three days after disease induction, rats were randomly treated either with everolimus or vehicle. Changes in progression of renal disease were investigated by immunohistochemistry on days 22 and 38 after disease induction. In the remnant kidney model, everolimus treatment worsened chronic disease progression as assessed by increased proteinuria, glomerulosclerosis, interstitial fibrosis, glomerular inflammation as well as decreased creatinine-clearance. This result was due to a markedly increased fraction of glomeruli with a defective glomerular architecture in the everolimus group. Everolimus apparently inhibited the chronic glomerular repair reaction via inhibition of the proliferative but not apoptotic activity of the glomerular endothelial and mesangial cells, which was associated with reduced glomerular vascular endothelial growth factor mRNA and protein. In contrast, the fraction of glomeruli with an intact glomerular architecture within the everolimus group showed clearly less glomerular enlargement compared to vehicle-treated nephrectomy rats. This study demonstrates potential mechanisms of mTOR inhibitor induced renal deterioration and proteinuria in the low nephron number remnant kidney model.

Increased mesangial cGMP levels prevent mesangial cell proliferation and matrix expansion in experimental glomerulonephritis

Introduction Mesangial cell proliferation is a prominent finding of various human diseases like membranoproliferative glomerulonephritis, lupus nephritis, IgAand diabetic nephropathy. Despite increasing knowledge on disease mechanisms therapeutical options are very limited. The NO-cGMP pathway has been linked with cell proliferation and matrix expansion. In our studies, we investigated the effects of direct sGC stimulation using BAY 41–2272 and PDE 5 inhibition using vardenafil on the early phase of experimental mesangial proliferative glomerulonephritis (anti-Thy1 nephritis) in the rat.

Activation of STAT3/Smad1 Is a Key Signaling Pathway for Progression to Glomerulosclerosis in Experimental Glomerulonephritis

Mesangial cell proliferation is a significant event in the development of progressive glomerular injuries. However, the issue of how cell proliferation is involved in the development of glomerulosclerosis is unclear. Recently, we showed that the overexpression of type IV collagen (Col IV), a major component of mesangial extracellular matrix, is transcriptionally regulated by Smad1 in diabetic glomerulosclerosis. In this study, we have demonstrated the effect of the administration of an anti-platelet-derived growth factor (PDGF) beta-receptor antibody (APB5) blocking activation by the PDGF-B chain on rat glomerulonephritis and have examined the signaling pathways that regulate both glomerular cell proliferation and glomerulosclerosis in vivo and in vitro. Experimental mesangial proliferative glomerulonephritis (Thy1 GN) was induced by a single intravenous injection of anti-rat Thy-1.1 monoclonal antibody. In Thy1 GN, mesangial cell proliferation and the expression of Col IV peaked at day 6. Immunohistochemical staining for the expression of Smad1, phospho-Smad1 (pSmad1), and phospho-STAT3 (pSTAT3) revealed that the peak for glomerular Smad1 expression occurred at day 6, consistent with the peak for mesangial proliferation. The expression of pSmad1 was up-regulated at day 1, and the peak for glomerular pSmad1 expression occurred at day 4 of the disease. When treated with APB5, both mesangial proliferation and sclerosis were reduced significantly. The expression of Smad1, pSmad1, and pSTAT3 was also significantly reduced by the administration of APB5. PDGF induced both mesangial cell replication and Col IV synthesis in association with an increased expression of pSTAT3 and pSmad1 on cultured mesangial cells. In addition, APB5 reduced mesangial cell proliferation in association with decreased pSmad1, pSTAT3, and Col IV protein expressions in vitro. The introduction of dominant negative STAT3 significantly decreased the expression of Col IV in cultured mesangial cells. These data suggest that the activation of STAT3 and Smad1 participates in the developing process of glomerulosclerosis in experimental glomerulonephritis.

Sulfated Hyaluronic Acid, a Potential Selectin Inhibitor, Ameliorates Experimentally Induced Crescentic Glomerulonephritis

Background/Aims: Sulfated polysaccharides are known to interfere with the binding of selectins and their ligands. Recently, we demonstrated that sulfated hyaluronic acid (SHA), a synthetic sulfated polysaccharide, showed preventive and therapeutic effects on experimental mesangial proliferative glomerulonephritis. Here we evaluated the protective potential of SHA on crescentic glomerulonephritis, using nephrotoxic serum (NTS) nephritis in Wistar-Kyoto (WKY) rats. Methods: Crescentic glomerulonephritis was induced by injection of NTS in WKY rats. Rats subsequently received intraperitoneal administration of SHA (0.5 or 1.5 mg/kg/day) or non-sulfated hyaluronic acid (HA) (1.5 mg/kg/day) for 14 days. The urinary protein excretion was measured, and expression of selectins, intraglomerular leukocytes and crescent formation were examined by immunohistochemistry. In addition, we examined the urinary protein excretion of SHA (1.5 mg/kg/day) administered from day 7 after the induction of crescentic glomerulonephritis. Results: The expression of P-selectin was increased in the glomerulus of crescentic glomerulonephritis. SHA reduced proteinuria, macrophage infiltration, and crescent formation in a dose-dependent manner. Furthermore, administration of SHA (1.5 mg/kg/day) from day 7 also reduced the urinary protein excretion on day 14 compared with that in saline and HA group. Conclusion: Our results suggest that SHA inhibits intraglomerular infiltration of macrophages, and prevents progression of experimental crescentic glomerulonephritis. Sulfated polysaccharides might be beneficial for the treatment of crescentic glomerulonephritis.

