Intraglomerular fibronectin in rat experimental glomerulonephritis.

To clarify the mechanisms of glomerular pericapillary fibronectin deposition in human membranous nephropathy and mesangial proliferative glomerulonephritis, intraglomerular fibronectin distribution was examined by light and electron microscopy using the experimental rat models of Heymann and nephrotoxic serum nephritis. As previously demonstrated by immunofluorescence microscopy (Pettersson and Colvin 1978; Ikeya et al. 1985, 1986), fibronectin was distributed in the mesangial areas and occasionally on percicapillary walls of normal glomeruli, while in nephrotoxic serum nephritis and Heymann nephritis, fibronectin was diffusely located along glomerular capillary walls as well as in the mesangium. By immunoelectron microscopy using the immunogold technique, fibronectin was also noted in the mesangial areas and the lamina densa of the glomerular basement membrane (GBM) in normal glomeruli. In nephrotoxic serum nephritis, fibronectin was seen around mesangial cells situated between endothelial cells and the GBM, suggesting that pericapillary fibronectin in nephrotoxic serum nephritis reflects mesangial extension. However, in Heymann nephritis, it was found uniformly in the lamina rara interna, lamina densa and lamina rara externa of the GBM, indicating no specific relation to glomerular cells. When sections of normal and both experimental nephritis kidneys were incubated with fluorescein isothiocyanate conjugated with rat plasma fibronectin, a linear pattern of fluorescein staining along the glomerular capillary walls was observed in Heymann nephritis but not in normal or nephrotoxic serum nephritic rats. The GBM in Heymann nephritis would thus appear to have an affinity for plasma fibronectin. Based on the above findings, fibronectin in the GBM of rats with Heymann nephritis may reasonably be concluded to originate from the plasma.     

IL-9 production by regulatory T cells recruits mast cells that are essential for regulatory T cell-induced immune suppression

Both mast cells (MCs) and regulatory T cells (Tregs) have gained attention as immunosuppressive cell populations. To investigate a possible interaction, we used the Th1- and Th17-dependent model of nephrotoxic serum nephritis (NTS), in which both MCs and Tregs have been shown to play a protective role. Transfer of wild-type (wt) Tregs into wt recipients almost completely prevents development of NTS and leads to a profound increase of MCs in the renal draining lymph nodes (LNs). By contrast, transfer of wt Tregs into animals deficient in MCs, which are characterized by an exaggerated susceptibility to NTS, no longer exhibited protective effects. Blocking the pleiotropic cytokine IL-9, known to be involved in MC recruitment and proliferation, by means of a mAb in mice receiving Tregs abrogated protection from NTS. Moreover, transfer of IL-9-deficient Tregs also failed to protect from NTS. In the absence of Treg-derived IL-9, MCs fail to accumulate in the LNs, despite the fact that IL-9 deficiency does not alter the general suppressive activity of Tregs. In summary, to our knowledge, we provide the first direct in vivo evidence that the nephroprotective, anti-inflammatory effects of Tregs critically depend on IL-9-mediated attraction of MCs into kidney-draining LNs.     

Increased severity of renal impairment in nephritic mice lacking the EP1 receptor

In experimental glomerulonephritis, inhibition of renal prostaglandin (PG) synthesis by nonsteroidal-anti-inflammatory drugs (NSAIDs) moderates proteinuria, yet can induce harmful effects on renal blood flow and Na+ - K+ - water balance thereby implicating 1 or more prostanoid receptor subtypes. We investigated the role of the PGE2 EP1 receptor in nephritis since it is expressed in the glomerulus, collecting duct and vasculature in which its activity might contribute to adaptive or maladaptive responses. Accordingly, a mouse model of accelerated antiglomerular basement membrane (anti-GBM) nephrotoxic serum (NTS) nephritis was induced in mice with targeted-deletion of the EP1 receptor (EP1-/-). Proteinuria was similar between wild-type (wt) and EP1-/- NTS groups, thus negating a role for this subtype in modulating the glomerular permeability barrier in this model of anti-GBM NTS. However, overall renal damage was more acute in NTS EP1-/- mice, as evidenced by the degree of glomerular mesangial matrix expansion and the frequency of tubular dilatations. These changes in renal pathology were accompanied by stronger impairment of renal function in NTS EP1-/- mice, such that levels of serum creatinine, urea, Na+, and K+ were each significantly higher than those observed in NTS wt mice. Lastly, compared with wt mice, induction of NTS more severely reduced urine osmolality and body mass in EP1-/- mice. Taken together, the increased renal impairment seen in NTS EP1-/- mice suggests that the EP1 subtype plays a compensatory role in the context of acute nephritis.     

