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Antibodies against macrophages that overlap in specificity with fibroblasts. Background Fibroblasts can be misidentified as macrophages because both cell types share antigens that are associated with popular antibodies targeting the monocyte/macrophage lineage. With the recent description of fibroblast-specific protein 1 (FSP1), we revisited the specificity of antibodies directed against macrophages to determine systematically which antibodies best distinguish both cell types in fibrotic tissues. Methods Tissue fibrosis was produced in mice carrying the GFP transgene encoding green fluorescent protein under the control of the FSP1 promoter. Single cell suspensions from these marked tissues were submitted for flow cytometry using antibodies against Mac-1, Mac-2, Mac-3, F4/80, CD68, major histocompatibility complex (MHC) class II, and CD45, and cDNA amplification of mRNA encoding the above target antigens was performed using specific primer sets in sorted pools of cells. Fibrotic tissues were also stained by immunohistochemistry with the same antibodies and examined under confocal microscopy. Results Comparison overlap between FSP1 + fibroblasts with each of the macrophage markers demonstrated that all antimacrophage antibodies (Mac-1, Mac-2, Mac-3, CD68, MHC class II, and CD45) except one (F4/80) recognize both cell types. Conclusion Antibodies directed against F4/80 clearly distinguish macrophages from FSP1 + fibroblasts in fibrotic tissues and is the preferred antibody in mice.

Selective depletion of fibroblasts preserves morphology and the functional integrity of peritoneum in transgenic mice with peritoneal fibrosing syndrome. Background A peritoneal fibrosing syndrome (PFS) can progressively reduce peritoneal ultrafiltration during chronic peritoneal dialysis in patients with renal failure. The pathogenesis of PFS is unclear and the role of peritoneal fibroblasts has not been evaluated experimentally. Methods We followed the fate of fibroblasts producing PFS in a mouse model using fibroblast-specific protein 1 (FSP1) as a marker. PFS was induced by daily peritoneal infusions of chlorhexidine gluconate (CHG) saline into transgenic mice expressing the thymidine kinase (∆tk) gene under the control of the FSP1 promoter (FSP1.∆tk mice). To demonstrate the role of fibroblasts in PFS, we treated these FSP1.∆tk mice with a nucleoside analogue to induce DNA chain termination and fibroblast death. Results Mice receiving peritoneal infusions of CHG saline every other day for 2 weeks developed increasing numbers of FSP1 + fibroblasts in the subserosal layers of the visceral peritoneum. Mac-3 + monocytes (macrophages) subsequently accumulated over the next 2 weeks in association with increased deposition of type I collagen and increased endothelial vascularity (CD31 + ) in these subserosal tissues. Since these peritoneal fibroblasts expressed monocyte chemoattractant protein-1 (MCP-1), heat shock protein 47 (HSP47), and vascular endothelial growth factor (VEGF), we suspect they were partially responsible for macrophage recruitment, matrix production, and the neoangiogenesis in the subserosal tissue. Treatment of PFS in FSP1.∆tk transgenic mice with a nucleoside analogue selectively reduced the numbers of peritoneal fibroblasts and attenuated the attendant changes in peritoneal histology. Rescuing the peritoneal membrane from chronic thickening and neoangiogenesis by reducing the number of fibroblasts also preserved ultrafiltration. Conclusion Peritoneal fibroblasts play a pivotal role in PFS, and their deletion using a fibroblasts-specific transgene was effective in preventing peritoneal fibrogenesis.

