Mesangioproliferative Glomerulonephritis Research Articles

Nephrin expression is increased in anti-Thy1.1-induced glomerulonephritis in rats

Nephrin is an important constituent of the glomerular filtration barrier and alteration of its expression is associated with severe proteinuria. In this study we show that injection of an anti-Thy1.1 antibody in rats not only induces a mesangioproliferative glomerulonephritis associated with increased proteinuria, but also leads to a sustained increase of nephrin mRNA and protein expression in renal glomeruli over a time period of 29 days. In contrast, podocin and CD2AP, two proteins shown to interact with nephrin in the slit diaphragm, are acutely downregulated at days 3–7 and, thereafter, recovered again to normal levels after 29 days. Interestingly, immunofluorescence staining of kidney sections at day 10 of the disease shows a highly heterogeneous pattern, in that some podocytes show complete absence of nephrin, whereas others show highly accumulated staining for nephrin compared to control sections, which in total results in an increased level of nephrin per glomerulus. In summary, our data show that in the course of mesangioproliferative glomerulonephritis in rats, an upregulation of nephrin expression occurs with a concomitant transient downregulation of podocin and CD2AP which may account for a highly dysregulated filtration barrier and increased proteinuria.

Role of mesangial Factor V expression in crescent formation in rat experimental mesangioproliferative glomerulonephritis.

It has been suggested that fibrin deposition participates in the development of crescents in active glomerulonephritis (GN). In human IgA nephropathy, which is a common form of mesangioproliferative GN (MsPGN), crescent formation is occasionally observed in active disease, leading to end-stage renal failure. Factor V is a membrane-bound potent cofactor for the conversion of prothrombin to thrombin by Factor Xa. An in vivo study was conducted to clarify the contribution of local fibrin production to crescent formation in MsPGN through mesangial Factor V expression. Wistar rats were injected intravenously with rabbit anti-rat thymocyte serum. Three days after injection, mesangiolysis with intense mesangial Factor V expression was observed and immunoelectron microscopy revealed fibrin localization in mesangiolytic lesions, which had spread into the glomerular basement membrane adjacent to the destroyed mesangium, accompanied by clots in Bowman's space. Marked glomerular fibrin deposition, together with its deposition in Bowman's space and cellular crescent formation, was noted with mesangial proliferation on day 8. Specific bands for Factor V mRNA were also detected from isolated glomeruli. Fibrin deposition and cellular crescent formation were significantly suppressed by treatment with anti-Factor V antibody. These results suggest that local fibrin production, following mesangial Factor V expression, together with mesangiolysis that spreads to the adjacent glomerular basement membrane, plays a role in crescent formation in MsPGN.

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

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

Mesangial cell expression of proto-oncogene Ets-1 during progression of mesangioproliferative glomerulonephritis

Ets-1 is a transactivator of matrix-associated genes and key factor in neoangiogenesis. The expression of Ets-1 mRNA and protein was analyzed in healthy rat kidney and in a model for mesangioproliferative disease without and with inhibition of platelet-derived growth factor-B (PDGF-B) activity. Immunohistochemistry was performed using a specific noncrossreacting anti-Ets-1 antibody and included a counterstain with alpha-smooth muscle actin (alpha-SMA). Nuclear proteins from isolated glomeruli were analyzed by Western blotting. Changes in Ets-1 mRNA levels were detected by in situ hybridization and Northern blotting. PDGF-B antagonism was performed in nephritic rats by specific aptamers. The distribution of Ets-1-positive cells in healthy rats was heterogenous with exclusively nuclear staining of glomerular, tubular, and vascular cells. Profound changes were detected in the anti-Thy 1.1 glomerulonephritis. Nuclear Ets-1 staining was intense in mesangial cells, whereas podocyte and endothelial cell staining was unchanged. The strongest signal was seen on days 2 to 7. By Western blotting of glomerular proteins a single 52 kD band was detected in healthy rats, which was increased 4.5-fold after disease induction. At the same time a 54 kD band appeared that most likely represents phosphorylated Ets-1. Ets-1 transcripts were detected in mesangial cells that include exon IV but lack exon VII. A concordant 6.4-fold up-regulation of mRNA was detected in glomeruli. Specific PDGF-B antagonism by aptamer treatment from days 3 to 7 after disease induction led to reduced Ets-1 expression on day 7, correlating with decreased mesangial cell numbers. A temporal increase of mesangial cell Ets-1 expression correlates with mesangial cell activation in mesangioproliferative glomerulonephritis. PDGF-B may partially contribute to the increased expression.

