Expression of a novel PDGF isoform, PDGF-C, in normal and diseased rat kidney

Platelet-derived growth factor-C (PDGF-C) is a new member of the PDGF family. Its expression in normal and diseased kidney is unknown. Rabbit antisera were generated against human full-length, core domain, and mouse PDGF-C, and their specificity was confirmed by Western blot analyses. Renal PDGF-C expression was analyzed by immunohistochemistry in normal rats (n = 8), mesangioproliferative anti-Thy 1.1 nephritis (n = 4 each at days 1, 4, 6, and 85), passive Heymann nephritis (PHN, n = 4), puromycin nephrosis (PAN, n = 2), Milan normotensive rats (MN, n = 2), and obese Zucker rats (n = 3). PDGF-C expression was also studied in anti-Thy 1.1 rats treated with PDGF-B aptamer antagonists (n = 5) or irrelevant control aptamers (n = 5). PDGF-C was constitutively expressed in arterial smooth muscle cells and collecting duct epithelial cells. Mesangial PDGF-C was markedly upregulated in anti-Thy 1.1 nephritis in parallel with the peak mesangial cell proliferation. Furthermore, PDGF-CC acted as a potent growth factor for mesangial cells in vitro. Inhibition of PDGF-B via specific aptamers reduced the injury in anti-Thy 1.1 nephritis but did not affect the glomerular PDGF-C overexpression or the mitogenicity of PDGF-CC in vitro. In PHN, PAN, and obese Zucker rats, glomeruli remained negative for PDGF-C despite severe glomerular injury. PDGF-C localized to podocytes at sites of focal and segmental sclerosis in MN. Interstitial PDGF-C expression was increased at sites of fibrosing injury in obese Zucker rats. The use of the different antisera resulted in virtually identical findings. It is concluded that PDGF-C is a novel mesangial cell mitogen that is constitutively expressed in the kidney and specifically upregulated in mesangial, visceral epithelial, and interstitial cells after predominant injury to these cells. PDGF-C may therefore be involved in the pathogenesis of renal scarring.     

Intrarenal injection of bone marrow-derived angiogenic cells reduces endothelial injury and mesangial cell activation in experimental glomerulonephritis

Loss of glomerular endothelial cells has been suggested to contribute to the progression of glomerular injury. Although therapeutic angiogenesis induced by administration of bone marrow-derived endothelial progenitor cells has been observed in disease models of endothelial injury, the effects on renal disease have not been clarified. Whether administration of culture-modified bone marrow mononuclear cells would mitigate the glomerular endothelial injury in anti-Thy1.1 nephritis was investigated. After cultivation under conditions that promote endothelial progenitor cell growth, bone marrow mononuclear cells were labeled with CM-DiI, a fluorescence marker, and injected into the left renal artery of Lewis rats with anti-Thy1.1 glomerulonephritis. The decrease in glomerular endothelial cells was significantly attenuated in the left kidney, as compared with the right, in nephritic rats that received the cell infusion. Glomerular injury score, the area positive for mesangial alpha-smooth muscle actin, and infiltration of macrophages were significantly decreased in the left kidney. CM-DiI-positive cells were distributed in glomeruli of the left kidney but not in those of the right kidney. Among CM-DiI-labeled cells incorporated into glomeruli, 16.5 +/- 1.2% of cells were stained with an endothelial marker, rat endothelial cell antigen-1. Culture-modified mononuclear cells secreted 281.2 +/- 85.0 pg of vascular endothelial growth factor per 10(5) cells per day. In conclusion, intra-arterial administration of culture-modified bone marrow mononuclear cells reduced endothelial injury and mesangial activation in anti-Thy1.1 glomerulonephritis. Incorporation into the glomerular endothelial lining and production of angiogenic factor(s) are likely to contribute to the protective effects of culture-modified mononuclear cells against glomerular injury.     

Thrombospondin peptides are potent inhibitors of mesangial and glomerular endothelial cell proliferation in vitro and in vivo.

