Rat Model Of Glomerulonephritis Research Articles

Protein Tyrosine Phosphatase Receptor-type Q: Structure, Activity, and Implications in Human Disease.

Protein tyrosine phosphatase receptor-type Q (PTPRQ), a member of the type III tyrosine phosphatase receptor (R3 PTPR) family, is composed of three domains, including 18 extracellular fibronectin type III (FN3) repeats, a transmembrane helix, and a cytoplasmic phosphotyrosine phosphatase (PTP) domain. PTPRQ was initially identified as a transcript upregulated in glomerular mesangial cells in a rat model of glomerulonephritis. Subsequently, studies found that PTPRQ has phosphotyrosine phosphatase and phosphatidylinositol phosphatase activities and can regulate cell proliferation, apoptosis, differentiation, and survival. Further in vivo studies showed that PTPRQ is necessary for the maturation of cochlear hair bundles and is considered a potential gene for deafness. In the recent two decades, 21 mutations in PTPRQ have been linked to autosomal recessive hearing loss (DFNB84) and autosomal dominant hearing loss (DFNA73). Recent mutations, deletions, and amplifications of PTPRQ have been observed in many types of cancers, which indicate that PTPRQ might play an essential role in the development of many cancers. In this review, we briefly describe PTPRQ structure and enzyme activity and focus on the correlation between PTPRQ and human disease. A profound understanding of PTPRQ could be helpful in the identification of new therapeutic targets to treat associated diseases.

Isotretinoin and the Kidney: Opportunities and Threats.

Retinoids are one of the most effective drugs in inducing complete or prolonged remission of severe acne vulgaris, but the adverse reactions associated with the use of them are raising a concern about the potential effect of these drugs on internal organs function such as the kidney. The aim of this review is to comprehensively gather data about isotretinoin, both potential adverse and beneficial effects on the kidney based on the current experimental and clinical findings. Very few studies, including five case reports, described that systemic oral isotretinoin within usual doses (40 mg/day or 0.5 mg/kg⁄day) within 1 to 4 months of treatment might be associated with different types of renal dysfunctions. These include acute interstitial nephritis, nephrotic syndrome, and hematuria with dysuria. The adverse reactions of systemic isotretinoin on the kidney and urinary system are unlikely and rare. In contrast, six experimental studies demonstrated the beneficial effects of either oral or parenteral low- (2 or 5 mg/kg/day) or high- (10, 20, 25, 40 mg/kg/day) dose isotretinoin on the kidney in the rat models of glomerulonephritis, obstructive nephropathy or allograft nephropathy. The nephroprotective functions of isotretinoin in these studies were attributed to its anti-proliferative, anti-fibrotic, and anti-inflammatory actions. However, clinical studies are warranted to elucidate the possible beneficial effects of isotretinoin in preventing or attenuating kidney injury in different settings.

Reno‐protective effects of everolimus vs. bone marrow‐derived stem cells in rat model of glomerulonephritis

