Tubular basement membrane immune deposits in association with BK polyomavirus nephropathy.

Tubular basement membrane immune deposits (TBMID) are rare in renal allografts and usually have been found in association with immune complex mediated glomerular injury. We report an association between TBMID and BK polyomavirus nephropathy (BKN). We reviewed clinical data and results of allograft biopsies of 30 patients with BKN (16 with and 14 without TBMID). TBMID were detected by immunofluorescence or electron microscopy. Initial and follow-up biopsies were assessed for degree of interstitial inflammation and fibrosis and severity of viral infection, and were correlated with patients' clinical data. Biopsies initially diagnostic for BKN with TBMID, compared to BKN biopsies without deposits, demonstrated more severe interstitial inflammation and fibrosis, and greater numbers of virally infected cells. Similar findings were present in follow-up biopsies. Utilizing three different antibodies directed against viral epitopes, viral antigens could not be detected within TBMID. Thirty percent of patients with TBMID and 70% without deposits had follow-up biopsies, in which virus could not be detected immunohistochemically. Treatment for all included decreasing immunosuppression, cidofovir and/or leflunomide. Clinical data correlated well with histological findings. We conclude that a significant proportion of patients with BKN show TBMID on kidney biopsy. The prognostic significance of this finding remains to be elucidated.

A clinicopathologic study of thrombotic microangiopathy in the setting of IgA nephropathy.

BACKGROUND: IgA nephropathy is the most common glomerulonephritis in the world. Thrombotic microangiopathy occurs in a number of clinical settings, including but not limited to thrombotic thrombocytopenic purpura/hemolytic uremic syndrome, malignant hypertension, anti-phospholipid antibody syndrome and radiation nephropathy. Renovascular complications, such as thrombotic microangiopathy, in the setting of IgA nephropathy may be overlooked and their significance as a concomitant histologic finding is unclear. METHODS: We conducted a clinicopathologic study to understand the possible relationship between IgA nephropathy and a concurrent thrombotic microangiopathy injury process. We identified 10 patients with an established diagnosis of IgA nephropathy and concurrent findings of thrombotic microangiopathy based on their renal biopsies. RESULTS: Six patients presented with malignant hypertension, while three others had severe hypertension (> or = 100 mmHg, diastolic). Five patients had nephrotic-range proteinuria. Seven patients had occasional arteriolar thrombi identified by light microscopy and prominent glomerular subendothelial space widening by electron microscopy, while three patients demonstrated only ultrastructural features of thrombotic microangiopathy. Other possible etiologic causes of thrombotic microangiopathy were not identified with the available clinical information. CONCLUSION: Our study suggests that a thrombotic microangiopathy injury, when present, is usually found in advanced stages of IgA nephropathy and can be associated with severe proteinuria. Although other possible causes of thrombotic microangiopathy, such as anti-phospholipid antibody syndrome, were excluded in only two patients, the thrombotic microangiopathy injury process may be a cause or a consequence of the severe hypertension encountered in most of the patients which, in turn, may be a consequence of the disease progression of IgA nephropathy.

PTPRQ is a novel phosphatidylinositol phosphatase that can be expressed as a cytoplasmic protein or as a subcellularly localized receptor-like protein.

PTPRQ (rPTP-GMC1) is a member of the type III receptor-like protein tyrosine phosphatase family. PTPRQ has very low activity against phosphotyrosine but is active against phosphatidylinositol phosphates that are involved in regulation of survival, proliferation, and subcellular architecture. Here, we report that PTPRQ can be expressed as a cytosolic or a receptor-like protein and that the form, subcellular localization, and cell types in which it is expressed are regulated by alternative promoter use and by alternative splicing. The first promoter drives expression of transcripts encoding a transmembrane protein in human podocytes and lung. PTPRQ protein is localized to the basal membrane of human podocytes, beginning when podocyte progenitors can first be identified in the embryonic kidney. A second promoter drives expression of a transcript that can encode a cytoplasmic protein containing the catalytic site. This is the major PTPRQ transcript in rat mesangial cells and human testis and is upregulated in mesangial cells in a rat model of mesangial proliferative glomerulonephritis. Differential regulation of expression of the transmembrane vs cytosolic forms, in different cell types during development or response to injury, may be a mechanism through which PTPRQ, with its activities against membrane phospholipids and against phosphotyrosine, can target specific substrates under different conditions.

Podocyte expression of the CDK-inhibitor p57 during development and disease.