Glomerulonephritis in water buffaloes ( Bubalus bubalis) naturally infected by Fasciola hepatica

Glomerulonephritis caused by Fasciola hepatica was observed in buffaloes. Renal biopsies of 20 buffaloes, 11 with F. hepatica and 9 uninfected buffaloes (controls), were examined by light microscopy, direct and indirect immunofluorescence, and immunohistochemical analysis. The biopsies of seven (63.6%) infected buffaloes revealed membranoproliferative glomerulonephritis, three biopsies (27.3%) showed mesangioproliferative glomerulonephritis, and one kidney presented normal biopsy specimens. In the control group, seven buffaloes (77.8%) presented normal biopsy specimens, while two (22.2%) revealed glomerulonephritis—one with a membranoproliferative pattern, and the other with a mesangioproliferative pattern—with extensive inflammatory cell infiltrate. Our conclusion is that glomerulopathy is associated with fascioliasis and that buffaloes are suitable as a naturally existing experimental model of renal injury by circulating immune complexes.

Coagulation in the mesangial area promotes ECM accumulation through factor V expression in MsPGN in rats.

It is well known that tissue factor starts the extrinsic coagulation pathway, which activates factor X to Xa, and factor V is a membrane-bound potent cofactor for the terminating stage of prothrombin activation by factor Xa. In a previous in vitro study, factor V was induced in cultured mesangial cells by inflammatory stimulation and increased expression of factor V promoted fibrin generation on the cultured mesangial cell surface. We report that extracellular matrix (ECM) accumulation is increased in association with coagulation in the mesangial area through factor V expression in mesangioproliferative glomerulonephritis (MsPGN). Wistar rats were intravenously injected with rabbit anti-rat thymocyte serum accompanied with or without simultaneous injection of rabbit anti-factor V antibody. Time course study in immunohistochemistry revealed that factor V expression was prominent on day 3 and fibrin-related antigen (FRA) deposition, then ECM accumulation, followed from day 3 to day 8. Massive fibronectin depositions and transforming growth factor (TGF)-beta expression were also noted in glomeruli from the disease control group, markedly higher than those in the normal group, and these depositions and expressions were significantly decreased in the anti-factor V neutralizing antibody-injected group. Northern blot analysis revealed that factor V mRNA expression was prominent on day 3 and was weak on day 8. Double-labeling experiments revealed the frequent colocalization of alpha-smooth muscle actin with factor V, FRA, and fibronectin in the same mesangial areas of glomeruli. TGF-beta, connective tissue growth factor (CTGF), collagen type IV, and fibronectin mRNA were upregulated in the disease control group, and anti-factor V-neutralizing antibody injection suppressed these mRNA expressions in glomeruli. The present results suggest that ECM components accumulation may progress in accordance with coagulation in the mesangial area through mesangial factor V expression and upregulated expression of TGF-beta and CTGF in MsPGN.

Histopathological diagnosis and outcome of paediatric nephrotic syndrome.

To determine the histological picture and outcome of treatment in cases of childhood nephrotic syndrome who needed renal biopsy. An analytical study. Department of Paediatrics, King Edward Medical College, and Mayo Hospital, Lahore from 1st June 1996 to 31st December, 2001 over a period of 5-1/2 years. Children suffering from nephortic syndrome who had atypical features at presentation were initially or late non-responders; frequent relapsers on < 1mg kg/day and were steroid dependent or frequently relapsed on < 1 mg kg/day but developed steroid toxicity were included. Renal biopsy was performed in these patients. Treatment was administered according to the histopathology reports. Prednisolone 60 mg /m2/day followed after response by 40 mg /m2 on alternate days (AD) which was later tapered off. In minimal change nephrotic syndrome (MCNS) with frequent relapses cyclophosphamide, cyclosporine and levimisole were used. For steroid resistant focal segmental glomerulosclerosis (FSGS) intravenous pulses of methyl-prednisolone and cyclosporine were also given. These patients were followed to see the response of the therapy. The commonest diagnosis was focal segmental glomerulosclerosis (FSGS) (42%) followed by minimal change disease (MCNS) (22%), membranoproliferative or mesangiocapillary glomerulonephritis (MPGN) (14%) and Mesangioproliferative glomerulonephritis (Mes PGN) (12%). There were 6% cases of membranous nephropathy and 4% of diffuse proliferative glomerulonephritis. On presentation, 40% had hematuria, 20% were found to be hypertensive, 12% patients had renal insufficiency and in 4% C3 level was low. Majority of the patients with MPGN and FSGS had atypical features whereas none of the patients with membranous nephropathy had any of these features. Thirty percent cases each of FSGS and MCNS were responders. Among non-responders there were 4 cases of FSGS and one of MPGN. FSGS was the commonest histology in cases of childhood nephrotic syndrome that needed renal biopsy. Highest frequency of atypical features was seen in MPGN and FSGS.