Glomerular endothelial injury and effectiveness of glomerular endothelial repair play a crucial role in the progression of renal disease. Recent studies suggest that progenitor cells could be an effective tool to enhance glomerular endothelial repair. We hypothesize that bone marrow‐derived stem cells (BMDSCs) attenuate experimental glomerulonephritis by restoring glomerular endothelial barrier function through improving the repair process. Anti‐Thy1 glomerulonephritis was induced in male Sprague Dawley rats by a single injection of an antibody against Thy1, which is mainly expressed on glomerular mesangial cells. Rats were then treated with the immunosuppressant everolimus daily or injected with bone marrow‐derived endothelial progenitor cells after one week of injection of anti‐Thy1 (n=6–7). After nine days of injection of anti‐Thy1, glomerular albumin permeability and albuminuria were significantly increased when compared to control group (P< 0.05). Everolimus was superior to BMDSCs in attenuating the increase in glomerular albumin permeability and albuminuria in anti‐Thy1 injected rats. Only everolimus treatment significantly reduced the markers of glomerular injury, nephrinuria and podocalyxin excretion levels, in anti‐Thy1 injected rats (nephrinuria was 0.4± 0.1 vs. 0.8± 0.02 mg/day and podocalyxin excretion was 7± 1 vs. 11± 1 μg/day in everolimus treated vs. untreated anti‐Thy1 injected rats, P< 0.05). Injection of Anti‐Thy1 induced hypercellular glomeruli with loss of Bowman space and variable number of lymphocyte when compared to control. Everolimus treatment partially restored Anti‐Thy1 renal pathophysiological changes whereas BMDSCs had no significant effect. Everolimus treatment also reduced the number of renal T cells and macrophage and lowered IL‐17 and caspase 3 expression levels in the anti‐Thy1 injected rat. These data suggest that everolimus early treatment preserves glomerular endothelial barrier function in anti‐Thy1‐induced glomerulonephritis via anti‐apoptotic and anti‐inflammatory mechanisms.Support or Funding InformationThis work was supported by American Heart Association scientist development grant and intramural grant from Augusta University To Dr. Ahmed A. ElmarakbyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A-306989, an inhibitor of adenosine kinase, is renoprotective in rodent models of podocyte, basement membrane, and obstructive injury

Adenosine (ADO) is an important regulatory purine nucleoside that accumulates at sites of inflammation and tissue injury including in diseases associated with renal pathology. Endogenous levels of ADO may be increased by inhibiting the ADO-metabolizing enzyme, ADO kinase (AK). AK inhibitors have demonstrated protection in rodent models of diabetic nephropathy. To further investigate AK inhibition as a potential mechanism for renal protection, A-306989, a potent non-nucleoside AK inhibitor, was examined in both in vitro and in vivo assays of renal injury. A-306989 prevented podocyte damage (disruption of actin cytoskeleton) and increased podocyte survival following puromycin aminonucleoside (PAN) application in both mouse and human conditionally immortalized podocytes. Prophylactic oral administration of A-306989 (1.5, 5 and 15mg/kg) reduced proteinuria in a dose-dependent manner and repressed pro-inflammatory/fibrotic gene up-regulation; A-306989 was also efficacious when administered two days following the PAN-insult. A-306989 (10 and 30mg/kg) also significantly reduced proteinuria and macrophage infiltration in a rat model of glomerulonephritis. Finally, A-306989 (15 and 50mg/kg) reduced the expression levels of pro-inflammatory/fibrotic genes, and reduced macrophage infiltration (50mg/kg), but did not affect the deposition of interstitial collagen in fibrotic kidneys from mice with unilateral ureter obstruction. A-306989 also had beneficial actions on “quality of life” measures including improving body weight loss. Thus, these data indicate that enhancement of endogenous ADO levels by A-306989 can positively modulate renal pathology and mimic some of the previously reported beneficial actions of ADO A2A receptor agonists.

Epoetin beta pegol alleviates oxidative stress and exacerbation of renal damage from iron deposition, thereby delaying CKD progression in progressive glomerulonephritis rats.

The increased deposition of iron in the kidneys that occurs with glomerulopathy hinders the functional and structural recovery of the tubules and promotes progression of chronic kidney disease (CKD). Here, we evaluated whether epoetin beta pegol (continuous erythropoietin receptor activator: CERA), which has a long half-life in blood and strongly suppresses hepcidin-25, exerts renoprotection in a rat model of chronic progressive glomerulonephritis (cGN). cGN rats showed elevated urinary total protein excretion (uTP) and plasma urea nitrogen (UN) from day 14 after the induction of kidney disease (day 0) and finally declined into end-stage kidney disease (ESKD), showing reduced creatinine clearance with glomerulosclerosis, tubular dilation, and tubulointerstitial fibrosis. A single dose of CERA given on day 1, but not on day 16, alleviated increasing uTP and UN, thereby delaying ESKD. In the initial disease phase, CERA significantly suppressed urinary 8-OHdG and liver-type fatty acid-binding protein (L-FABP), a tubular damage marker. CERA also inhibited elevated plasma hepcidin-25 levels and alleviated subsequent iron accumulation in kidneys in association with elevated urinary iron excretion and resulted in alleviation of growth of Ki67-positive tubular and glomerular cells. In addition, at day 28 when the exacerbation of uTP occurs, a significant correlation was observed between iron deposition in the kidney and urinary L-FABP. In our study, CERA mitigated increasing kidney damage, thereby delaying CKD progression in this glomerulonephritis rat model. Alleviation by CERA of the exacerbation of kidney damage could be attributable to mitigation of tubular damage that might occur with lowered iron deposition in tubules.