Screening for potential serum biomarkers in rat mesangial proliferative nephritis

Mesangial proliferative nephritis (MesPGN) is a common kidney disease worldwide. The main feature of the disease is mesangial cell proliferation‐induced injury to kidney function. In this study, we explored serum biomarkers for kidney function injury in anti‐Thy1 nephritis. We found that mesangial proliferation were increased on days 5 and 7, and recovered by day 14 in anti‐Thy1 nephritis. 24‐h urine protein, the ratio of urine protein to urine creatine, serum creatine, and blood urea nitrogen, were increased at days 5 and 7 in the model. We found that TXN, BET1, PrRP, VGF, and NPS differed strongly from controls on days 5 and, associated with kidney injury when detected by SELDI‐TOF MS. Moreover, we applied LC‐MS to detect differential protein expression and found A2M, C3, ITIH4, ITIH3, VDBP, AFM, and SERPINF2 to be upregulated, and ES1, HPX, SERPINC1, SERPINA1F, SERPINA4, SERPINA3K, SPI, TF, VNN3, SERPINF1, and PON1 to be downregulated, on days 5 and 7, associated with kidney injury. The levels of VNN3 and VDBP were validated by Western blotting. Overall, this study explored a group of candidate biomarkers of mesangial proliferation inducing kidney injury, to provide the basis of an assessment model for MesPGN in the future.     

应用高脂饮食联合维生素D3建立慢性系膜增殖性肾炎血管病变大鼠模型

目的 探讨应用高脂饮食建立慢性系膜增殖性肾炎血管病变模型的方法.方法 雄性Wistar大鼠行单侧肾切除后随机分为单纯肾切除组、单纯肾炎组、单纯高脂组、肾炎高脂组.单纯肾炎组、肾炎高脂组在单侧肾切除后3d尾静脉注射OX7抗体(100 mg/kg),1周后尾静脉连续注射OX7抗体(每次100 mg/kg,1次/周,共3次),单纯肾切除组和单纯高脂组在同一时间尾静脉注射PBS,注射抗体后第2天单纯高脂组、肾炎高脂组腹腔注射维生素D3(6万U/kg,1次/4周),同时给予高脂饲料.分别于第4、8、10周观察各组大鼠的一般情况、体重、血压、尿蛋白、血浆白蛋白、血脂、血钙、肾功能以及肾脏病理改变.结果 模型组(肾炎高脂组)大鼠第8周肾小球外的小动脉出现管壁增厚,管腔变小,平滑肌细胞减少,细胞排列紊乱,纤维组织增生.第10周单纯肾炎组和单纯高脂组肾小球外小动脉管壁轻度增厚,管腔变化不明显,模型组血管病变积分明显高于单纯肾炎组和单纯高脂组(P＜0.05).结论 通过对慢性抗Thy1肾炎大鼠加用高脂饲料并腹腔注射维生素D3的方法,可以成功建立慢性系膜增殖性肾炎血管病变模型.     

Bioinformatics analysis of proteomic profiles during the process of anti-Thy1 nephritis

Anti-Thy1 nephritis is a well-established experimental mesangial proliferative nephritis model. Exploring the molecular mechanisms of pathophysiology in anti-Thy1 nephritis may elucidate the pathogeneses of mesangial proliferation. We examined the roles and acting mechanisms of differentially expressed proteins (DEPs) by bioinformatics analysis of glomeruli proteomic profiles during the course of anti-Thy1 nephritis. In total, 108 DEPs were found by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), and 40 DEPs were identified by matrix-assisted laser desorption ionization/time of flight and liquid chromatography-MS. DEPs were classified into five clusters (Clusters 1-5), according to their expression trends using Cluster 3.0 software, involved in regulating biological processes such as the stress response, cell proliferation, apoptosis, energy metabolism, transport, and the actin cytoskeleton. The expression patterns of ten DEPs, distributed across five clusters, including AKR1A1, AGAT, ATP6V1B2, HIBADH, MDH1, MPST, NIT2, PRDX6, PSMB7, and TPI1, were validated by Western blotting. Based on Western blotting and immunohistochemistry, we also found that the DEP FHL2, which was primarily expressed in the mesangial region, was down-regulated on days 3 and 5, and up-regulated on day 10. In vitro, we found that FHL2 overexpression induced mesangial cell proliferation by increasing the number of S-phase cells and decreasing G2/M-phase cells, whereas inhibiting FHL2 had the opposite effect. This study explored novel DEPs and their expression patterns during anti-Thy1 nephritis, and elucidated FHL2's effect on mesangial cell proliferation. These results will contribute to our understanding of the pathogenesis of mesangial proliferation.     