BACKGROUND: The mature podocyte is a terminally differentiated cell with a limited proliferative capacity. The precise cell cycle proteins necessary for establishing podocyte quiescence during development or permitting podocyte cell cycle re-entry in disease states have not been fully defined. Accordingly, we studied the role of the cyclin dependent kinase (CDK)-inhibitor p57Kip2 (p57) in modulating these processes. METHODS: The expression of p57 protein in relation to markers of DNA synthesis was examined in developing mouse kidneys, and in the passive Heymann nephritis (PHN) and anti-glomerular antibody models of glomerular disease by immunohistochemistry. The role of p57 in glomerulogenesis was explored by examining renal tissue from embryonic p57-/- mice, and the expression of p21, p27 and p57 protein and mRNA was examined in podocytes in vitro. RESULTS: The de novo expression of p57 during glomerulogenesis coincides with the cessation of podocyte proliferation, and the establishment of a mature phenotype, and p57 is expressed exclusively in podocytes in mature glomeruli. However, p57 knockout mice have normal glomerular podocyte development. In addition, mRNA but not protein levels of p57 increased upon differentiation of podocytes in vitro. There was a marked decrease in p57 expression in both animal models of podocyte injury. This was diffuse in PHN, whereas in the murine model, loss of expression of p57 occurred predominantly in proliferating podocytes, expressing proliferating cell nuclear antigen (PCNA). CONCLUSION: Despite the de novo expression of p57 protein coinciding with the cessation of primitive podocyte proliferation during glomerulogenesis, embryonic p57-/- mice glomeruli were histologically normal. Cultured podocytes did not require changes in p57 protein levels to undergo differentiation. These data suggest that p57 alone is not required for podocyte differentiation, and that other cell cycle regulators may play a role. Furthermore, although injury to mature podocytes in experimental glomerular disease is associated with a decrease in p57, the levels of all three members of the Cip/Kip family of CDK inhibitors appear to determine the capability of podocytes to proliferate.

Cryoglobulinemic glomerulonephritis in thymic stromal lymphopoietin transgenic mice.

Mixed cryoglobulins are complexes of immunoglobulins that reversibly precipitate in the cold and lead to a systemic disease in humans. Renal involvement usually manifests as a membranoproliferative glomerulonephritis with marked monocyte infiltration and, at times, intracapillary thrombi. Thymic stromal lymphopoietin (TSLP) is a recently cloned cytokine that supports differentiation and long-term growth of B cells. Here we report that TSLP overexpression in mice results in the development of mixed cryoglobulins, with renal involvement closely resembling cryoglobulinemic glomerulonephritis as it occurs in humans. One hundred twenty-three mice were sacrificed at monthly intervals, with at least five TSLP transgenic mice and five controls in each group. Blood, kidneys, spleen, liver, lung, and ear were collected and studied by routine microscopy, immunofluorescence, immunohistochemistry, and electron microscopy. TSLP transgenic animals developed polyclonal mixed cryoglobulinemia (type III) and a systemic inflammatory disease involving the kidney, spleen, liver, lung, and ears. Renal involvement was of a membranoproliferative type demonstrating thickened capillary walls with cellular interposition and double contours of the basement membrane, expansion of the mesangium because of increased matrix and accumulation of immune-deposits, subendothelial immune-deposits, focal occlusion of capillary loops, and monocyte/macrophage influx. In contrast to the severe glomerular lesions, the tubulointerstitium was not involved in the disease process. The renal lesions and the disease course were more severe in females when compared to males. We describe a mouse strain in which a B-cell-promoting cytokine leads to formation of large amounts of mixed cryoglobulins and a systemic inflammatory injury that resembles important aspects of human cryoglobulinemia. This is the first reproducible mouse model of renal involvement in mixed cryoglobulinemia, which enables detailed studies of a membranoproliferative pattern of glomerular injury.

Osteopontin expression in human cyclosporine toxicity.

BACKGROUND: Osteopontin is a secreted phosphoprotein that has a number of diverse biological functions, including cell signaling, mediation of cell adhesion, migration, and chemoattraction of monocytes/macrophages. Up-regulation of osteopontin expression by proximal tubular epithelium has been demonstrated in both human and rodent models of renal injury in association with macrophage influx. METHODS: We studied the expression of osteopontin protein and mRNA in renal donor biopsies (N = 7) and renal transplant biopsies with cyclosporine A toxicity (N = 23) by immunohistochemistry and in situ hybridization. Serial tissue sections were immunostained with a monocyte/macrophage marker, CD68, to demonstrate the pattern of macrophage infiltration. RESULTS: Strong osteopontin expression was observed in the majority of pretransplant donor biopsies in the absence of any macrophage infiltration. In the biopsies with cyclosporine toxicity, osteopontin expression was widespread and demonstrated moderate immunohistochemical signal intensity that did not correlate with the number of interstitial macrophages present. CONCLUSIONS: Strong osteopontin protein and mRNA expression by tubular epithelium was observed in pretransplant donor biopsies and in biopsies with cyclosporine toxicity without an inflammatory cell infiltration. Therefore, osteopontin expression alone is insufficient to serve as the principal mediator of intrarenal monocyte/macrophage influx in the transplant setting.