Effects of a Tricaprylin Emulsion on Anti-glomerular Basement Membrane Glomerulonephritis in Rats: In Vivo and in Silico Studies.

Glomerulonephritis (GN) is a set of pathological conditions that result in the destruction of glomeruli and loss of renal function, commonly leading to the development of end-stage renal disease. Current pharmacotherapy is limited to immunosuppressive therapy. In the present study, we found a novel antinephritic effect of a tricaprylin emulsion in the anti-glomerular basement membrane (anti-GBM) GN rat model. We evaluated the treatment in vivo by comparing administration of the emulsion with administration of a casein kinase II (CK2) inhibitor in this rat model, and performed a gene ontology-based microarray analysis to reveal in silico the detailed mechanism of action. Our results showed that administration of the tricaprylin emulsion, or even tricaprylin alone, significantly ameliorated the anti-GBM antibody-induced renal dysfunction in these rats. We believe that tricaprylin is the key active antinephritic component of the emulsion and might be a promising drug for the effective treatment of nephritis. Moreover, with respect to microarray analysis, we developed a generally applicable and rapid method to compare gene expression profile data for multiple models of nephritis and clinical samples from a public domain microarray database.

The Effects of a Selective CK2 Inhibitor on Anti-glomerular Basement Membrane Glomerulonephritis in Rats.

Protein kinase CK2 ("casein kinase II") is a protein serine/threonine kinase that plays critical roles in biological processes such as cell growth, cell cycle progression, and apoptosis. So far, we have identified that one catalytic isozyme of CK2, CK2α, is over-expressed in the kidney during the progression of glomerulonephritis (GN). Moreover, we have shown that in vivo inhibition of CK2 by administration of CK2 inhibitors was effective in the treatment of experimental GN. Hence the development of potent CK2 inhibitors should be considered in therapeutic strategies for GN. In the present study we identified compound 13, a pyrazine derivative, as a potent CK2 inhibitor. By performing enzyme kinetics analysis in vitro, we characterized the inhibition of compound 13 toward each CK2 catalytic isozyme. Furthermore, in vivo, we demonstrated that compound 13 is effective in attenuating proteinuria, decreasing the enhanced level of blood urea nitrogen and serum creatinine, and ameliorating glomerular crescent formation in an experimental GN rat model. On the other hand, cellular apoptosis was detected in the rat testis following administration of compound 13. This study provides clues for new strategies for developing applicable compounds into CK2-targeted GN treatments.

Abstract 433: Renal Effects of Everolimus Versus Bone Marrow-derived Stem Cells in Rat Model of Glomerulonephritis