The Rapamycin derivative RAD inhibits mesangial cell migration through the CDK-inhibitor p27KIP1

The link between mesangial cell (MC) proliferation and migration during glomerular repair in the experimental mesangial proliferative glomerulonephritis suggests that cell cycle regulation and cell migration require similar pathways, such as cell cycle proteins. The immunosuppressant RAD inhibits mesangial cell (MC) proliferation via G1/S arrest. Moreover, RAD dramatically impairs glomerular healing in the anti-Thy1 model. We tested the hypothesis that RAD alters MC migration in vitro and that this effect was mediated by the CDK-inhibitors p21CIP1 and p27KIP1. Using a modified Boyden chamber in vitro migration assay, our results showed that RAD dose dependently (1–50 nM) inhibited fibronectin-induced chemotaxis in wild-type (wt) MC. RAD treatment prevented the decrease in p27KIP1 induced by mitogenic growth factors, but had no effect on p21CIP1 by Western blot analysis. The antimigratory effect of RAD in wt MC was substantially dependent on p27KIP1, but not p21CIP1, since the inhibitory effects of 1–10 nM RAD on MC migration were similar in p21CIP1 deficient and wild-type MC. The effect of RAD on MC migration was also examined in the anti-Thy1 model by BrdU-labeling of proliferating MC on day 3 that typically repopulate the glomerulus from the hilus. A control biopsy on day 3 was taken to define the starting point prior to the initiation of RAD (3 mg/kg or placebo). MC migration was determined on day 7 by measuring the distances of BrdU-labeled MC (OX-7+/BrdU+cells) from the glomerular hilus using computerized morphometry. RAD significantly reduced the migratory response of BrdU-labeled MC compared to controls.We conclude that the immunosuppressant RAD effectively inhibits MC migration in vivo and in vitro thereby limiting the normal glomerular repair process after severe injury. Moreover, RAD-induced inhibition of MC migration in vitro is partially mediated by the CDK-inhibitor p27KIP1, but not p21CIP1.

Antisense Oligonucleotides Against Thrombospondin-1 Inhibit Activation of TGF-β in Fibrotic Renal Disease in the Rat in Vivo

Specific treatment of chronic progressive renal disease is very limited. TGF-beta, considered as the major cytokine causing tissue scarring, must be activated extracellularly before it can bind to its receptors. Thrombospondin-1 (TSP1) has been identified as an activator of latent TGF-beta in in vitro systems and in pancreas and lung homeostasis in mouse pups in vivo, but whether this is also true in inflammatory fibrotic disease is unknown. We examined a rat model of mesangial proliferative glomerulonephritis, where TGF-beta has been demonstrated to mediate renal fibrosis. In this study, antisense phosphorothioate oligonucleotides against TSP1 were successfully transferred into almost all glomeruli of perfused diseased kidneys and markedly inhibited de novo synthesis of TSP1. This effect was accompanied by decreased activation but not expression of TGF-beta and by the inhibition of the TGF-beta-dependent smad-signaling pathway, as well as transcription of TGF-beta target genes such as EDA-fibronectin, resulting in a markedly suppressed accumulation of extracellular matrix. In sharp contrast, neither glomerular cell proliferation nor influx of macrophages was affected by this therapy in experimental mesangial proliferative glomerulonephritis. These results demonstrate that TSP1 is the major endogenous activator of TGF-beta in experimental inflammatory kidney disease.

Pentoxifylline attenuated the renal disease progression in rats with remnant kidney.