Expression of chemokines and chemokine receptors during human renal transplant rejection.

Infiltration of renal allografts by leukocytes is a hallmark of acute transplant rejection. Chemokines attract leukocytes bearing specific chemokine receptors, and the specific leukocyte chemokine receptor phenotype is associated with types of immune responses, ie, T helper subtype 1 (Th1; CXC chemokine receptor 3 [CXCR3], CC chemokine receptor 5 [CCR5]) versus Th2 (CCR3, CCR4, CCR8). We studied the expression of the chemokine monocyte chemoattractant protein-1 and the chemokine receptors CCR2B and CXCR4 messenger RNA (mRNA) by in situ hybridization, as well as the chemokine receptors Duffy antigen receptor for chemokines (DARC) and CCR5 protein by immunohistochemistry in renal biopsy specimens with acute cellular rejection (n = 12) and acute vascular rejection (n = 8), transplant nephrectomy specimens (n = 6), and normal areas of tumor nephrectomy specimens (n = 5). CC chemokines and CC chemokine receptor mRNA expression were evaluated by ribonuclease protection assay in specimens from four transplant nephrectomies and one tumor nephrectomy. Upregulation of mRNAs for the chemokines, interferon-inducible protein-10 (IP-10); regulated on activation normal T-cell expressed and secreted; macrophage inflammatory protein-1alpha (MIP-1alpha); MIP-1beta; and lymphotactin, as well as the chemokine receptors, CCR2 and CCR5, were documented during allograft rejection. CCR1 mRNA was detectable in both allografts and controls, but CCR3 and CCR8 were absent. The number of CXCR4, CCR5, and CCR2B mRNAs expressing leukocytes and DARC-positive vessels increased during rejection episodes. CXCR4 mRNA was the most widely expressed. Leukocytes in diffuse interstitial infiltrates were mainly CCR5 positive, but in areas in which leukocytes formed nodular aggregates of infiltrating cells, the number of CCR5-positive cells was low. Instead, leukocytes in these nodular aggregates mainly expressed CXCR4. DARC was expressed on peritubular capillaries, where it was upregulated in areas of interstitial infiltration. Induction of chemokines during renal allograft rejection is accompanied by infiltration of leukocytes bearing the respective chemokine receptors. The upregulation of the CXCR3 ligand IP-10, as well as CCR5 and its ligands, in the absence of CCR3 and CCR8 is indicative that renal allograft rejection is primarily the result of a Th1-type immune response.

Up-regulation of extracellular matrix proteoglycans and collagen type I in human crescentic glomerulonephritis.

BACKGROUND: The pathogenesis of crescentic glomerulonephritis (CGN) involves cellular migration and proliferation in the urinary space, frequently followed by fibrous organization. Extracellular matrix proteoglycans (PGs) may regulate these events via effects on cellular migration, interactions with growth factors, including transforming growth factor-beta (TGF-beta), and control of collagen fibrillogenesis. The expression of PG in human CGN is unknown. METHODS: Renal tissues from 18 patients with CGN were examined immunohistochemically for versican, decorin, biglycan and collagen type I, and were compared with morphologically normal tissues from six tumor nephrectomies. Synthesis of decorin, biglycan, and procollagen type I mRNAs was evaluated by in situ hybridization. RESULTS: Versican was strongly expressed in cellular crescents and periglomerular areas, whereas decorin and biglycan accumulated in collagen type I-enriched regions, including fibrocellular and fibrous crescents, and interstitial fibrosis. PG and collagen type I accumulation colocalized with myofibroblasts in crescents, periglomerular areas, and interstitium. CONCLUSIONS: The temporal and spatial patterns of expression demonstrated in this study provide evidence to support pathogenic roles for PG in the evolution of CGN. Based on known biological properties of this molecule, versican may facilitate migration of cells in developing crescents. Decorin and biglycan may contribute to progression of CGN, perhaps via interactions with collagen type I in the remodeled extracellular matrix.