The progression of renal disease is mainly influenced by degree of glomerular endothelial injury and effectiveness of glomerular endothelial repair. Considerable research efforts have recently been undertaken to examine glomerular endothelial repair by progenitor cells. We hypothesize that bone marrow-derived stem cells (BMDSCs) ameliorate experimental glomerulonephritis by restoring glomerular endothelial barrier function through improving the repair process. Anti-Thy1-glomerulonephritis was induced in Sprague Dawley rats by a single injection of an antibody against Thy1, which is mainly expressed on glomerular mesangial cells. Rats were then treated with the immunosuppressant everolimus (EV) daily or injected with isolated BMDSCs after one week of injection of anti-Thy1. After nine days of Injection of anti-Thy1, glomerular albumin permeability and albuminuria were significantly increased when compared to control group (P< 0.05). EV was superior than BMDSCs in attenuating the increase in glomerular albumin permeability and albuminuria in anti-Thy1 injected rats. However, only EV treatment significantly reduced the markers of glomerular injury, nephrinuria and podocalyxin excretion levels in anti-Thy1 injected rats (nephrinuria was 0.4± 0.1 vs. 0.8± 0.02 mg/day and podocalyxin excretion was 7± 1 vs. 11± 1 μg/day in EV treated vs. untreated anti-Thy1 injected rats, P< 0.05). Anti-Thy1 injected rats also showed hypercellular glomeruli with loss of Bowman space and variable number of lymphocyte and EV treatment partially restored these pathophysiological changes whereas BMDSCs had no significant effect. EV treatment also reduced number of renal CD3+ and macrophage and lowered IL-17 and caspase 3 levels in the anti-Thy1 injected rat kidneys. These data suggest that EV early treatment preserve glomerular endothelial barrier function in Anti-Thy1-induced glomerulonephritis via anti-apoptotic and anti-inflammatory mechanisms.

Factor inhibiting HIF limits the expression of hypoxia-inducible genes in podocytes and distal tubular cells

The two hypoxia-inducible factors (HIF-1α and HIF-2α) are transcription factors that regulate the response to hypoxia. Recently, the factor inhibiting HIF (FIH1) was identified as a molecular oxygen-dependent dioxygenase that blunts the transcriptional activity of HIF and has also been implicated in HIF-dependent and -independent hypoxia responses. Interestingly, HIF accumulation in the kidney has been shown to confer renal protection and to also cause glomerular injury or enhance renal fibrosis. In order to better understand the regulation of hypoxia-inducible genes, we determined the expression of FIH1 in the kidney and its functional role in isolated renal cells. FIH1 was expressed only in distal tubules and in podocytes, thus showing a very distinct expression pattern, partially overlapping with sites of HIF-1α expression. In tubular cells, RNA silencing of FIH1 caused transcriptional activation of HIF target genes during hypoxia. In contrast, FIH1 silencing in podocytes enhanced transcription of hypoxia-inducible genes in an HIF-independent manner. Using the anti-Thy.1 rat model of glomerulonephritis, we found a gradual decrease of glomerular FIH1 expression during disease progression paralleled by an increase in hypoxia-inducible genes including CXCR4, a mediator of glomerular inflammation. Thus, FIH1 appears to be a suppressor of oxygen-dependent genes in the kidney, operating through HIF-dependent and -independent mechanisms.

A new pentapeptide compound, PLNPK, ameliorates anti-glomerular basement membrane nephritis in Wistar rats

PLNPK is a pentapeptide compound extracted from pig spleen with a Pro-Leu-Asn-Pro-Lys molecular structure. The spleen is the biggest immune organ in the body, in which there are lots of immunocytes and immune molecules. Our pilot study showed that PLNPK could suppress the transformation and proliferation of T lymphocytes and the production of antibodies in mice. It is widely accepted that most types of glomerulonephritis are immunological diseases caused by the reaction of antigen and antibody. Both humoral immunity and cell-mediated immunity contribute to the progress of these diseases, and suppression of immunoreactions and inflammation is important to ameliorate nephritis. After the immunosuppressive effects of this compound were discovered, this study also examined whether PLNPK had beneficial effects on a rat model of glomerulonephritis. The results suggested PLNPK (200 μg/kg/d and 400 μg/kg/d) reduced urinary protein excretion, lessened the deposit of autoantibodies along the glomerular basement membrane (GBM), reduced formation of crescent and protein casts, and ameliorated glomerular fibrosis and GBM injury. After treatment with PLNPK (200 μg/kg/d and 400 μg/kg/d) for 7 days, macrophage infiltration in the glomeruli was markedly reduced. Our results suggest that PLNPK has a beneficial effect on rat anti-GBM nephritis.