Decorin in renal inflammation and fibrosis – more than an inhibitor of TGF‐β

Introduction Decorin, a small leucine‐rich proteoglycan, participates in extracellular matrix assembly and influences cell behaviour by interacting with signalling membrane receptors and TGF‐β. Treatment with decorin has been shown to have beneficial effects in an acute model of mesangioproliferative glomerulonephritis due to interaction with TGF‐β. The underlying mechanisms, however, remain unclear because upon complex formation with TGF‐β, the cytokine's activity may become increased, decreased or not influenced at all. Hence, the objectives of the present study were twofold: firstly, to provide evidence that decorin influences the course and final outcome of renal inflammation and secondly, to find anti‐fibrotic mechanisms of decorin both related and unrelated to the regulation of TGF‐β activity.Results Based on our studies in human diabetic nephropathy, we postulate two regulatory mechanisms by which decorin modulates TGF‐β‐mediated fibrosis: (1) increased quantities of glomerular decorin are synthesized and form complexes with TGF‐β, which then are removed via glomerular capillaries or the urinary tract, and (2) in the presence of type‐I collagen, decorin is able to sequester TGF‐β in the extracellular matrix, thereby withdrawing the cytokine from its cell‐surface receptors. Furthermore, we have compared the evolution of tubulointerstitial fibrosis in wild‐type (WT) and decorin–/– mice in a model of unilateral ureteral obstruction. Without obstruction, kidneys from decorin–/– mice did not differ in any aspect from their WT counterparts. However, already 12 h after obstruction, decorin–/– animals showed lower levels of p27KIP1 and soon thereafter a more pronounced up‐regulation and activation of initiator and effector caspases, followed by enhanced apoptosis of tubular epithelial cells. At later stages, a higher increase of TGF‐β1 became apparent. After 7 days, there was a 15‐fold transient up‐regulation of the related proteoglycan biglycan, which was mainly caused by the appearance of biglycan‐expressing mononuclear cells. Other small proteoglycans showed no similar response. Owing to enhanced degradation of type‐I collagen and increased tubular epithelial cell apoptosis, end‐stage kidneys from decorin–/– animals were more atrophic than WT kidneys.Conclusion These data suggest that decorin exerts beneficial effects on renal inflammation, primarily by influencing the expression of a key cyclin‐dependent kinase inhibitor, thereby limiting the degree of apoptosis and tubular atrophy. In later stages, anti‐fibrotic effects of decorin are based on the regulation of TGF‐β1 expression, mononuclear cell infiltration and collagen turnover.

The benefit of plasma exchange in anuric patient with steroid-resistant nephrotic syndrome - long-term follow-up