A human PKD1 transgene generates functional polycystin-1 in mice and is associated with a cystic phenotype.

Three founder transgenic mice were generated with a 108 kb human genomic fragment containing the entire autosomal dominant polycystic kidney disease (ADPKD) gene, PKD1, plus the tuberous sclerosis gene, TSC2. Two lines were established (TPK1 and TPK3) each with approximately 30 copies of the transgene. Both lines produced full-length PKD1 mRNA and polycystin-1 protein that was developmentally regulated, similar to the endogenous pattern, with expression during renal embryogenesis and neonatal life, markedly reduced at the conclusion of renal development. Tuberin expression was limited to the brain. Transgenic animals from both lines (and the TPK2 founder animal) often displayed a renal cystic phenotype, typically consisting of multiple microcysts, mainly of glomerular origin. Hepatic cysts and bile duct proliferation, characteristic of ADPKD, were also seen. All animals with two copies of the transgenic chromosome developed cysts and, in total, 48 of the 100 transgenic animals displayed a cystic phenotype. To test the functionality of the transgene, animals were bred with the Pkd1(del34) knockout mouse. Both transgenic lines rescued the embryonically lethal Pkd1(del34/del34) phenotype, demonstrating that human polycystin-1 can complement for loss of the endogenous protein. The rescued animals were viable into adulthood, although more than half developed hepatic cystic disease in later life, similar to the phenotype of older Pkd1(del34/+) animals. The TPK mice have defined a minimal area that appropriately expresses human PKD1. Furthermore, this model indicates that over-expression of normal PKD1 can elicit a disease phenotype, suggesting that the level of polycystin-1 expression may be relevant in the human disease.

Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: role in podocyte proliferation and maturation.

BACKGROUND: Normal human podocytes are terminally differentiated and quiescent cells. It is not known why podocytes fail to proliferate in response to most forms of injury. Proliferation is regulated by cell cycle proteins and their inhibitors. The Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) in general prevent proliferation by inhibiting cyclin-CDK complexes. In the current study, we determined the expression and possible role of specific CDK inhibitors in podocyte proliferation in human disease characterized by podocyte injury. METHODS: Immunostaining was performed for the CDK inhibitors p21, p27, and p57 and the proliferation marker Ki-67 on renal biopsies from patients with minimal change disease (MCD; N = 6), membranous glomerulopathy (MGN; N = 19), cellular variant of focal segmental glomerulosclerosis (FSGS; N = 12), collapsing glomerulopathy (CG; N = 9), and HIV-associated nephropathy (HIVAN; N = 16). Adult nephrectomy specimens without evidence of glomerular disease served as controls (N = 9). RESULTS: Normal quiescent podocytes express p27 and p57, but not p21. In diseases without podocyte proliferation (MCD, MGN), p21, p27, and p57 expression did not change. In contrast, there was a uniform decrease in p27 and p57 immunostaining in diseases with podocyte proliferation (cellular FSGS, CG, and HIVAN). This was accompanied by the de novo expression of p21 in podocytes. CONCLUSIONS: Our results show that podocyte quiescence may require the presence of the CDK inhibitors p27 and p57. In human glomerular diseases, a decrease in p27 and p57 may be permissive for the altered proliferative podocyte phenotype. p21 may have a multifactorial role in podocyte cell cycle regulation.

C5b-9 membrane attack complex mediates endothelial cell apoptosis in experimental glomerulonephritis.

We studied the role of the C5b-9 membrane attack complex in two models of inflammatory glomerulonephritis (GN) initiated by acute glomerular endothelial injury in Piebold-viral-Glaxo (PVG) complement-sufficient rats (C+), C6-deficient rats (C6-), and rats systematically depleted of complement with cobra venom factor (CVF). GN was induced by performing a left nephrectomy and selectively perfusing the right kidney with either 1) the lectin concanavalin A (Con A) followed by complement-fixing anti-Con A (Con A GN) or 2) purified complement-fixing goat anti-rat glomerular endothelial cell (GEN) antibody [immune-mediated thrombotic microangiopathy (ITM)]. Comparable levels of GEN apoptosis were detected in C+ animals in both models. CVF administration reduced GEN apoptosis by 10- to 12-fold. GEN apoptosis was C5b-9 dependent because PVG C6- rats were protected from GEN loss. Furthermore, functional inhibition of the cell surface complement regulatory protein CD59 by renal perfusion with anti-CD59 antibody in ITM resulted in a 3.5-fold increase in GEN apoptosis. Last, in Con A GN, abrogation of GEN apoptosis preserved endothelial integrity and renal function. This study demonstrates the specific role of C5b-9 in the induction of GEN apoptosis in experimental inflammatory GN, a finding with implications for diseases associated with the presence of antiendothelial cell antibodies.