Adenosine A2Areceptor activation and macrophage‐mediated experimental glomerulonephritis

In immune-induced inflammation, leukocytes are key mediators of tissue damage. Since A(2A) adenosine receptors (A(2A)Rs) are endogenous suppressors of inflammation, we examined cellular and molecular mechanisms of kidney damage to determine if selective activation of A(2A)R would suppress inflammation in a rat model of glomerulonephritis. Activation of A(2A)R reduced the degree of kidney injury in both the acute inflammatory phase and the progressive phase of glomerulonephritis. This protection against acute and chronic inflammation was associated with suppression of the glomerular expression of the MDC/CCL22 chemokine and down-regulation of MIP-1alpha/CCL3, RANTES/CCL5, MIP-1beta/CCL4, and MCP-1/CCL2 chemokines. The expression of anti-inflammatory cytokines, interluekin (IL)-4 and IL-10, also increased. The mechanism for these anti-inflammatory responses to the A(2A)R agonist was suppression of macrophages function. A(2A)R expression was increased in macrophages, macrophage-derived chemokines were reduced in response to the A(2A)R agonist, and chemokines not expressed in macrophages did not respond to A(2A)R activation. Thus, activation of the A(2A)R on macrophages inhibits immune-associated inflammation. In glomerulonephritis, A(2A)R activation modulates inflammation and tissue damage even in the progressive phase of glomerulonephritis. Accordingly, pharmacological activation of A(2A)R could be developed into a novel treatment for glomerulonephritis and other macrophage-related inflammatory diseases.

Mesenchymal Stem Cells Prevent Progressive Experimental Renal Failure but Maldifferentiate into Glomerular Adipocytes

Glomerulonephritis (GN) is a major cause of renal failure. This study sought to determine whether intrarenal injection of rat mesenchymal stem cells (MSC) can preserve renal function in a progressive rat model of GN. Early in GN (day 10), fluorescently labeled rat MSC localized to more than 70% of glomeruli, ameliorated acute renal failure, and reduced glomerular adhesions. Fifty days later, proteinuria had progressed in controls to 40 +/- 25 mg/d but stayed low in MSC-treated rats (13 +/- 4 mg/d; P < 0.01). Renal function on day 60 in the MSC group was better than in medium controls. Kidneys of the MSC group as compared with controls on day 60 contained 11% more glomeruli per 1-mm(2) section of cortex but also significantly more collagen types I, III, and IV and alpha-smooth muscle actin. Approximately 20% of the glomeruli of MSC-treated rats contained single or clusters of large adipocytes with pronounced surrounding fibrosis. Adipocytes exhibited fluorescence in their cytoplasm and/or intracellular lipid droplets. Lipid composition in these adipocytes in vivo mirrored that of MSC that underwent adipogenic differentiation in vitro. Thus, in this GN model, the early beneficial effect of MSC of preserving damaged glomeruli and maintaining renal function was offset by a long-term partial maldifferentiation of intraglomerular MSC into adipocytes accompanied by glomerular sclerosis. These data suggest that MSC treatment can be a valuable therapeutic approach only if adipogenic maldifferentiation is prevented.

Increased Production of Adrenomedullin in Glomeruli from Anti-Glomerular Basement Membrane Glomerulonephritis Rats Treated with Methylprednisolone

Background/Aims: Adrenomedullin (AM) has anti-proliferative and apoptotic effects on mesangial cells (MCs). Both effects play an important role in the progression of glomerulonephritis (GN). Glucocorticoids are widely used for the treatment of GN; however, the relationship between AM regulation in MCs or glomeruli and glucocorticoid treatment has not been clarified. Methods: Using the cultured rat MCs, AM secretion induced by methylprednisolone (m-PSL), and MC proliferation and apoptosis caused by AM were examined. In addition, the role of AM receptor antagonist, AM(22-52), was also investigated. Then, we made an anti-glomerular basement membrane (GBM) GN rat model and compared the AM expression and production in each glomeruli obtained from the control or m-PSL-treated anti-GBM GN rats. Results: In the cultured rat MCs, AM secretion was increased by m-PSL. MC proliferation was inhibited, while MC apoptosis was increased by AM. MC apoptosis was inhibited by the addition of AM(22-52). M-PSL therapy ameliorated the progression of anti-GBM GN rats. AM expression and production were increased in the glomeruli from m-PSL-treated rats compared to the controls. Conclusion: Considering the anti-proliferative and apoptotic effects of AM on MCs, increased AM in the glomeruli might participate in the improvement of glomerular lesions in anti-GBM GN rats treated with m-PSL.