Human antiglomerular basement membrane autoantibody disease in XenoMouse II. Background Previous studies have identified regions within α3(IV) collagen in human antiglomerular basement membrane (anti-GBM) disease, however, information pertaining to the nature of the pathogenic human autoantibodies has been limited by a lack of a relevant disease model. Availability of engineered mice that produce antibodies (that is, XenoMouse II™ strains) provides an ideal opportunity to examine the human antibody response. Methods XenoMouse II™ mice that produce human IgG2 (γ2κ) in response to antigenic challenge were immunized with various forms of α3(IV)NC1 GBM collagen, including native bovine α3(IV) NCl collagen, E. coli expressed r α3(IV)NCl, and mammalian fetal kidney 293 cell expressed r α3(IV)NC1 preparations. The mice were evaluated for autoantibody (Ab) production and nephritis. Results All immunized XenoMouse II animals produced human anti-GBM Ab associated with proliferative glomerulonephritis, linear IgG deposits along the murine GBM and tubular basement membrane (TBM), C3 deposits (weaker). A fully human mAb (Ig γ 2 κ), produced from a mouse immunized with native bovine α3(IV)NCl collagen produced basement membrane deposits, nephritis and proteinuria on transfer to normal XenoMouse II. Furthermore, monoclonal antibodies (mAb) shared idiotypic properties with polyclonal autoantibodies derived from patients with anti-GBM disease, supporting a structural relationship among the antibodies. Conclusions The results further support the importance of α3(IV)NCl collagen in the pathogenesis of anti-GBM disease. Moreover, to our knowledge this is the first demonstration that experimentally induced, pathogenic human autoantibodies result in disease. This new model of anti-GBM disease, therefore, provides the means and unique reagents to both decipher the molecular basis of the human anti-GBM autoantibody response and the opportunity to test specific therapies aimed at modulation of either B cells producing human autoantibodies or the human pathogenic antibodies themselves, in vivo, prior to trial in patients with the spontaneous form of the disease.

Expression of apoptosis regulatory proteins in tubular epithelium stressed in culture or following acute renal failure.BackgroundWhile tubular cell death is a characteristic of acute renal failure (ARF), the molecular mechanisms that modulate this cell death are unclear. Cell fate in acute renal failure hinges on a balance of survival and mortality factors in a changing environment. We further explored this issue by studying selected cell death-related proteins in experimental renal failure.MethodThe expression of genes that promote (c-myc, Bax, BclxS) or protect (Bcl2, BclxL) from cell death was studied by Northern blot, Western blot, and immunohistochemistry in murine kidneys following ARF induced by folic acid or in renal tubular epithelial cells (MCT) stressed in culture.ResultsRenal mRNA levels encoding for c-myc and BclxL were elevated in ARF while the Bcl2/Bax ratio was decreased (Bcl2 decreased and Bax increased; P < 0.05). Protein levels of BclxL increased and Bcl2 protein decreased. Expression of tumor necrosis factor (TNF-α), a mediator of ARF, was also increased. Immunohistochemistry further demonstrated that BclxL was increased in some tubuli and absent in others, while Bcl2 expression decreased diffusely. Bax staining was also patchy among tubuli and individual cells in the tubular wall and lumen. As a relative deficit of survival factors is present in ARF, MCT epithelium were deprived of serum survival factors. This resulted in apoptosis, decreased Bcl2/Bax and BclxL/Bax ratios (P < 0.05) and sensitization to TNF-α-induced apoptosis (P < 0.05). The latter was prevented by enforced overexpression of BclxL (P < 0.01). TNF-α increased the mRNA levels encoding for c-myc and decreased BclxL expression. Neither MCT cells nor the kidney expressed BclxS.ConclusionsA relative deficit of survival factors likely contributes to changes in levels of BclxL and Bax in ARF. These deficits predispose to cell death induced by persistent lethal factors such as TNF-α that is increased in ARF and a potential source of increased c-myc, a downstream facilitator of cell death. These findings implicate members of the Bcl2 family of proteins as regulators of tubular cell death in ARF and single them out as potential therapeutic targets.