Recently, plasma exchange (PE) has been added to the treatment regimen for patients with steroid-, cyclophosphamide-, and cyclosporine-resistant nephrotic syndrome. This is a case report of a female patient with severe acute renal failure (ARF) during the relapse of steroid-resistant nephrotic syndrome (SRNS) who recovered completely after PE and became steroid-sensitive in further follow-up of 48 months. An 8-year-old girl was referred to Nephrology Department of the University Children's Hospital due to relapse of SRNS complicated with ARF. Her nephrotic syndrome (mesangioproliferative glomerulonephritis) was diagnosed at the age of 17 months. During the following 6 years, she was given several therapeutic regimens including pulse prednisolone, cyclophosphamide, Cyclosporine (CyA), but she continued to have frequent relapses and during the last six months she was steroid- and cyclosporine-resistant. Three days before admission, she was febrile, had cellulites of the lower abdominal wall, diarrhea, vomiting, hypovolemic shock with generalized edema, severe hypoproteinemia and hypoalbuminemia. In a local hospital, she was treated with fresh frozen plasma, albumin, methylprednisolone, furosemide and antibiotics, but she became anuric and was referred to our hospital. There were no signs of hemolysis. Anuria lasted for 12 days. She was discharged after 42 days in remission with normal GFR. Principal treatment included: 13 sequential hemodialysis sessions (30% of body weight was removed as excess volume), 6 PE, corticosteroids, CyA, ACE inhibitor, antibiotics, antimycotics, and cimetidine. Six PE sessions were performed every other day. In further 48-month follow-up, while under the treatment of CyA the patient had a few steroid-sensitive relapses, the first being 6 months after PE. The second kidney biopsy showed focal segmental glomerulosclerosis with no signs of apparent CyA nephrotoxicity. "Malignant" course of disease in our patient was a good reason to introduce PE into the treatment. Since PE was the only additional mode of treatment, it is believed that its effect was crucial for milder activity of the disease.

Management of idiopathic nephrotic syndrome in childhood

The management of idiopathic nephrotic syndrome (INS) in children includes immunosuppressive and symptomatic treatment. The response to corticosteroid therapy is the best prognostic marker of the disease. The majority of children with INS (about 85%) are steroid-sensitive as they normalize proteinuria within 4 weeks of daily, oral prednisone administration. The most of steroid-sensitive patients (94%) has minimal change of nephrotic syndrome, while the majority (80.5%-94.4%) of those who are steroid-resistant has focal segmental glomerulosclerosis or mesangioproliferative glomerulonephritis. Initial therapy of INS consists of 60 mg/m2/day prednisone daily for 4 weeks followed by 40 mg/m2 on alternate days for 4 weeks, thereafter decreasing alternate day therapy every month by 25% over the next 4 months. Thus, the overall duration of the initial corticosteroids course is 6 months that may be significantly protective against the future development of frequent relapses. Approximately 30% of patients experience only one attack and are cured after the first course of therapy; 10-20% have only 3 or 4 steroid-responsive episodes before permanent cure; the remaining 40-50% of patients are frequent relapsers, or steroid-dependent. Standard relapse therapy consists of 60 mg/m2/ day prednisone until urine is protein free for at least 3 days, followed by 40 mg/m2 on alternate days for 4 weeks. The treatment of frequent-relapses and steroid-dependent INS includes several different regimens: maintenance (6 months) alternate steroid therapy just above steroid threshold (0.1-0.5 mg/kg/48h), levamisole, alkylating agents (cyclophosphamide or chlorambucil) or cyclosporine. The worse prognosis is expected in steroid-resistant patients who are the most difficult to treat. Renal biopsy should be performed in them. At present, there is no consensus on therapeutic regimen for steroid-resistant patients. The following immunosuppressive drugs have been used with varying success: cyclosporine, intravenous methyl prednisone pulses alone or combined with or followed by alkylating agents, plasma-exchange, and angiotensin-converting enzyme (ACE) inhibitors. Symptomatic treatment includes: 1) dietary regimen with normal protein intake and salt restriction, 2) calcium and vitamin D are prescribed with steroids, 3) diuretics should be used in case of severe edema, 4) infusion of albumin in case of severe hypovolemia, 5) treatment of hypertension, 6) anticoagulant therapy, and 7) prophylactic antibiotics in high-risk patients.