Nonparathyroid hormone-mediated calcium resorption in a rat model of immune glomerulonephritis

Skeletal demineralization is a frequent accompaniment of chronic renal disease and is likely multifactorial. We studied the role of inflammation in stimulating bone resorption in a rat model of glomerulonephritis (GN). Three-week-old Sprague-Dawley rats received either saline (n = 8) or horse spleen apoferritin and lipopolysaccharide (HSA/LPS, n = 8) by intraperitoneal injection, for 6 weeks; afterward, they were observed for either an additional 3 weeks (9 weeks total; n = 4 from each group) or 14 weeks (20 weeks total; n = 4 from each group). Kidneys were analyzed by histomorphometry, and blood and urine samples were obtained to assess bone resorption. Whole-body and isolated femur Dual-Energy X-ray Absorptiometry (DEXA) scans were performed at the end of each study. HSA/LPS-treated animals developed a proliferative GN by 9 weeks, which is associated with proteinuria but no change in renal function. Between 9 and 20 weeks, there was evidence of an increasing interstitial inflammation (1381 +/- 67 interstitial cells/mm(2) at 9 weeks and 1818 +/- 28 interstitial cells/mm(2) at 20 weeks.) There was also evidence of bone resorbing activity as assessed by experimental/control (E/C) < 1.0 at 9 (E/C plasma = 0.66 +/- 0.05) and 20 (E/C plasma = 0.52 +/- 0.04) weeks. Parathyroid hormone (PTH) levels were normal at all time points, and no differences in bone mineral density were found. This model produces not only an immune glomerular/tubular injury, but also a stimulus for bone resorption that is related to objective measures of inflammation severity. The bone resorption is not caused by renal insufficiency, hyperparathyroidism, or steroid therapy. This model will prove useful in other studies of the role of renal inflammation in skeletal disorders.

Modulation of Inflammation by Slit Protein In Vivo in Experimental Crescentic Glomerulonephritis

A basic conservation of cell migration guidance mechanisms in the nervous and immune systems was proposed when Slit, known for its role in axon guidance, was found to inhibit chemokine-induced leukocyte chemotaxis in vitro. These studies examined the role of Slit2 in modulating inflammation in vivo. In a rat model of glomerulonephritis, endogenous glomerular Slit2 expression fell after disease induction, and its inhibition during the early disease period accelerated inflammation. Ex vivo glomerular leukocytes showed decreased chemokine and chemoattractant-induced chemotaxis in response to Slit2, suggesting an anti-inflammatory role for glomerular Slit2. In contrast to the effect of inhibition, glomerulonephritis was ameliorated by systemic Slit2 administration. Slit2 treatment improved disease histologically and also improved renal function when given early in the disease course. Leukocytes harvested from rats receiving Slit2 showed decreased monocyte chemoattractant protein-1 (MCP)-1-mediated migration, consistent with a peripheral Slit2 effect. In keeping with this functional alteration, Slit2-mediated inhibition of RAW264.7 cell chemotaxis was associated with decreased levels of active cdc42 and Rac1, implicating GTPases in leukocyte Slit2 signaling. These findings suggest a role for endogenous Slit2 in the inhibition of chemoattractant-mediated signals, demonstrate a potentially important anti-inflammatory effect for Slit2 in vivo, and provide further evidence for conserved mechanisms guiding the process of migration in distinct cell types.

Counteractive effects of HGF on PDGF-induced mesangial cell proliferation in a rat model of glomerulonephritis.