BACKGROUND: Thrombospondin 1 (TSP1), a multifunctional, matricellular glycoprotein, is expressed de novo in many inflammatory disease processes, including glomerular disease. Short peptide fragments derived from the type I properdin repeats of the TSP1 molecule mimic anti-angiogenic and/or transforming growth factor-beta (TGF-beta)-activating properties of the whole TSP1 glycoprotein. We investigated the effects of D-reverse peptides derived from the type I domain of TSP1 in experimental mesangial proliferative glomerulonephritis in the rat (anti-Thy1 model), as well as their effects on cultured mesangial and glomerular endothelial cells. METHODS: Effects of TSP peptides on proliferation of mesangial or glomerular endothelial cells in culture after growth arrest or growth factor stimulation (fibroblast growth factor-2, platelet-derived growth factor-BB, 10% fetal calf serum) were measured by [3H]thymidine incorporation assay. Adhesion of rat mesangial cells (MCs) to a TSP-peptide matrix was assayed using an attachment-hexosaminidase assay. TSP peptides were intraperitoneally injected daily in rats that had received an intravenous injection of polyclonal anti-Thy1 antibody to induce mesangial proliferative glomerulonephritis. On biopsies from days 2, 5, and 8 of anti-Thy1 disease, mesangial and glomerular endothelial proliferation, matrix expansion, mesangial activation, and microaneurysm formation were assessed. Functional parameters such as blood pressure and proteinuria were also measured. RESULTS: An 18-amino acid peptide (type I peptide) with anti-angiogenic and TGF-beta-activating sequences decreased mesangial and glomerular endothelial cell proliferation in vitro and in vivo and reduced microaneurysm formation and proteinuria in experimental glomerulonephritis. Analogues lacking the TGF-beta-activating sequence mimicked most effects of the type I peptide. The mechanism of action of these peptides may include antagonism of fibroblast growth factor-2 and alteration of MC adhesion. The TGF-beta-activating sequence alone did not have significant effects on mesangial or glomerular endothelial cells in vitro or in experimental kidney disease in vivo. CONCLUSION: Peptides from TSP1 may be promising therapeutics in treating glomerular disease with mesangial and endothelial cell injury.     

Inducible nitric oxide synthase-derived nitric oxide promotes glomerular angiogenesis via upregulation of vascular endothelial growth factor receptors

The vascular endothelial growth factor (VEGF) system is of major importance for glomerular endothelial repair in glomerulonephritis (GN) and is significantly affected by nitric oxide (NO) release. For investigating whether glomerular upregulation of inducible NO synthase (iNOS) in GN might affect VEGF-mediated repair, a selective iNOS inhibitor, L-N6-(1-iminoethyl)-lysin (L-NIL), was administered to rats with anti-Thy 1.1 GN from day -2 until day 5 after GN induction. Compared with untreated nephritic rats, L-NIL-treated nephritic rats showed similar mean arterial BP, significantly decreased de novo peak nitrate production, and increased albuminuria on day 6. This was preceded by a significant decrease of glomerular endothelial cell proliferation and endothelial area on day 2 in L-NIL-treated nephritic rats. Upregulation of glomerular VEGF mRNA and protein expression, in particular of the VEGF(164) splicing variant, occurred similarly in L-NIL-treated and untreated nephritic rats on days 2 and 7. However, the upregulation of glomerular VEGF receptor 1 and 2 mRNA expression on day 2 was reduced by 77 and 67%, respectively, in L-NIL-treated nephritic rats as compared with untreated nephritic rats. In parallel, glomerular VEGF(165) binding was reduced by 34% in L-NIL-treated nephritic rats on day 2. Glomerular upregulation of the VEGF(164) co-receptor neuropilin-1 mRNA in nephritic rats was reduced by L-NIL treatment only on day 7. Healthy untreated or L-NIL-treated controls showed no significant differences in any parameter analyzed. In conclusion, impaired repair of glomerular endothelium and downregulation of glomerular VEGF receptor expression was observed after selective iNOS inhibition in experimental GN. These data identify iNOS-derived NO production as the first in vivo regulator of the glomerular VEGF system and as an important mechanism promoting glomerular healing.     