Glomerular gene and protein expression of angiogenic factors in mesangioproliferative glomerulonephritis in the rat

The distinct roles of angiopoietin (Ang)-1 and Ang2, counteracting ligands for the endothelium-specific Tie2 receptor, in tumor development and progression have remained poorly understood. We investigated the expression of Ang1 and Ang2 during multistep mouse skin carcinogenesis and in human squamous cell carcinoma (SCC) xenografts. Expression of Ang2, but not of Ang1, was up-regulated in angiogenic tumor vessels already in early stages of skin carcinogenesis and was also strongly increased in SCCs. Stable overexpression of Ang1 in human A431 SCCs resulted in a more than 70% inhibition of tumor growth, associated with enhanced Tie2 phosphorylation levels, as compared with low levels in control transfected tumors. No major changes in the vascular density, vascular endothelial growth factor mRNA and protein expression, and vascular endothelial growth factor receptor-2 phosphorylation levels were observed in Ang1-expressing tumors. However, the fraction of tumor blood vessels with coverage by α-smooth muscle actin-positive periendothelial cells was significantly increased, indicative of an increased vascular maturation status. These findings identify an inhibitory role of Ang1/Tie2 receptor-mediated vessel maturation in SCC growth and suggest that up-regulation of its antagonist, Ang2, during early-stage epithelial tumorigenesis contributes to the angiogenic switch by counteracting specific vessel-stabilizing effects of Ang1.

Expression of Ras GTPases in normal kidney and in glomerulonephritis.

Small monomeric Ras GTPases play critical and specific roles in the control of cellular proliferation and apoptosis but the expression of the three Ras isoforms (Ha-Ras, Ki-Ras and N-Ras) in human renal tissue is unknown. This work is an immunohistochemical study of Ras expression in normal renal tissue and in membranous glomerulonephritis (MGN), IgA nephropathy (IgAN) and IgA-negative mesangioproliferative glomerulonephritis (MPGN). Formalin-fixed, paraffin-embedded tissue was stained using pan-Ras monoclonal antibody (mAb) and Ras isoform-specific mAb. Detection employed a (DAKO Envision) modified polymer system. The expression of Ras isoforms in normal human kidney was cell-specific. For example, N-Ras was detected in tubule epithelial cells but not in glomerular or interstitial cells. Ki-Ras was expressed in mesangial cells, interstitial cells and in proximal convoluted tubule cells (PCT) (particularly localized at brush borders) and in collecting duct cells (CD) (localized to cell membranes) but not in podocytes. Cytoplasmic Ha-Ras was detected in all the above cell types except podocytes. MGN was associated with podocyte expression of all three Ras isoforms and with reduced mesangial cell expression of Ha-Ras and Ki-Ras. IgAN was characterized by podocyte expression of Ha-Ras (but not Ki-Ras) and reduced mesangial cell expression of Ki-Ras without alterations in mesangial Ha-Ras expression. MPGN was associated with reduced mesangial cell Ha-Ras and Ki-Ras expression without significant podocyte Ras expression. These disease-specific and isoform-specific alterations in Ras expression may be of significance in pathogenesis and warrant further functional investigation.

A fully human monoclonal antibody (CR002) identifies PDGF-D as a novel mediator of mesangioproliferative glomerulonephritis.

PDGF-B is of central importance in mesangioproliferative diseases. PDGF-D, a new PDGF isoform, like PDGF-B, signals through the PDGF betabeta-receptor. The present study first determined that PDGF-D is mitogenic for rat mesangial cells and is not inhibited by a PDGF-B antagonist. Low levels of PDGF-D mRNA were detected in normal rat glomeruli. After induction of mesangioproliferative nephritis in rats by anti-Thy 1.1 mAb, glomerular PDGF-D mRNA and protein expression increased significantly from days 4 to 9 in comparison with nonnephritic rats. Peak expression of PDGF-D mRNA occurred 2 d later than peak PDGF-B mRNA expression. In addition, PDGF-D serum levels increased significantly in the nephritic animals on day 7. For investigating the functional role of PDGF-D, neutralizing fully human mAb were generated using the XenoMouse technology. Rats with anti-Thy 1.1-induced nephritis were treated on days 3 and 5 with different amounts of a fully human PDGF-DD-specific neutralizing mAb (CR002), equal amounts of irrelevant control mAb, or PBS by intraperitoneal injection. Specific antagonism of PDGF-D led to a dose-dependent (up to 67%) reduction of glomerular cell proliferation. As judged by double immunostaining for 5-bromo-2'-deoxyuridine and alpha-smooth muscle actin, glomerular mesangial cell proliferation was reduced by up to 57%. Reduction of glomerular cell proliferation in the rats that received CR002 was not associated with reduced glomerular expression of PDGF-B mRNA. PDGF-D antagonism also led to reduced glomerular infiltration of monocytes/macrophages (day 5) and reduced accumulation of fibronectin (day 8). In contrast, no effect was noted in normal rats that received an injection of CR002. These data show that PDGF-D is overexpressed in mesangioproliferative states and can act as an auto-, para-, or even endocrine glomerular cell mitogen, indicating that antagonism of PDGF-D may represent a novel therapeutic approach to mesangioproliferative glomerulonephritides.