Activation and proliferation of glomerular mesangial cells play an important role in the development of mesangioproliferative glomerulonephritis. We investigated the role of hepatocyte growth factor (HGF) in regulating activated mesangial cell proliferation. In glomeruli of normal rats, mesangial cells barely expressed the c-Met/HGF receptor. However, when mesangioproliferative glomerulonephritis was induced in rats by the administration of an anti-Thy 1.1 antibody, glomerular HGF expression transiently decreased along with mesangiolysis, and activation of mesangial cells was associated with upregulation of the c-Met receptor. Activated mesangial cells in culture also expressed the c-Met/HGF receptor. Although addition of HGF to cultured mesangial cells did not increase DNA synthesis, HGF did diminish PDGF-induced DNA synthesis. PDGF induced activation of ERK, which continued for at least 48 h. When PDGF and HGF were simultaneously added, HGF inhibited the prolonged activation of ERK, which suggests that early inactivation of PDGF-induced ERK may be involved in the inhibitory effect of HGF on mesangial cell proliferation. Furthermore, administration of HGF to rats with anti-Thy 1.1 nephritis resulted in a selective suppression of activated mesangial cell proliferation, and this suppressive effect was associated with attenuation of phosphorylated glomerular ERK. These results indicate that HGF counteracts PDGF-induced mesangial cell proliferation and functions as a negative regulator of activated mesangial cell proliferation.

Long‐term anti‐glomerular basement membrane disease in the rat: a model of chronic giomerulonephritis with nephrosis, hypertension and progressive renal failure

SUMMARY: Current experimental models of glomerulonephritis focus on the acute phases of renal injury. Models replicating the features of human glomerulonephritis, including hypertension, protein‐uria, hyperlipidaemia, renal fibrosis and renal failure, are lacking. the aim of this experiment was to define a rat model of glomerulonephritis that replicates the features of progressive glomerulonephritis in humans. Passive accelerated antiglomerular basement membrane disease was induced in inbred Sprague‐Dawley rats. Age‐matched control rats received pre‐immunisation, but were given saline rather than nephrotoxic serum. Groups of diseased rats (n=4–6) were killed at 4, 6 and 7 weeks, and tissues were extracted for histological assessment. Diseased rats developed renal failure over 7 weeks (91% reduction in creatinine clearance vs controls at week 7, P&lt; 0.001). Proteinuria reached a plateau from week 2 to week 7 (269.1 ± 107.9 mg/24h vs 1.00 ± 0.18 mg/mL for controls at week 7, P&lt; 0.001). Diseased rats became hyperlipidaemic (108% increase in cholesterol vs control, P&lt;0.05) and hypertensive (38% increase in systolic blood pressure vs controls, P&lt;0.001). Histology revealed progressive renal fibrosis and scarring, with fibrocellular crescent formation (93% of glomeruli by week 7), glomerulosclerosis and tubulointerstitial damage. α‐Smooth muscle actin expression and interstitial matrix (collagen III) deposition increased progressively. Urinary transforming growth factor‐β excretion was increased by over eightfold versus controls. This model of passive accelerated antiglomerular basement membrane disease simulates many clinical and pathological features of chronic giomerulonephritis in humans, and may provide a good model to test new therapies.

Nitric oxide induces MIP-2 transcription in rat renal mesangial cells and in a rat model of glomerulonephritis.