Role of the secretory PLA2-IB and PLA2R in human podocyte injury and its possible mechanism

Objective To investigate the role of the group IB secretory phospholipase A2 (sPLA2-IB) and M-type phospholipase A2 receptor (PLA2R) in human podocyte injury and its possible signal transduction pathway. Methods Differentiated human podocytes were exposed to PBS or different sPLA2-IB concentration conditions (10-9, 10-7, 10-5 mol/L) for 2 hours. The wound healing assay was used to measure cell migration rate; Apoptosis in cultured human podocytes was assessed by Hoechst 33342 staining and flow cytometry; Western blot was used to analyze the protein expression of p-cPLA2α, p-p38, and p53. Then control siRNA or PLA2R siRNA were transfected to podocytes. Podocytes were divided into normal control group, negative control siRNA group and PLA2R siRNA group. Twenty four hous later, the cells were stimulated by 10-5 mol/L sPLA2-IB for 2 hours. The protein expression of p-cPLA2α, p-p38, and p53 were detected by Western blot. Results Compared to PBS control group, the migration ability of podocytes decreased when stimulated with sPLA2-IB (10-7 mol/L-10-5 mol/L), and the apoptosis of podocytes increased in a concentration-dependent manner, the protein level of p-cPLA2α, p-p38 and p53 protein increased too. After the knockdown of PLA2R by PLA2R siRNA transfection, stimulated the podocytes with the same dosage of sPLA2-IB, the protein expression of p-cPLA2α, p-p38 and p53 all decreased. Conclusion sPLA2-IB stimulation can increase human podocyte apoptosis and decrease its migration ability. The possible mechanism might be through p38-cPLA2α-p53 pathway.     

Angiogenic protein Cyr61 is expressed by podocytes in anti-Thy-1 glomerulonephritis

Dynamic recovery of glomerular structure occurs after severe glomerular damage in anti-Thy-1 glomerulonephritis (Thy-1 GN), but its mechanism remains to be investigated. To identify candidate genes possibly involved in glomerular reconstruction, screening was performed for genes that are specifically expressed by podocytes and are upregulated in glomeruli of Thy-1 GN. Among them, cysteine-rich protein 61 (Cyr61 or CCN1), a soluble angiogenic protein belonging to the CCN family, was identified. By Northern blot analysis, Cyr61 mRNA was markedly upregulated in glomeruli of Thy-1 GN from day 3 through day 7, when mesangial cell migration was most prominent. By in situ hybridization and immunohistochemistry, Cyr61 mRNA and protein were expressed by proximal straight tubules and afferent and efferent arterioles in normal rat kidneys and were intensely upregulated at podocytes in Thy-1 GN. Platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1), of which the gene expression in the glomeruli of Thy-1 GN was upregulated in similar time course as Cyr61, induced Cyr61 mRNA expression in cultured podocytes. Furthermore, supernatant of Cyr61-overexpressing cells inhibited PDGF-induced mesangial cell migration. In conclusion, it is shown that Cyr61 is strongly upregulated at podocytes in Thy-1 GN possibly by PDGF and TGF-beta. Cyr61 may be involved in glomerular remodeling as a factor secreted from podocytes to inhibit mesangial cell migration.     

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: These data strongly suggest an in vivo role for extracellular nucleotides in mediating early MC proliferation after MC injury.     

In vivo identification of the mitogen-activated protein kinase cascade as a central pathogenic pathway in experimental mesangioproliferative glomerulonephritis

Evidence was recently provided for the activation of extracellular signal-regulated kinase (ERK), the best characterized mitogen-activated protein kinase, as an intracellular convergence point for mitogenic stimuli in animal models of glomerulonephritis (GN). In this study, in vivo ERK activity was blocked, with a pharmacologic inhibitor (U0126) of the ERK-activating kinase, in rats with mesangioproliferative GN. After injection of the monoclonal anti-Thy1.1 antibody (OX-7), the rats were treated (days 3 to 6) with low (10 mg/kg body wt) or high (100 mg/kg body wt) doses of U0126 administered intraperitoneally twice daily. On day 6 after induction of the disease, whole cortical tissue and isolated glomeruli were examined by using kinase activity assays, Western blot analyses, and immunohistochemical assays. Treatment with U0126 significantly reduced glomerular stimulation of ERK in anti-Thy1 GN. In the high dose-treated group, this downregulation was accompanied by a reduction in the number of glomerular mitotic figures, back to healthy control levels, and significant decreases in the numbers of total (P < 0.05) and 5-bromo-2'-deoxyuridine-positive (P < 0.05) glomerular cells. Immunohistochemical double-staining of renal sections demonstrated that mesangial cells were the major glomerular targets of U0126 in anti-Thy1 GN. These observations point to ERK as a putative intracellular mediator of the proliferative response in GN and suggest that pharmacologic treatments that interfere with the activation of ERK may be of potential therapeutic interest.     