Autoreactive T-cells in Goodpasture's syndrome recognize the N-terminal NC1 domain on α3 type IV collagen. Goodpasture's syndrome is mediated by immunopathogenic autoantibodies to the α3 NC1 domain of type IV collagen. It is not known whether collaborating T-cells participate in this autoreactive response. Here we describe the first T-cell clone isolated from a Goodpasture patient autoreactive to α3 type IV collagen of glomerular basement membrane. To investigate cellular autoreactivity, T-cells from Goodpasture patients or controls were isolated and stimulated by purified native or recombinant type IV collagen proteins and synthetic oligopeptides. Cell surface markers, the T-cell receptor repertoire, and MHC-restriction were analyzed. T-cell clones specific for the α3(IV) NC1 domain were established in two Goodpasture patients, but not in controls. One of the three CD8 + T-cell clones was characterized further. It was MHC class I restricted (HLA-A11) and expressed the T-cell receptor Vβ 5.1. chain. This clone specifically recognized a motif at the N-terminal area of the α3(IV) NC1 domain (AA 51 to 59: GSPATWTTR). We conclude that autoreactive T-cells exists in Goodpasture patients and may play a crucial role in the inflammatory process. T-cell clones are autoreactive to the α3(IV) NC1 domain. At least for one of the clones, the T-cell epitope is different from the putative antibody-binding site.

Mutations in the COL4A5 gene encoding the alpha 5(IV) chain of type IV collagen have been implicated as the primary defect in X-linked Alport syndrome. Several kinds of mutations have been reported so far, spanning point mutations to complete gene deletions. About 5% of Alport patients, who undergo renal transplantation, develop anti-glomerular basement membrane (GBM) nephritis, causing loss of allograft function. In one such patient, COL4A5 gene deletion was recently identified. In the present study, the GBM constituent, targeted by the anti-GBM alloantibodies from the patient who had complete COL4A5 gene deletion was identified. Its identity was determined on the basis of circulating antibody binding to various GBM constituents, domains of bovine type IV collagen and recombinant NC1 domain of human type IV collagen. These results establish, for the first time, the absence of the alpha 5(IV) chain in Alport GBM and, in the same patient, the production of an alloantibody that is targeted to a different chain of type IV collagen, the alpha 3(IV) chain. These findings provide further support for the hypothesis that: (1) anti-alpha 3(IV) collagen alloantibodies mediate the allograft glomerulonephritis; and (2) COL4A5 gene mutations cause defective assembly of the alpha 3(IV) collagen alloantibodies mediate the allograft glomerulonephritis; and (2) COL4A5 gene mutations cause defective assembly of the alpha 3(IV) chain in Alport GBM, as reflected by the production of anti-alpha 3(IV) alloantibodies.

TNFα induces expression of the chemoattractant cytokine RANTES in cultured mouse mesangial cells. We investigated the effect of several immune-relevant cytokines on expression of the chemoattractant intercrine/chemokine RANTES in a mouse mesangial cell line (MMC). Fifty ng/ml recombinant tumor necrosis factor alpha (TNFα) induced a marked increase in RANTES transcripts after two hours. RANTES mRNA remained elevated for 24 to 48 hours after stimulation, and could be abolished by co-incubation with 30 µg/ml of a neutralizing rabbit anti-TNFα antibody. Protein expression of RANTES, as assessed by indirect immunofluorescence and Western blotting, increased in MMCs 24 hours after TNFα stimulation. Interleukin-1β, tumor necrosis factor beta (TNFβ), and lipopolysaccharide (LPS) also increased expression of RANTES mRNA. In addition, RANTES mRNA expression was stimulated in glomeruli harvested from rats following renal in vivo perfusion with TNFα. Our results indicate that mesangial cells produce the small cytokine RANTES. This factor, in concert with other chemoattractants, may play a role in the glomerular recruitment of inflammatory cells like macrophages/monocytes.