Renal cyclo-oxygenase and lipoxygenase products in health and disease

Renal glomeruli have cyclo-oxygenase and lipoxygenase enzymes which convert arachidonic acid to prostaglandins, thromboxane and 12-hydroxyeicosatetraenoic acid. Glomerular epithelial and mesangial cells, in culture, also synthesize these arachidonate products. Angiotensin and vasopressin contract mesangial cells and stimulate mesangial synthesis of PGE2. PGE2, in the glomerulus, antagonizes the actions of angiotensin on the mesangium and hence reduces angiotensin-mediated glomerular contraction. Glomerular immune injury (nephrotoxic serum nephritis) augments glomerular production of prostaglandins and thromboxane. Thromboxane reduces glomerular function and inhibition of thromboxane synthesis preserves glomerular filtration rate and renal plasma flow in this disease model. Spontaneously hypertensive rats also have enhanced glomerular prostaglandin and thromboxane synthesis. Although acute inhibition of thromboxane synthesis will vasodilate the hypertensive rat kidney, chronic inhibition does not reduce blood pressure or increase renal blood flow.     

Activated macrophages direct apoptosis and suppress mitosis of mesangial cells

During inflammation in the glomerulus, the complement of resident myofibroblast-like mesangial cells is regulated by mitosis and apoptosis, but the cellular mechanisms controlling the size of mesangial cell populations have remained obscure. Prompted by studies of development, we sought evidence that macrophages regulate mesangial cell number. Rat bone marrow-derived macrophages primed with IFN-gamma then further activated in coculture with LPS or TNF-alpha elicited a 10-fold induction of rat mesangial cell apoptosis and complete suppression of mitosis, effects inhibitable by the NO synthase inhibitors L-monomethyl arginine and L-N(6)-(1-iminoethyl) lysine dihydrochloride. Complete dependence upon macrophage-derived NO was observed in comparable experiments employing activated bone marrow macrophages from wild-type and NO synthase 2(-/-) mice. Nevertheless, when mesangial cells were primed with IFN-gamma plus TNF-alpha, increased induction by activated macrophages of mesangial apoptosis exhibited a NO-independent element. The use of gld/gld macrophages excluded a role for Fas ligand in this residual kill, despite increased expression of Fas and increased susceptibility to soluble Fas ligand exhibited by cytokine-primed mesangial cells. Finally, activated macrophages isolated from the glomeruli of rats with nephrotoxic nephritis also induced apoptosis and suppressed mitosis in mesangial cells by an L-monomethyl arginine-inhibitable mechanism. These data demonstrate that activated macrophages, via the release of NO and other mediators, regulate mesangial cell populations in vitro and may therefore control the mesangial cell complement at inflamed sites.     

The biology of mesangial cells in glomerulonephritis

It is likely that a complex bidirectional interaction occurs between mesangial cells and the immune cells which infiltrate the mesangium during nephritis. Macrophages and other immune cells liberate a series of mediators, including substances such as IL-1, beta-endorphin, TNF, and PDGF--all of which promote the growth of mesangial cells. The end result is mesangial cell proliferation and increased matrix production, both of which are seen in nephritis. The proliferating mesangial cells liberate autocoids such as IL-1 and PDGF, thereby setting up an amplifying loop. Simultaneously, suppressive factors such as TGF-beta are released which antagonize the actions of these growth-promoting substances. The proliferating mesangial cells also produce immunomodulatory peptides, which will in turn act on the infiltrating macrophages to stimulate their replication and activation. Such activated macrophages continue to amplify the inflammatory lesion and also promote the phagocytosis of localized antigen-antibody complexes. The net effect of all of these interactions will depend on the dominance of substances which persist and override the roles of other molecules. Studies of the controls which regulate the production of these growth factors/immune modulators will yield insights into the fundamental mechanisms which determine the outcome in glomerulonephritis.     

Masugi's nephritis: preparation of an active nephrotoxic serum

An active nephrotoxic serum (NTS) was prepared by immunization of rabbits with isolated renal glomeruli of rats and Freund's complete adjuvant. The glomeruli were isolated from the renal cortex by the sieving procedure. Intravenous injection of the NTS to rats induced the development of marked proteinuria and the nephrotic syndrome. Histological and electron microscopic studies of renal tissue performed by days 5 and 15 since the first injection of the NTS revealed extracapillary proliferative glomerulonephritis. IgG depositions on the basal membranes of the glomerular capillaries were linear on day 5 and granular on day 15 since the first injection of the NTS.     