Pathways to recovery and loss of nephrons in anti-Thy-1 nephritis.

The present histopathologic study of anti-Thy-1.1 models of mesangioproliferative glomerulonephritis in rats provides a structural analysis of damage development and of pathways to recovery and to nephron loss. As long as the disease remains confined to the endocapillary compartment, the damage may be resolved or recover with a mesangial scar. Irreversible lesions with loss of nephrons emerge from extracapillary processes with crucial involvement of podocytes, leading to tuft adhesions to Bowman's capsule (BC) and subsequent crescent formation. Two mechanisms appeared to be responsible: (1) Epithelial cell proliferation at BC and the urinary orifice and (2) misdirected filtration and filtrate spreading on the outer aspect of the nephron. Both may lead to obstruction of the tubule, disconnection from the glomerulus, and subsequent degeneration of the entire nephron. No evidence emerged to suggest that the kind of focal interstitial proliferation associated with the degeneration of injured nephrons was harmful to a neighboring healthy nephron.

Biglycan, a Nitric Oxide-regulated Gene, Affects Adhesion, Growth, and Survival of Mesangial Cells

During glomerular inflammation mesangial cells are the major source and target of nitric oxide that pro-foundly influences proliferation, adhesion, and death of mesangial cells. The effect of nitric oxide on the mRNA expression pattern of cultured rat mesangial cells was therefore investigated by RNA-arbitrarily-primed polymerase chain reaction. Employing this approach, biglycan expression turned out to be down-regulated time- and dose-dependently either by interleukin-1beta-stimulated endogenous nitric oxide production or by direct application of the exogenous nitric oxide donor, diethylenetriamine nitric oxide. There was a corresponding decline in the rate of biglycan biosynthesis and in the steady state level of this proteoglycan. In vivo, in a model of mesangioproliferative glomerulonephritis up-regulation of inducible nitric-oxide synthase mRNA was associated with reduced expression of biglycan in isolated glomeruli. Biglycan expression could be normalized, both in vitro and in vivo, by using a specific inhibitor of the inducible nitric-oxide synthase, l-N6-(l-iminoethyl)-l-lysine dihydrochloride. Further studies showed that biglycan inhibited cell adhesion on type I collagen and fibronectin because of its binding to these substrates. More importantly, biglycan protected mesangial cells from apoptosis by decreasing caspase-3 activity, and it counteracted the proliferative effects of platelet-derived growth factor-BB. These findings indicate a signaling role of biglycan and describe a novel pathomechanism by which nitric oxide modulates the course of renal glomerular disease through regulation of biglycan expression.

Laser capture microdissection and real-time PCR for analysis of glomerular endothelin-1 gene expression in mesangiolysis of rat anti-Thy 1.1 and murine Habu Snake Venom glomerulonephritis.

Molecular analysis of pathologic changes in glomeruli requires methods allowing rapid and exact detection of alterations in gene expression. Here, we analyzed endothelin-1 (ET-1) mRNA expression in mesangiolytic glomeruli during the course of a rat and murine model of mesangioproliferative glomerulonephritis (GN). A novel method combining laser capture microdissection (LCM), which permits the precise removal of selected mesangiolytic glomeruli, with a highly sensitive real-time RT-PCR technique was used. Anti-Thy 1.1. GN was introduced in male Sprague-Dawley rats (1.0 mg/kg body weight of OX-7 IV) and Habu Snake Venom GN was introduced in C57BL6 mice (habu snake venom toxin 6 mg/kg body weight IV). The degree of mesangiolysis during both GNs was analyzed using a semiquantitative scoring system. Mesangiolytic glomeruli were microdissected at different days of the diseases (day 2, 6, and 12 in anti-Thy 1.1 GN and days 1, 3, 7, and 14 in Habu Snake Venom GN) and from normal control animals. After RNA extraction and cDNA synthesis, ET-1 gene expression was measured by real-time RT-PCR. In parallel, in anti-Thy 1.1. GN ET-1 mRNA expression was analyzed using semiquantitative nonradioactive in situ hybridization; ET-1 protein expression was investigated by immunohistochemistry. Mesangiolysis peaked at day 6 in anti-Thy1.1 GN and at day 1 in Habu Snake Venom GN. Mesangiolytic glomeruli were easily microdissected on cryostat sections in both models; quantification of mRNA with RT-PCR was reliable and reproducible. Glomerular ET-1 mRNA expression increased during the course of anti-Thy 1.1 GN and Habu Snake Venom GN peaked when mesangiolysis was most pronounced. This was seen by RT-PCR after glomerular LCM and by in situ hybridization; in parallel, glomerular ET-1 protein expression was increased. Combination of LCM and RT-PCR is a reliable method for quantification of localized gene expression in isolated renal structures. The above data argue for an important role of ET-1 in pathogenesis and/or repair of mesangiolysis in experimental mesangioproliferative GN.