The various theories discussed here suggest that somatic renal cells are susceptible to biologic modulation by the immune system independent of an inflammatory effect. (1) The mode of repression of type IV collagen synthesis by novel, soluble antigen-binding proteins, the down-regulation of class II MHC expression with interruption of antigen presentation to epithelia after selective gene regulation by antibody, and the diverse interactions of antibody with renal glomerular cells producing functional disturbances in endocytosis and permselectivity; (2) modification of surface-antigen composition; (3) alteration of matrix deposition, remodeling and composition; (4) biophysical perturbation of cytoskeletal and cell membrane components; (5) and lastly, alterations in cell adhesion through cell-surface alterations, all lend testimony to the richness of the signal transduction pathways in somatic cells. Although the preceding examples represent only a small fraction of those which may take place within the glomerular and tubular microenvironments, these paradigms may nevertheless serve as new models upon which one can consider the multitude of potential communications between disparate biologic systems that connect in complex organisms.

Isolation and characterization of cDNA from renal tubular epithelium encoding murine Rantes. We have been interested in identifying proinflammatory molecules which might play a role in attracting monocytes and T cells to the kidney. Some of the new intercrines are potential candidates. In this report we have isolated cDNA encoding murine Rantes (MuRantes) from renal tubular epithelium (MCT cells). MuRantes is a 91 amino acid member of the -C-C- or intercrine β subgroup of the Scy superfamily. The amino acid sequence for mature MuRantes was deduced from its coding cDNA and was found to be 90% homologous to its mature human counterpart (HuRantes). MCT epithelium expresses a single mRNA transcript for MuRantes of ∼1100 bp. The MuRantes protein could be detected in cell lysates of MCT epithelium by western blotting and in the cytoplasm of MCT cells by immunofluorescence using a polyclonal antibody generated against HuRantes fusion protein. A search protocol using MuRantes-specific primers and cDNA amplification revealed that mRNAs for MuRantes are expressed additionally in syngeneic mesangial cells (MMC cells), whole kidney, liver, and spleen, as well as in nephritogenic antigen-specific CD4 + helper and CD8 + effector T cells. cDNA amplification studies also demonstrated a significant elevation in mRNA transcripts encoding MuRantes in response to the stimulation of MCT epithelium with TNFα and IL-1α in culture, but not with TGFβ, γIFN, or IL-6. Our findings indicate that proximal tubular epithelium is an authentic source of MuRantes, and that transcripts encoding MuRantes are responsive to the modulating influence of paracrine factors having a known role in the development of parenchymal injury.

Analysis of the cDNA sequence encoding MHC-Aβ in tubular epithelium from mouse kidney. Class II gene products of the major histocompatibility complex (MHC) are not expressed usually in abundance on normal epithelium. The cell surface visibility of such proteins for the immune system is thought to be limited protectively in order to minimize inflammation consequent to the recognition of self-antigens in parenchymal structures by T lymphocytes. In the current experiments we investigated whether the previously recognized sparseness of Aβ on the surface of tubular epithelial cells might be accounted for by a protein coding difference deduced from the primary structure of its transcript compared with sequence from lymphoid cells that normally express Aβ in generous amounts. We demonstrate, however, using clones obtained from a cDNA library prepared from tubular epithelium harvested from H-2 S (Aβ/α + Eβ/α - ) mice susceptible to autoimmune interestitial nephritis, that the nucleotide sequence encoding the class II Aβ chain in cells from both compartments is essentially identical. Our findings suggest that there is no primary structural aberrancy in the coding region of parenchymal Aβ that would contribute to its low expression. The protective tolerance afforded by reduced numbers of class II molecules in normal tissues is, therefore, more likely the result of repressive regulatory processes.