Transplanted mesenchymal stem cells accelerate glomerular healing in experimental glomerulonephritis

Bone marrow-derived cells contribute to glomerular cell turnover and repair, but the cell types involved are unknown. Whether rat mesenchymal stem cells (MSC) can accelerate recovery from damage in rat mesangioproliferative anti-Thy1.1 glomerulonephritis was studied. After injection into the left renal artery on day 2 after disease induction, fluorescently labeled MSC were detected in 20 to 50% of glomeruli and rare intrarenal vessels but not in the tubulointerstitium, in contralateral kidneys, or in medium controls. In control experiments, injected mesangial cells were detected less frequently in glomeruli in comparison with injected MSC. In nephritic outbred Wistar rats, MSC injection led to an approximately 50% reduction of mesangiolysis on days 4 and 6 after disease induction, accompanied by three- to four-fold higher intraglomerular cell proliferation on day 4 and more rapid mesangial reconstitution as detected by alpha-smooth muscle actin expression. Injection of MSC into tail veins or intra-arterial injection of mesangial cells instead of MSC failed to reproduce any of these findings. In inbred Lewis rats, anti-Thy1.1 nephritis followed an aggravated course with transient acute renal failure. Acute renal failure was ameliorated by MSC injection into the left renal artery on day 2 after disease induction. Again, MSC led to more rapid recovery from mesangiolysis, increased glomerular cell proliferation, and reduction of proteinuria by 28%. Double immunostaining of 5-bromo-2'-deoxyuridine-labeled MSC for endothelial, mesangial, or monocyte/macrophage antigens showed that 85 to 95% of MSC that localized in glomeruli on day 6 failed to express these markers. In vitro, MSC secreted high amounts of vascular endothelial growth factor and TGF-beta1 but not PDGF-BB. In conclusion, even low numbers of MSC can markedly accelerate glomerular recovery from mesangiolytic damage possibly related to paracrine growth factor release and not to differentiation into resident glomerular cell types or monocytes/macrophages.     

Effects of leukocytosis and macrophage activation on anti-Thy 1.1 glomerulonephritis in the rat

OBJECTIVE: In anti-Thy 1.1 proliferative glomerulonephritis (GN), glomerular infiltration of polymorphonuclear leukocytes (PMNs) reaches a peak level after 1 h and that of macrophages does so 24 h after induction. However, the roles of PMNs and macrophages in the pathogenesis of anti-Thy 1.1 GN remain unclear. We examined the effects on this model of leukocytosis induced by granulocyte colony-stimulating factor (G-CSF) and of macrophages stimulated by macrophage colony-stimulating factor (M-CSF). MATERIAL AND METHODS: Anti-Thy 1.1 GN was induced in male Wistar rats by intravenous injection of OX-7, a monoclonal antibody to the Thy 1 antigen. G-CSF (10 microg/kg/day), M-CSF (20 microg/kg/day) or vehicle was administered intraperitoneally for 7 days starting 24 h before the injection of OX-7. Histological examination of renal biopsy specimens was performed on Days 1, 5 and 14 after induction. RESULTS: Circulating and glomerular-infiltrating PMNs (RP-3-positive cells) were increased at Day 5 in G-CSF-treated rats compared with controls receiving vehicle, and glomerular mesangiolysis continued at Day 5. The number of proliferating cells positive for proliferating cell nuclear antigen at Day 5 and matrix scores at Day 14 were smaller in G-CSF-treated rats than in control rats. The mesangiolysis score was significantly higher in the G-CSF group than in the control group at Days 5 and 14, but not at Day 1. ED-1-positive cells were increased in number at Day 5 and matrix accumulation decreased at Day 14 in M-CSF-treated rats compared with controls. Serum creatinine level at Day 14 was lower in the M-CSF group, but not in the G-CSF group, compared with the control group. CONCLUSIONS: Activated macrophages may inhibit excess matrix accumulation and ameliorate the recovery of renal function, whereas leukocytosis inhibits the repair of mesangial cell injury in this model.     