Incidence of latent mesangial IgA deposition in renal allograft donors in Japan

Mesangial immunoglobulin A (IgA) deposition is incidentally encountered in asymptomatic individuals, but its precise frequency and significance had not been clarified. The background of the latent IgA deposition is related to the epidemiology and pathogenesis of IgA nephropathy. Zero-hour allograft biopsies were performed in 510 renal transplantations (446 living donors, and 64 cadaveric donors) at the Kidney Center of Tokyo Women's Medical University. Mesangial IgA and C3 deposition were analyzed immunohistochemically, and the frequency and clinicopathologic features of mesangial IgA deposition were investigated. Mesangial IgA deposition was present in 82 (16.1%) of the total 510 allografts with no statistical difference between living donors (72/446, 16.1%) and cadaveric donors (10/64, 15.6%) or between blood-related donors (66/392, 16.8%) and nonblood-related donors (16/110, 14.5%). Mesangial C3 deposition was present in 16 (19.5%) of the 82 allografts with mesangial IgA deposition. The grade of hematuria in IgA(+) donors was significantly higher than IgA(-) donors (1.30 +/- 1.17 vs. 0.86 +/- 0.89, P = 0.025). Histologic investigation of IgA(+) allografts revealed the frequency of mesangioproliferative glomerulonephritis (PGN) was significantly higher in IgA(+)/C3(+) allografts (8/16, 50%) than in IgA(+)/C3(-) allografts (11/66, 16.7%) (P = 0.0084). Moreover, the number of infiltrated macrophages to glomerulus (cells/glomerular cross section) was significantly higher in the IgA(+)/C3(+) allografts than in IgA(+)/C3(-), IgA(-)/C3(+) and IgA(-)/C3(-) allografts (1.10 +/- 0.62 vs. 0.61 +/- 0.42, P = 0.0008; 0.47 +/- 0.34, P = 0.023; and 0.37 +/- 0.23, P = 0.002, respectively). The latent mesangial IgA deposition was a relatively common phenomenon in the healthy Japanese donors. This phenomenon was associated with mild degree of microhematuria, mesangial proliferation and glomerular macrophage infiltration in some of the affected individuals, especially with combined IgA and C3 deposition.

Histopathological spectrum of childhood nephrotic syndrome in Indian children.

Nephrotic syndrome in children is a clinical manifestation of different histopathological subtypes. There is a paucity of recent large studies dealing with the histopathological spectrum from developing countries. A prospective study was performed from January 1990 to December 2000 at our center, involving 600 children (with age of onset up to 16 years) with idiopathic nephrotic syndrome (INS). The objectives were: (1) to study the histopathological distribution of different subtypes of INS and (2) to compare the clinical and biochemical parameters at the time of diagnosis of minimal change disease (MCD) with non-MCD subtypes. For the purpose of this study we analyzed only those children with INS who underwent biopsies. The study group included 290 children in which adequate biopsy reports were available. There were 213 males and 77 females. Mean age at onset of INS was 7.9+5.1 years. Facial edema was found in 286 (98.6%), microhematuria in 120 (41.3%), gross hematuria in 7 (2.5%), and hypertension in 77(26.8%) patients. All patients of the study group were seronegative for HBsAg and HIV. Focal and segmental glomerulosclerosis (FSGS) was the most common histopathological subtype, occurring in 110 of 290 children (38%). Other subtypes included MCD in 95 children (32%), membranoproliferative glomerulonephritis (MPGN) in 44 children (15%), mesangioproliferative glomerulonephritis in 33 children (11%), membranous glomerulonephritis in 5 children (2%), and diffuse mesangial sclerosis in 3 children (1%). In children under 8 years of age, MCD was the most common entity, whereas FSGS predominated in children with age at onset greater than 8 years. The age at onset of nephrotic syndrome was significantly higher in the non-MCD group than the MCD group. The incidence of hypertension, microhematuria, and gross hematuria was significantly lower in the MCD group. MCD remains the most common histopathological subtype in Indian children with INS and onset under 8 years of age. The incidence of MPGN continues to be high. MCD can be differentiated from non-MCD subtype by younger age at onset, absence of hypertension, and absence of microscopic hematuria.