Rapid shift from virally infected cells to germinal center-retained virus after HIV-2 infection of macaques.

Lymphoid tissues are the primary target during the initial virus dissemination that occurs in HIV-1-infected individuals. Recent advances in antiretroviral therapy and techniques to monitor virus load in humans have demonstrated that the early stages of viral infection and host response are major determinants of the outcome of individual infections. Relatively little is known about immunopathogenic events occurring during the acute phase of HIV infection. We analyzed viral dissemination within lymphoid tissues by in situ hybridization and by combined immunohistochemistry/in situ hybridization during the acute infection phase (12 hours to 28 days) in pig-tailed macaques (Macaca nemestrina), challenged intravenously with a virulent strain of HIV-2, HIV-2(287). Two stages in viral dissemination were clearly evident within the first 28 days after HIV-2(287) infection. First, a massive increase in individual HIV-2-infected cells, mostly CD3+ T lymphocytes and a smaller percentage of macrophages and interdigitating dendritic cells, was identified within lymph nodes which peaked on the 10th day after HIV-2 infection. A shift of HIV-2 distribution was demonstrable between day 10 and day 14 after HIV-2 infection. Coincident with a marked reduction in individual HIV-2 RNA+ cells by day 14 postinfection, there was a dramatic increase in germinal center-associated HIV-2 RNA. High concentrations of HIV-2 RNA persisted in germinal centers in all animals by days 21 and 28 postinfection. Thus, HIV-2 appears to go through an initial, highly disseminated cellular phase followed by localization in the follicular dendritic cell network with relatively few infected cells. In this nonhuman primate model of HIV-associated immunopathogenesis, using a virus derived from a human pathogen, we identified a significant shift in the pattern of HIV-2 localization within a narrow time frame (day 10 to day 14). This shift in virus localization and behavior indicates that there may be a discrete but remarkably narrow window for therapeutic interventions that interrupt this stage in the natural course of HIV infection. Reproducibility and the accelerated time course of disease development make this model an excellent candidate for such intervention studies.

Osteopontin expression in human crescentic glomerulonephritis.

UNLABELLED: Osteopontin expression in human crescentic glomerulonephritis. BACKGROUND: Osteopontin is a molecule with diverse biological functions, including cell adhesion, migration, and signaling. The expression of osteopontin has been demonstrated in a number of models of renal injury in association with accumulations of monocyte/macrophages, including recent reports of osteopontin expression in glomerular crescents in a rat model of anti-glomerular basement membrane glomerulonephritis. METHODS: Glomerular expression of osteopontin in biopsies of human crescentic glomerulonephritis (N = 25), IgA nephropathy with crescents (N = 2), and diffuse proliferative lupus glomerulonephropathy with crescents (N = 1) was studied by immunohistochemistry, in situ hybridization, and combined immunohistochemistry/in situ hybridization. Additionally, antibodies to cell-specific phenotypic markers were used to identify cellular components of the glomerular crescent, which express osteopontin protein and mRNA. RESULTS: All of the crescents present in the biopsies studied contained a significant number of cells that expressed osteopontin protein and mRNA, demonstrated by immunohistochemistry and in situ hybridization, respectively. Using replicate tissue sections and combined immunohistochemistry/in situ hybridization, we showed that the majority of the strongly osteopontin-positive cells are monocyte/macrophages. In addition to the very strong and cell-associated localization, a weaker and more diffuse pattern of osteopontin protein and mRNA expression could be seen in a number of crescents. None of the osteopontin mRNA-expressing cells could be identified as parietal epithelial cells, CD3-positive T cells, or alpha-smooth muscle actin-positive myofibroblasts. Interstitial monocyte/macrophages did not express osteopontin, except when located in a periglomerular inflammatory infiltrate. CONCLUSIONS: Macrophages present in the human glomerular crescent express osteopontin protein and mRNA at a high level. This expression supports a role for osteopontin in the formation and progression of the crescentic lesion via chemotactic and signaling properties of the molecule.

Expression of decorin, biglycan, and collagen type I in human renal fibrosing disease.