HMGB1 mediates IFN-γ-induced cell proliferation in MMC cells through regulation of cyclin D1/CDK4/p16 pathway

Previous studies have revealed the elevated serum levels of High-mobility group box-1(HMGB1) and the interferon-γ (IFN-γ)-induced proliferation of renal mesangial cells in patients or experimental animals with systemic lupus erythematosus (SLE). However, it is still not elucidated whether HMGB1 involves in the pathogenesis of lupus nephritis (LN) and mediates IFN-γ-induced mesangial cell proliferation. Therefore, in the present study we demonstrated HMGB1 mRNA and protein levels were increased in the glomeruli of LN patients and BXSB mice. HMGB1 increased the proliferation index of mouse mesangial cells (MMC) that was accompanied with the up-regulation of cyclin D1, CDK4 and the down-regulation of p16, subsequently promoting the transition from the G0/G1 to S stage. Inhibition of HMGB1 by a specific short hairpin RNA vector prevented cyclin D1/CDK4/p16 up-regulation and attenuated IFN-γ-induced MMC cell proliferation and PCNA (proliferating cell nuclear antigen, PCNA) expression. These findings indicate that HMGB1 mediates IFN-γ-induced cell proliferation in MMC cells through regulation of cyclin D1/CDK4/p16 pathway and promoting the cell cycle transition from G1 to S stage.     

Immune reponses in human mesangial cells regulated by emodin from Polygonum hypoleucum Ohwi

In the hope of identifying agents of therapeutic value in glomerulonephritis from Chinese herbs, we found that methanolic extracts of Polygonum hypoleucum Ohwi (P. hypoleucum Ohwi) inhibit human mesangial cells proliferation activated with interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) previously. This study was designed to identify bioactive components from P. hypoleucum Ohwi and elucidate their action mechanisms. We tested four anthraquinones emodin, emodin 1-O-beta-D-glucoside (49A), physcion (62A), and physcion 1-O-beta-D-glucoside (50A) purified from P. hypoleucum Ohwi for their effects on human mesangial cell proliferation and cytokines production in vitro. On a percentage basis, emodin had the highest suppressing activity on the human mesangial cells proliferation activated by IL-1beta and IL-6. The IC50 of emodin on human mesangial cells proliferation were 17.9+/-1.2 microM. In contrast to 49A, 50A, and 62A, emodin also decreased IL-1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha) production in human mesangial cells activated with IL-1beta and IL-6. The IC50 of emodin on IL-1beta, IL-6 and TNF-alpha production in activated human mesangial cells were 16.6+/-1.8 microM, 8.2+/-1.3 microM, and 9.5+/-1.6 microM, respectively. Moreover, IL-1beta and TNF-alpha mRNA expression in activated human mesangial cells was impaired by emodin. The intracellular free Ca2+ concentration ([Ca2+]i) in IL-1beta and IL-6 activated human mesangial cells was decreased by emodin. It is unlikely that cytotoxicity was involved because no cell deaths were observable. We hypothesize that the inhibitory mechanisms of emodin on activated human mesangial cells proliferation may be related to the impairments of gene expression and production of cytokines and [Ca2+]i in the cells.     

Respective roles of decay-accelerating factor and CD59 in circumventing glomerular injury in acute nephrotoxic serum nephritis