Cloning of rodent megsin revealed its up-regulation in mesangioproliferative nephritis

BACKGROUND: We recently cloned a new human mesangium-predominant gene, megsin. Megsin is a novel member of the serine protease inhibitor (serpin) superfamily. To elucidate functional roles of this gene, we cloned megsin in rodents and investigated its role in a rat nephritis model. METHODS: Megsin homologues were cloned from cultured rat and mouse mesangial cDNAs utilizing polymerase chain reaction (PCR) with degenerative primers. Expression of megsin in three different types of resident glomerular cells was investigated by PCR. Levels of megsin mRNA expression at various time points in the anti-Thy1 rat nephritis model were studied by semiquantitative PCR and Northern blotting analysis. In order to investigate megsin protein expression in anti-Thy1 nephritis rats, we raised antibody against rat megsin-specific synthetic peptide, with which immunohistochemical studies were performed. RESULTS: Rat and mouse megsins were composed of 380 amino acids, which revealed 75.3 and 73.9% identity, respectively, with human megsin at the amino acid level. Characteristic features as an inhibitory serpin were conserved in both rat and megsin megsins. PCR analysis revealed expression of megsin in cultured mesangial cells but not in glomerular epithelial or endothelial cells. In anti-Thy1 nephritis rats, semiquantitative PCR and Northern blotting showed that expression of megsin mRNA was up-regulated in glomeruli at day 8. Immunohistochemical studies demonstrated the prominent accumulation of megsin in glomeruli at the same time point. Megsin was mainly localized in mesangial area. The megsin expression level returned to the basal level at day 28. CONCLUSION: Sequences of megsin were well conserved among different species. Rat megsin was also predominantly expressed in mesangial cells. Expression of megsin was up-regulated at the peak of hypercellularity and matrix accumulation in the mesangioproliferative glomerulonephritis model, suggesting that megsin may participate in the process of glomerulosclerosis by modulating extracellular matrix deposition or cell survival.     

Nitric oxide synthase isoform expression in acute versus chronic anti-Thy 1 nephritis

BACKGROUND: Two inbred Lewis rat substrains (LEW/Moe, LEW/Maa) were identified responding differently to induction of anti-Thy 1 glomerulonephritis (aThy 1-GN). LEW/Moe rats show an acute mesangioproliferative glomerulonephritis with rapid healing of glomerular lesions within four weeks, while LEW/Maa rats develop severe glomerular injury followed by chronic glomerular sclerosis and persistent albuminuria. We investigated whether the glomerular expression pattern of nitric oxide synthase (NOS) isoforms could explain these substrain-related differences. METHODS: Rats (N = 5 to 7 per group) were investigated in a time course experiment. Severity of aThy 1-GN was determined by albuminuria measurements, glomerular matrix score and microaneurysm formation. Glomerular gene expression of NOS isoforms was determined by semiquantitative RT-PCR. Inducible NOS (iNOS) activity was determined in cultured glomeruli and peritoneal macrophages. Neuronal NOS (nNOS) protein expression was detected by Western blotting and enzyme histochemistry. Plasma renin activity (PRA) was measured by RIA. RESULTS: Induction of iNOS expression and activity was found significantly increased and sustained in LEW/Maa vs. LEW/Moe rats associated with an increased number of infiltrating macrophages and with an increased capacity of iNOS-expression and iNOS-activation by isolated macrophages in LEW/Maa rats. Glomerular nNOS mRNA and nNOS protein expression were constitutively increased in LEW/Maa rats. Renal nNOS localization was restricted to the macula densa region in both substrains and associated with increased PRA in LEW/Maa rats. No difference in glomerular endothelial NOS-mRNA expression between the substrains was observed. CONCLUSIONS: Increased glomerular iNOS and nNOS expression were associated with chronic anti-Thy 1 glomerulonephritis in LEW/Maa rats and may contribute to glomerular damage by separate mechanisms.     