BACKGROUND: The extracellular matrix proteoglycans decorin and biglycan may have a pathogenic role in renal fibrosing disease via regulation of the activity of growth factors, such as transforming growth factor-beta, and effects on collagen type I fibrillogenesis. The expression of decorin and biglycan in human glomerular diseases characterized by mesangial sclerosis is unknown. METHODS: Decorin, biglycan, and collagen type I were localized immunohistochemically in human renal biopsy cases of amyloidosis (N = 18), diabetic nephropathy (N = 11), fibrillary glomerulonephritis (N = 5), immunotactoid glomerulopathy (N = 5), light-chain deposition disease (N = 4), idiopathic mesangial sclerosis (N = 4), and nephrosclerosis (N = 6), and in morphologically normal tissues obtained from tumor nephrectomies (N = 8). Decorin and biglycan mRNA synthesis was evaluated by in situ hybridization. RESULTS: Decorin and biglycan protein were not identified in normal glomeruli. Decorin accumulated in amyloid deposits, but not in deposits of fibrillary glomerulonephritis or immunotactoid glomerulopathy. Biglycan weakly accumulated in amyloid deposits, and both decorin and biglycan weakly stained mesangial nodules in cases of morphologically advanced light-chain deposition disease and diabetic nephropathy. In all analyzed cases, irrespective of the underlying disease, decorin and biglycan accumulated in glomeruli in areas of fibrous organization of the urinary space and in areas of tubulointerstitial fibrosis. Biglycan, but not decorin, accumulated in the neointima of arteriosclerotic blood vessels. Decorin and biglycan mRNA synthesis was detected at sites of proteoglycan accumulation in glomeruli, interstitium, and neointima. Collagen type I colocalized with decorin and biglycan deposits. CONCLUSIONS: Differences in extracellular matrix proteoglycan composition may be diagnostically useful in distinguishing morphologically similar diseases. Distinct patterns of proteoglycan expression may be related to modulation of specific growth factor activity in different glomerular diseases.

The cyclin kinase inhibitor p21WAF1/CIP1 is required for glomerular hypertrophy in experimental diabetic nephropathy.

BACKGROUND: Diabetic nephropathy is characterized by glomerular hypertrophy. We have recently shown that experimental diabetes mellitus is associated with an increase in glomerular expression of the cyclin kinase inhibitor p21WAF1/CIP1 (p21). Furthermore, in vitro glucose-induced mesangial cell hypertrophy is also associated with an up-regulated expression of p21. In this study, we tested the hypothesis that p21 mediates diabetic glomerular hypertrophy in vivo. METHODS: Experimental diabetes mellitus was induced by streptozotocin in mice in which p21 was genetically deleted (p21 -/-) and in wild-type mice (p21 +/+). Kidney biopsies were obtained from diabetic and control (citrate injected) p21 +/+ and p21 -/- mice at day 60. The tissue was used for morphologic studies of glomerular size (measured by computer image-analysis system), glomerular cellularity (cell count), glomerular matrix expansion (silver stain), apoptosis (TUNEL), and expression of transforming growth factor-beta1 (TGF-beta1) by in situ hybridization. RESULTS: The glomerular tuft area increased 11.21% in diabetic p21 +/+ mice at day 60 compared with control (3329.98 +/- 244.05 micrometer(2) vs. 2994. 39 +/- 176.22 micrometer(2), P = 0.03), and the glomerular cell count did not change in diabetic p21 +/+ mice at day 60 compared with the control. These findings are consistent with glomerular hypertrophy. In contrast, the glomerular tuft area did not increase in diabetic p21 -/- mice at day 60 compared with the control (3544.15 +/- 826.49 vs. 3449.15 +/- 109.65, P = 0.82), nor was there an increase in glomerular cell count (41.41 +/- 13.18 vs. 46.95 +/- 3.00, P = 0.43). Diabetic p21 +/+ mice, but not p21 -/- mice, developed an increase in proteinuria at day 60 compared with the control. Tubular cell proliferation, measured by proliferating cell nuclear antigen immunostaining, was increased in both diabetic p21 +/+ (2.1-fold) and p21 -/- (7.61-fold) mice compared with controls. Glomerular cell apoptosis did not increase in diabetic mice. Although glomerular TGF-beta1 mRNA levels increased in both strains of diabetic mice at day 60, the glomerular matrix did not expand. CONCLUSIONS: Hyperglycemia was associated with glomerular hypertrophy in p21 +/+ mice. Despite the increase in TGF-beta1 mRNA, diabetic p21 -/- mice did not develop glomerular hypertrophy, providing evidence that the cyclin kinase inhibitor p21 may be required for diabetic glomerular hypertrophy induced by TGF-beta1. The loss of p21 increases tubular but not glomerular cell proliferation in diabetic nephropathy. The absence of glomerular hypertrophy appears protective of renal function in diabetic mice.