Decay-accelerating factor (DAF or CD55) and CD59 are regulators that protect self cells from C3b deposition and C5b-9 assembly on their surfaces. Their relative roles in protecting glomeruli in immune-mediated renal diseases in vivo are unknown. We induced nephrotoxic serum (NTS) nephritis in Daf1(-/-), CD59a(-/-), Daf1(-/-)CD59a(-/-), and wild-type (WT) mice by administering NTS IgG. After 18 h, we assessed proteinuria, and performed histological, immunohistochemical, and electron microscopic analyses of kidneys. Twenty-four mice in each group were studied. Baseline albuminuria in the Daf1(-/-), CD59a(-/-), and Daf1(-/-)CD59a(-/-) mice was 82, 83, and 139 as compared with 92 microg/mg creatinine in the WT controls (p > 0.1). After NTS, albuminuria in CD59a(-/-) and WT mice (186 +/- 154 and 183 +/- 137 microg/mg creatinine, p > 0.1) was similar. In contrast, Daf1(-/-) mice developed severe albuminuria (378 +/- 520, p < 0.05) that was further exacerbated in Daf1(-/-)CD59a(-/-) mice (577 +/- 785 micro g/mg creatinine, p < 0.05). Glomerular histology showed essentially no infiltrating leukocytes in any group. In contrast, electron microscopy revealed prominent podocyte foot process effacement in Daf1(-/-) mice with more widespread and severe damage in the double knockouts compared with only mild focal changes in CD59a(-/-) or WT mice. In all animals, deposition of administered (sheep) NTS Ig was equivalent. This contrasted with marked deposition of both C3 and C9 in Daf1(-/-)CD59a(-/-) and Daf1(-/-) mice, which was evident as early as 2 h post-NTS injection. The results support the proposition that in autoantibody-mediated nephritis, DAF serves as the primary barrier to classical pathway-mediated injury, while CD59 limits consequent C5b-9-mediated cell damage.     

Inhibition of mesangial cell nitric oxide in MRL/lpr mice by prostaglandin J2 and proliferator activation receptor-gamma agonists

MRL/Mp-lpr/lpr (MRL/lpr) mice develop immune complex glomerulonephritis similar to human lupus. Glomerular mesangial cells are key modulators of the inflammatory response in lupus nephritis. When activated, these cells secrete inflammatory mediators including NO and products of cyclooxygenase perpetuating the local inflammatory response. PGJ2, a product of cyclooxygenase, is a potent in vitro inhibitor of macrophage inflammatory functions and is postulated to function as an in vivo inhibitor of macrophage-mediated inflammatory responses. We hypothesized that in lupus, a defect in PGJ2 production allows the inflammatory response to continue unchecked. To test this hypothesis, mesangial cells were isolated from MRL/lpr and BALB/c mice and stimulated with IL-1beta or LPS plus IFN-gamma. In contrast to the 2- to 3-fold increase in PGJ2 production by stimulated BALB/c mesangial cells, supernatant PGJ2 did not increase in MRL/lpr mesangial cell cultures. NO production in stimulated MRL/lpr and BALB/c mesangial cells, was blocked by PGJ2 and pioglitazone. These studies suggest that abnormalities in PGJ2 production are present in MRL/lpr mice and may be linked to the heightened activation state of mesangial cells in these mice.     

The regulation of the extracellular matrix turnover in the experimental glomerulopathies

Pathogenic mechanisms invoved in glomerulopathies were investigated on the passive form of accelerate model of nephrotoxic nephritis and puromycin aminonucleoside nephrosis. The aim of the present study was to examine the interaction of some cytokines produced by mononuclear leukocytes and mesangial cells in a regulation of synthesis and degradation of the extracellular matrix components and adhesion molecules expression. In acute stage of nephrotoxic nephritis and puromycin aminonucleoside nephrosis the mononuclear leukocyte infiltration into glomeruli was noted. Mononuclear leukocytes produce cytokines that control mesangial cells proliferation and extracellular matrix accumulation. In summary the current study showed a key role of mononuclear leukocytes in inflammation and extracellular matrix turnover. The absence of mononuclear leukocytes or their abundance induce the disturbance of extracellular matrix synthesis and progression factors in glomerulosclerosis.     

An ultrastructural study of proliferative nephritis induced experimentally by a monoclonal antibody against mesangial cells: replacement of mesangial cells by cells of the monocyte-macrophage system

An experimental nephritis accompanied by transient proteinuria can be produced by an intravenous injection of the monoclonal antibody, 1-22-3, raised against isolated rat glomeruli. The present study deals with the ultrastructural changes in the glomeruli in rats after the injection of this antibody. At 2 h after injection, all the mesangial cells had completely degenerated and neutrophils invaded most mesangial areas. Monocytes occupied the vacant mesangial areas at 24 h and gradually increased in number over the next 4 days. At 4 and 6 days, macrophage-like cells, possibly derived from monocytes, underwent frequent mitosis, resulting in a remarkable proliferation of these cells. The interpretation of these cells as macrophages was strongly supported by the fact that they contained previously injected latex particles in large numbers. From 2 to 4 weeks after injection, the macrophage-like cells gradually transformed into cells indistinguishable from normal mesangial cells. In the present experimental nephritis where all mesangial cells were initially destroyed, cells of the monocyte-macrophage system appear to play a leading role in the pathogenesis of the ensuing proliferative glomerulonephritis, and represent the source of the replacing mesangial cells.