Nitric oxide is a crucial mediator of several forms of glomerulonephritis. We examined the effects of NO on the mRNA expression pattern in glomerular mesangial cells by using a low-stringency reverse transcriptase-polymerase chain reaction method and detected a cDNA fragment that was induced by interleukin 1b (IL-1b) and further up-regulated by the NO donor diethylenetriamine-nitric oxide (DETA-NO). Each respective cDNA fragment was found to match with the cDNAs of rat macrophage inflammatory protein 2 (MIP-2) and GRO/cytokine-induced neutrophil chemoattractant 2b (CINC-2b). Further characterization of MIP-2 regulation by Northern blot analysis confirmed an NO- and IL-1b-dependent increase in MIP-2 mRNA levels. Moreover, inhibition of IL-1b-induced endogenous NO formation by the NO-synthase (NOS) inhibitor L-NMMA markedly attenuated MIP-2 protein expression. We cloned 770 bp of the 5'-flanking region of rat MIP-2 and fused this fragment to a luciferase reporter gene. Transfection of the construct into mesangial cells resulted in a 3.5-fold increase in luciferase activity in cells treated with DETA-NO when compared to controls, suggesting a transcriptional mechanism for NO-induced MIP-2 expression. Deletion and mutational analysis identified critical nuclear factor (NF)-kB and NF-IL-6 binding sites required for NO regulation of MIP-2. In vivo, inhibition of NO synthesis in the Thy-1.1 model of mesangioproliferative glomerulonephritis by the specific inducible-NOS inhibitor L-NIL resulted in a marked reduction of MIP-2 mRNA expression. Furthermore, infiltration of neutrophils into the glomerulus was dramatically attenuated in L-NIL-treated rats.

Induction of TGF-β1 by the matricellular protein SPARC in a rat model of glomerulonephritis

Induction of TGF-beta1 by the matricellular protein SPARC in a rat model of glomerulonephritis. SPARC has been implicated as a counteradhesive and antiproliferative protein associated with deposits of extracellular matrix in renal disease. We have examined the effect of recombinant SPARC containing a C-terminal His tag (rSPARC) in an acute model of mesangial cell injury that is induced in the rat by an antibody against the Thy1 antigen on the mesangial cell membrane. The recombinant protein was administered 24 hours after the induction of nephritis and was infused through day 4. rSPARC was localized to the renal glomeruli of rats treated with anti-Thy1 antibody. Type I collagen and fibronectin, as well as transforming growth factor-beta1 (TGF-beta1), were increased at day 5 in rats treated with rSPARC (N = 4, P < 0.05 vs. delivery buffer), but only minimal effects were seen on mesangial cell and endothelial cell proliferation. In primary cultures of rat mesangial cells, infusion of rSPARC was associated with increases in TGF-beta1 mRNA and in total, secreted TGF-beta1 protein. rSPARC stimulates expression of TGF-beta1 both in vitro and in vivo. Given the closely regulated expression of SPARC, TGF-beta1, and type I collagen in several animal models of glomerulonephritis, we propose that SPARC could be one of the major mediators of the induction of TGF-beta1 in renal disease.

SPARC Regulates the Expression of Collagen Type I and Transforming Growth Factor-β1 in Mesangial Cells

The matricellular protein SPARC is expressed at high levels in cells that participate in tissue remodeling and is thought to regulate mesangial cell proliferation and extracellular matrix production in the kidney glomerulus in a rat model of glomerulonephritis (Pichler, R. H., Bassuk, J. A., Hugo, C., Reed, M. J., Eng, E., Gordon, K. L., Pippin, J., Alpers, C. E., Couser, W. G., Sage, E. H., and Johnson, R. J. (1997) Am. J. Pathol. 148, 1153-1167). A potential mechanism by which SPARC controls both cell cycle and matrix production has been attributed to its regulation of a pleiotropic growth factor. In this study we used primary mesangial cell cultures from wild-type mice and from mice with a targeted disruption of the SPARC gene. SPARC-null cells displayed diminished expression of collagen type I mRNA and protein, relative to wild-type cells, by the criteria of immunocytochemistry, immunoblotting, and the reverse transcription-polymerase chain reaction. The SPARC-null cells also showed significantly decreased steady-state levels of transforming growth factor-beta1 (TGF-beta1) mRNA and secreted TGF-beta1 protein. Addition of recombinant SPARC to SPARC-null cells restored the expression of collagen type I mRNA to 70% and TGF-beta1 mRNA to 100% of wild-type levels. We conclude that SPARC regulates the expression of collagen type I and TGF-beta1 in kidney mesangial cells. Since increased mitosis and matrix deposition by mesangial cells are characteristics of glomerulopathies, we propose that SPARC is one of the factors that maintains the balance between cell proliferation and matrix production in the glomerulus.