Localization of fibroblast growth factor-2 (basic FGF) and FGF receptor-1 in adult human kidney.

BACKGROUND: The expression pattern of fibroblast growth factor-2 (FGF-2; basic FGF), a pleiotrophic growth factor, as well as one of its receptors (FGFR1), in the kidney is highly controversial. METHODS: Using an approach that combines multiple antibodies for immunohistochemistry and correlative in situ hybridization, we assessed the intrarenal expression of both FGF-2 and FGFR1 in 13 specimens of adult kidney removed during tumor nephrectomy. RESULTS: The FGF-2 expression pattern in the kidneys as detected by immunohistochemistry was variable and depended on the antibody used. The most consistent expression of FGF-2 protein was demonstrated in glomerular parietal epithelial cells, tubular cells (mainly of the distal nephron), as well as arterial endothelial cells. These locations also corresponded to areas of FGF-2 mRNA expression. Additionally, by immunohistochemistry, FGF-2 protein was detected in arterial smooth muscle cells and occasional podocytes. The expression of FGFR1 protein and mRNA was most consistently present in tubular cells of the distal nephron and in vascular smooth muscle cells. In situ hybridization, but not immunohistochemistry, also suggested FGFR1 expression in cells that could not be precisely identified within the glomerular tuft as well as some interstitial cells. CONCLUSION: These data suggest potential autocrine and paracrine pathways within the FGF-2 system, particularly within the vascular walls and in the distal nephron, and thereby provide information for further mechanistic understanding of the role of the FGF-2 system in human renal disease.

Obstructive uropathy in the mouse: role of osteopontin in interstitial fibrosis and apoptosis.

BACKGROUND: Osteopontin is a macrophage adhesive protein that is expressed by renal tubules in tubulointerstitial disease. METHODS: To investigate the function of OPN, we induced tubulointerstitial disease in OPN null mutant (OPN-/-) and wild-type (OPN+/+) mice by unilateral ureteral ligation. Tissue was analyzed for macrophages (ED-1), types I and IV collagen deposition, TGF-beta expression, and for tubular and interstitial cell apoptosis. RESULTS: Obstructed kidneys from both OPN-/- and OPN+/+ mice developed hydronephrosis, tubular atrophy, interstitial inflammation and fibrosis. OPN was absent in OPN-/- kidneys but was increased in obstructed OPN+/+ kidneys. Macrophage influx, measured by computer-assisted quantitative immunostaining, was less in OPN-/- mice compared to OPN+/+ mice at day 4 (threefold, P < 0.02), day 7 (fivefold, P < 0.02), but not at day 14. Interstitial deposition of types I and IV collagen were also two- to fourfold less in obstructed OPN-/- kidneys (P < 0.02). There was also a reduction of TGF-beta mRNA expression in the interstitium at day 7 (by in situ hybridization) and a near significant 34% reduction in cortical TGF-beta activity (P = 0.06) compared to obstructed OPN+/+ kidneys at day 14. Obstructed kidneys from OPN-/- mice also had more interstitial and tubular apoptotic cells (TUNEL assay) compared to obstructed OPN+/+ mice at all time points. The ability of OPN to act as a cell survival factor was also documented by showing that the apoptosis of serum-starved NRK52E renal epithelial cells was markedly enhanced in the presence of neutralizing anti-OPN antibody. CONCLUSION: OPN mediates early interstitial macrophage influx and interstitial fibrosis in unilateral ureteral obstruction. OPN may also function as a survival factor for renal tubulointerstitial cells.

Thrombotic microangiopathy in the HIV-2-infected macaque.