Previous studies have reported that pentoxifylline, a phosphodiesterase inhibitor, attenuates experimental mesangial proliferative glomerulonephritis. This study hypothesized that pentoxifylline could also attenuate the renal disease progression in rats with remnant kidney. After 5/6 subtotal nephrectomy, rats developed progressively elevated proteinuria and plasma creatinine, glomerulosclerosis, interstitial inflammation, and fibrosis, all of which were attenuated by 40 to 60% by pentoxifylline. However, the elevated BP was not changed by pentoxifylline. Pentoxifylline reduced the upregulation of monocyte chemoattractant protein-1 gene by 60% in the cortex of remnant kidney, as well as in a dose-dependent manner in the albumin- or angiotensin II-stimulated proximal tubular cells. It also reduced the upregulation of mitogenic and profibrogenic genes by 50%, including platelet-derived growth factor, fibroblast growth factor-2, transforming growth factor-beta(1), connective tissue growth factor, and types I and III collagen in the cortex of remnant kidney. Furthermore, pentoxifylline was found to decrease the numbers of interstitial myofibroblasts by 60% in the cortex of remnant kidney and suppress the proliferation of cultured interstitial fibroblasts. It also reduced the angiotensin II-induced or transforming growth factor-beta(1)-induced expression of connective tissue growth factor gene in cultured fibroblasts and mesangial cells. Combining pentoxifylline with an angiotensin-converting enzyme inhibitor, cilazapril, almost completely attenuated the renal disease progression in rats with remnant kidney. In conclusion, pentoxifylline alone can attenuate the chronic renal disease progression. Its combination with cilazapril has the potential to prevent the renal disease progression almost completely.

P2 receptor antagonist PPADS inhibits mesangial cell proliferation in experimental mesangial proliferative glomerulonephritis

Although extracellular nucleotides have been shown to confer mitogenic effects in cultured rat mesangial cells through activation of purinergic P2 receptors (P2Y receptors), thus far the in vivo relevance of these findings is unclear. Virtually all cells and in particular the dense granules of platelets contain high levels of nucleotides that are released upon cell injury or platelet aggregation. In experimental mesangial proliferative glomerulonephritis in the rat (anti-Thy1 model), mesangiolysis and glomerular platelet aggregation are followed by a pronounced mesangial cell (MC) proliferative response leading to glomerular hypercellularity. Therefore, we examined the role of extracellular nucleotides and their corresponding receptors in nucleotide-stimulated cultured mesangial cells and in inflammatory glomerular disease using the P2 receptor antagonist PPADS. The effects of PPADS on nucleotide- or fetal calf serum (FCS)-stimulated proliferation of cultured MC were measured by cell counting and [3H]thymidine incorporation assay. After induction of the anti-Thy1 model, rats received injections of the P2-receptor antagonist PPADS at different doses (15, 30, 60 mg/kg BW). Proliferating mesangial and non-mesangial cells, mesangial cell activation, matrix accumulation, influx of inflammatory cells, mesangiolysis, microaneurysm formation, and renal functional parameters were assessed during anti-Thy1 disease. P2Y-mRNA and protein expression was assessed using RT-PCR and real time PCR, Northern blot analysis, in situ hybridization, and immunohistochemistry. In cultured mesangial cells, PPADS inhibited nucleotide, but not FCS-stimulated proliferation in a dose-dependent manner. In the anti-Thy1 model, PPADS specifically and dose-dependently reduced early (day 3), but not late (day 8), glomerular mesangial cell proliferation as well as phenotypic activation of the mesangium and slightly matrix expansion. While no consistent effect was obtained in regard to the degree of mesangiolysis, influx of inflammatory cells, proteinuria or blood pressure, PPADS treatment increased serum creatinine and urea in anti-Thy1 rats. P2Y receptor expression (P2Y2 and P2Y6) was detected in cultured MC and isolated glomeruli, and demonstrated a transient marked increase during anti-Thy1 disease. These data strongly suggest an in vivo role for extracellular nucleotides in mediating early MC proliferation after MC injury.

PDGF signal transduction inhibition ameliorates experimental mesangial proliferative glomerulonephritis

Platelet-derived growth factor (PDGF) has been consistently implicated in the cell proliferation and extracellular matrix accumulation, which characterize progressive glomerular disease. In the present study, the effects of a potent and selective inhibitor of PDGF receptor tyrosine kinase, STI 571, were examined in vitro and in vivo. Cultured mesangial cells were incubated with PDGF (50 ng/mL) and fibroblast growth factor-2 (FGF-2; 50 ng/mL) and treated with STI 571 (0.13 to 2.0 micromol/L). Experimental mesangial proliferative glomerulonephritis was induced in male Wistar rats with monoclonal OX-7, anti-rat Thy-1.1 antibody with rats randomized to receive either STI 571 (50 mg/kg intraperitoneally daily) or vehicle. Animals were examined six days later. In vitro, both PDGF and FGF-2 induced a threefold increase in mesangial cell 3H-thymidine incorporation. STI 571 reduced PDGF but not FGF-2-stimulated mesangial cell proliferation in a dose-dependent manner, with complete abolition at 0.4 micromol/L. In animals with Thy-1.1 glomerulonephritis, PDGF receptor tyrosine kinase blockade was associated with significant reductions in mesangial cell proliferation (P < 0.001), the number of activated (alpha-smooth muscle positive) mesangial cells, and glomerular type IV collagen deposition (P < 0.001). The amelioration of the pathological findings of experimental mesangial proliferative glomerulonephritis by blockade of PDGF receptor activity suggests the potential clinical utility of this approach as a therapeutic strategy in glomerular disease.