Specific inhibition of Egr-1 prevents mesangial cell hypercellularity in experimental nephritis

Mesangial cell proliferation is a frequent finding in glomerulonephritis. In cultured mesangial cells, we demonstrated that inhibition of the zinc finger transcription factor, early growth response gene-1 (Egr-1), by specific antisense oligonucleotides (AS ODN) blocks mesangial cell proliferation. Therefore, we here investigated the effect of Egr-1 inhibition on the course of an experimental mesangioproliferative glomerulonephritis in vivo. On day 3 after induction of anti-Thy-1.1 nephritis, specific glomerular oligonucleotide transfer was achieved by injection of an oligonucleotide/hemagglutinating virus of Japan/liposome mixture into the left renal artery. The right kidney was left untreated. Induction of nephritis led to a sixfold induction of Egr-1 protein on day 6 of disease. This increase in Egr-1 expression was reduced by 48% in the left kidney by transfer of specific AS ODN. In parallel, the increases in glomerular cellularity, number of mitoses, and glomerular tuft area observed in day 6 nephritic animals were inhibited in the left kidney by 60%, 53%, and 50%, respectively. Changes in the right kidney were not significantly influenced. Likewise, control oligonucleotides showed no effect. Finally, the expression of platelet-derived growth factor-B (PDGF-B), a known target gene of Egr-1, was repressed by transfer of specific AS ODN against Egr-1. We conclude that the transcription factor Egr-1 plays a critical role for mesangial cell proliferation in vivo. Interfering with the induction of Egr-1 or with its target genes could give rise to novel therapeutic principles in mesangioproliferative glomerulonephritis.

Cux-1 transgenic mice develop glomerulosclerosis and interstitial fibrosis

Cux-1 is a murine homeobox gene that is highly expressed in the nephrogenic zone of the developing kidney. Transgenic mice ectopically expressing Cux-1 develop renal hyperplasia associated with down-regulation of the cyclin kinase inhibitor p27. Because the reduction of p27 has been associated with mesangial cell proliferation and glomerular disease, we evaluated glomerular changes in Cux-1 transgenic mice. Adult kidneys from Cux-1 transgenic mice were analyzed morphologically for changes in glomerular cell number and for changes in mesangial and interstitial extracellular matrix deposition. Mesangial matrix expansion was identified by light microscopy. Glomerular cell number was performed following immunohistochemistry. Type IV collagen deposition was analyzed by immunofluoresence and Western blotting. Renal function was evaluated by serum protein, blood urea nitrogen (BUN), creatinine, and electrolyte analysis, and by urine protein and creatinine analysis. In adult transgenic glomeruli, Cux-1 was ectopically expressed in mesangial cells, and this was associated with an increase in mesangial cell number, resulting from an increase in proliferation. There was a marked increase in mesangial matrix area in transgenic mice compared to non-transgenic littermates, related to an increase in type IV collagen. Podocyte foot process effacement was observed in transgenic mice, and this was related to an increase in urinary albumin. Interstitial fibrosis was also observed in transgenic kidneys. These observations indicate that increased expression of Cux-1 in mesangial cells results in cell proliferation and mesangial expansion. In addition, these changes are potentially related to disruption of podocyte architecture leading to loss of filtration. These results suggest that expression of Cux-1 is sufficient to induce the early events of mesangioproliferative glomerulonephritis.