Thrombotic microangiopathy (TMA) has been increasingly reported in human immunodeficiency virus (HIV)-infected humans over the past decade. The pathogenesis is unknown. We prospectively analyzed the renal pathology and function of 27 pigtailed macaques (Macaca nemestrina), infected intravenously with a virulent HIV-2 strain, HIV-2(287), in addition to that of four uninfected control macaques. Necropsies were performed between 12 hours and 28 days after infection. HIV-2 antigen was detectable in peripheral blood mononuclear cell (PBMC) cocultures in all animals after 10 days of HIV-2 infection; a rapid decline in CD4(+) PBMC (<350/microliter) was seen in five of six animals 21 days and 28 days after infection. No macaque developed features of clinical AIDS. Typical lesions of human HIV-associated nephropathy were undetectable. Six of the 27 HIV-2-infected macaques demonstrated both histological TMA lesions (thrombi in glomerular capillary loops and small arteries, mesangiolysis) and ultrastructural lesions (mesangiolysis, subendothelial lucency, platelet thrombi in glomerular capillary lumina). Extrarenal thrombi were detected in the gastrointestinal and adrenal microvasculature of macaques that had developed renal TMA. None of the control animals demonstrated features of renal TMA at necropsy. In a retrospective analysis of kidneys obtained from 39 additional macaques infected with HIV-2(287), seven cases demonstrated TMA. In situ hybridization showed no detectable HIV-2 RNA in kidney sections of 65/66 HIV-2-infected macaques, including all 13 TMA cases. Expression of the chemokine receptor CXCR4, the putative coreceptor for HIV-2(287), was absent in intrinsic renal cells in all HIV-2-infected macaques. The HIV-2-infected macaque may be a useful model of human HIV-associated TMA. Our data do not support a role of direct HIV-2 infection of intrinsic renal cells as an underlying mechanism.

The cyclin kinase inhibitor p21CIP1/WAF1 limits glomerular epithelial cell proliferation in experimental glomerulonephritis.

BACKGROUND: During glomerulogenesis, visceral glomerular epithelial cells (VECs) exit the cell cycle and become terminally differentiated and quiescent. In contrast to other resident glomerular cells, VECs undergo little if any proliferation in response to injury. However, the mechanisms for this remain unclear. Cell proliferation is controlled by cell-cycle regulatory proteins where the cyclin-dependent kinase inhibitor p21Cip1,WAF1 (p21) inhibits cell proliferation and is required for differentiation of many nonrenal cell types. METHODS: To test the hypothesis that p21 is required to maintain a differentiated and quiescent VEC phenotype, experimental glomerulonephritis was induced in p21 knockout (-/-) and p21 wild-type (+/+) mice with antiglomerular antibody. DNA synthesis (proliferating cell nuclear antigen, bromodeoxyuridine staining), VEC proliferation (multilayers of cells in Bowman's space), matrix accumulation (periodic acid-Schiff, silver staining), apoptosis (TUNEL), and renal function (serum urea nitrogen) were studied on days 5 and 14 (N = 6 per time point). VECs were identified by location, morphology, ezrin staining, and electron microscopy. VEC differentiation was measured by staining for Wilms' tumor-1 gene. RESULTS: Kidneys from unmanipulated p21-/- mice were histologically normal and did not have increased DNA synthesis, suggesting that p21 was not required for the induction of VEC terminal differentiation. Proliferating cell nuclear antigen and bromodeoxyuridine staining was increased 4.3- and 3.3-fold, respectively, in p21-/- mice with glomerulonephritis (P < 0.0001 vs. p21+/+ mice). At each time point, VEC proliferation was also increased in nephritic p21-/- mice (P < 0.0001 vs. p21+/+ mice). VEC re-entry into the cell cycle was associated with the loss of Wilms' tumor-1 gene staining. Nephritic p21-/- mice had increased extracellular matrix protein accumulation and apoptosis and decreased renal function (serum urea nitrogen) compared with p21+/+ mice (P < 0.001). CONCLUSION: These results show that the cyclin kinase inhibitor p21 is not required by VECs to attain a terminally differentiated VEC phenotype. However, the loss of p21, in disease states, is associated with VEC re-entry into the cell cycle and the development of a dedifferentiated proliferative phenotype.

Collapsing glomerulopathy in renal allografts: a morphological pattern with diverse clinicopathologic associations.

We reviewed the clinical and pathological characteristics of seven patients with collapsing glomerulopathy (CG) in renal allograft biopsy specimens. All patients underwent biopsies for graft dysfunction. Two patients had nephrotic proteinuria (protein, >3.5 g/24 h), whereas all others had only modest or insignificant proteinuria. In five of seven patients, additional pathological processes, including microvascular injury, acute rejection, recurrent diabetic nephropathy, and immune complex glomerulonephritis, were present, each of which likely contributed to graft dysfunction and proteinuria. None of the patients in this series had nephrotic syndrome solely attributable to CG. Three biopsy specimens had features consistent with chronic rejection. The development of CG in renal allograft biopsy specimens was associated with graft dysfunction and a high rate of graft loss. These findings emphasize the prognostic significance of CG in renal allografts and suggest that CG may result from diverse pathogenic mechanisms.