Adrenomedullin reduces mesangial cell number and glomerular inflammation in experimental mesangioproliferative glomerulonephritis

BACKGROUND: Adrenomedullin (ADM) is a vasodilator peptide that is abundantly expressed in the kidney. ADM has antiproliferative effects on glomerular mesangial cells (MC) in vitro. Whether or not treatment with ADM can reduce MC proliferation in vivo [i.e., in mesangioproliferative glomerulonephritis (GN)] is unknown. We tested the hypothesis that ADM substitution reduces MC proliferation in GN. METHODS: GN in rats was induced by injection of an anti-Thy-1.1 antibody. Rats received osmotic minipumps, which continuously delivered rat ADM (500 ng/hour, N = 11), or vehicle (N = 13) from day 3 to day 6 after GN induction. Rats were sacrificed 6 days after induction of GN. On kidney sections, cells staining positive for proliferating cell nuclear antigen, mesangial cells, monocytes, and apoptotic cells were counted. Parameters of inflammation and fibrosis were measured in renal cortex and sieved glomeruli by real-time polymerase chain reaction (PCR). RESULTS: Systolic blood pressure, diuresis, albuminuria, creatinine clearance, microaneurysm formation, and mesangial matrix expansion were not influenced by ADM infusion. However, ADM treatment significantly reduced the number of MC, showed a tendency to reduce total glomerular cell proliferation, and significantly increased apoptosis. ADM-treated GN animals showed significantly less glomerular monocyte infiltration. ADM treatment normalized transforming growth factor (TGF)-beta1 mRNA expression and reduced monocyte chemoattractant protein-1 (MCP-1), osteopontin, plasminogen activator inhibitor-1 (PAI-1), collagen I, and collagen III mRNA expression significantly. CONCLUSION: Exogenous ADM infusion reduces MC number and glomerular monocyte infiltration in the state of mesangial proliferation during acute experimental mesangioproliferative GN. These findings indicate that ADM can influence the course of mesangioproliferative GN.     

Fractalkine expression and the recruitment of CX3CR1+ cells in the prolonged mesangial proliferative glomerulonephritis

BACKGROUND: We established the reversible and the prolonged models of mesangial proliferative glomerulonephritis (GN) with anti-Thy 1 antibody 1-22-3. However, the essential factors leading to the prolonged glomerular alterations have not been identified. METHODS: The expressions of several chemokines and cytokines were compared in the reversible and the prolonged models. Expression of fractalkine and the number of the fractalkine receptor CX3CR1-positive cells in the glomeruli in the prolonged model were significantly higher than those in the reversible model. Then, the localization of fractalkine and the characteristics of CX3CR1+ cells were analyzed in glomeruli. To elucidate the significance of the fractalkine expression, we analyzed the expression in the model treated with angiotensin II receptor antagonist, candesartan. RESULTS: Immunostaining of fractalkine was detected on endothelial cells on the fifth day, and fractalkine staining also was detected in the mesangial area on day 14. Major parts of the CX3CR1+ cells in the glomeruli were macrophages, especially ED3+ cells. Candesartan treatment ameliorated the glomerular morphological findings at six weeks after disease induction. Although the treatment did not ameliorate the morphological finding at two weeks, decreased expression of fractalkine and CX3CR1+ were already detected at two weeks in rats treated with candesartan. CONCLUSIONS: Fractalkine expression and the recruitment of CX3CR1+ cells in glomeruli might play an important role in the development of the prolonged disease. These expressions could be predictors of the prolonged disease of the mesangial proliferative glomerulonephritis.     

Antigen expression in different parenchymal cell types of rat kidney and heart

Rat glomerular epithelial, and mesangial and tubular cells were isolated by steel meshes of different pore sizes and enzymatic treatment. Rat heart endothelial cells were isolated by enzymatic disaggregation. Endothelial, glomerular mesangial, glomerular epithelial and tubular cells were cultured in Minimum essential medium until they reached confluency (5 to 9 days). These different rat parenchymal cell types were characterized by morphology and antibody stainings. Endothelial cells were characterized by factor VIII positivity and mesangial cells were desmin positive. Both glomerular epithelial and tubular cells expressed the brush border and Tamm-Horsfall antigens, but in vivo injected trypan blue accumulated selectively to proximal tubular cells. Class I expression was high (84 to 95% of about 10(5) cells grown in Lab-Tek culture chambers were reactive with anti-class I antibody) in unstimulated endothelial, glomerular epithelial and tubular cells and was even higher (100%) after incubation with gamma-interferon for three days. Mesangial cells expressed class I considerably less in normal state (34%), but gamma-interferon induction upregulated it to 95%. Both the surface and intracytoplasmic expression of class I antigens were upregulated with three-day gamma-interferon treatment. Class II expression was low in all unstimulated cells (5 to 10%). The three-day gamma-interferon treatment upregulated both surface as well as cytoplasmic expression of class II antigens in all cell types.