Expression of homeobox genes in a proximal tubular cell line derived from adult mice. We have been studying the expression of several homeobox genes in cultures of proximal tubular epithelium (MCT cells) harvested from adult mus musculus . Hox genes 2.1, 2.3, and 3.3, in particular, are all expressed at low levels in resting MCT cells. The expression of Hox 2.1 and 3.3 were not influenced by mitogenic (epidermal growth factor; EGF, and platelet-derived growth factor; PDGF) nor by hypertrophogenic cytokines (angiotensin II; Ang II) in serum-free media. Transcripts for Hox 2.3, however, were elevated in MCT cells by Ang II, EGF, and serum treatment, as early as 30 minutes after their addition, whereas no change, or slight reductions were observed with transforming growth factor β (TGFβ), PDGF, and gamma-interferon (γIFN). Hox 2.3 was also super-induced by serum, in the presence of cycloheximide, in cells rested previously in serum-free media, suggesting that new protein synthesis was not required for expressive augmentation. The induction of Hox 2.3, moreover, was not specific for tubular epithelium, since the gene could be activated in tubulointerstitial fibroblasts after treatment with EGF. These experiments collectively represent a first report regarding the characterization of transcripts encoding homeoboxes in adult cells derived from renal tissue. The putative DNA-binding properties of homeobox proteins in general, the prompt and rapid induction of Hox 2.3 by morphogenic cytokines in tubulointerstitial cells, and the observed effect of cycloheximide on this gene, all indicate that Hox 2.3 might have a role in the general activation of mature somatic cells, as an immediate early event, probably in the capacity of a nuclear trans-acting factor.

Aside from the normal, sporadic turnover of epithelial cells along the adult nephron, most somatic cells in the kidney do not divide, or if they are obligated to respond to an external stimulus, there seems to be less hyperplasia in preference for cellular hypertrophy. A variety of pathophysiologic stimuli which provide temporary or permanent fixed reductions in renal function often result, for example, in compensatory enlargement of the kidney, principally in the tubulointerstitium [1]. Such adaptive responses are thought to provide a mechanism for redressing initial damage, as well as providing for a limited restoration of function. The renal hemodynamic alterations associated with these events, like increases in single nephron glomerular filtration rate (SNGFR) and glomerular blood flow, have been extensively reviewed elsewhere [2, 3], Changes in the size of the tubular nephron, as well as subsequent, late renal fibrosis have traditionally been considered a response to modifications in the mechanics of filtration—the so-called work hypothesis [4]. There is, however, alternative evidence in animals [5, 6], and humans [7], that the cellular processes of compensatory renal enlargement, themselves might share in the responsibility for the continued, long-term loss in remaining nephrons. Careful assessment of early enlargement parameters, like de novo synthesis of phosphatidylcholine, reveal for example, that some biochemical alterations occur earlier than the increase in SNGFR, or can be dissociated from semi-concurrent hemodynamic changes [8, 9]. Recent studies also provide evidence that growth factors can directly modulate renal hemodynamics, consistent with a contemporary view that initial compensatory responses might be responsible for subsequent alterations in hemodynamic parameters [10, 11]. The nature of the adaptive response of nephrons depends on the age of the individual, and whether the underlying initial insult is chronic or more acute. Tubular epithelial cells, for example, respond to acute tubular necrosis with proliferation and mitogenic activity, as defined by an increase in DNA synthesis. The renal growth response in neonatal animal following injury is also hyperplastic. Chronic damage of the adult kidney, in contrast, leans more towards compensatory renal hypertrophy with an increase in size and a rise in the protein/DNA ratio. In the 1960s, the view that approximately 75 to 80% of compensatory renal enlargement in adults was cellular hypertrophy, and the remaining 25 to 20% hyperplasia, is still widely held today [12]. While most anatomical parts of the nephron are probably involved in compensatory hypertrophy, the fact that a major constituent of kidney mass is proximal tubules suggests that an increase in size and protein content of proximal tubular cells may principally contribute to compensatory renal hypertrophy [13]. The present article, therefore, will examine some of these early-onset mechanisms of tubulointerstitial changes. Table 1 gives an overview of currently used experimental models of compensatory renal enlargement. Most models, particularly the ablative ones, result mainly in renal hypertrophy. Others, like the induction of acute tubular necrosis in animals by folate injection, however, are characterized by a mitogenic response producing hyperplasia [1, 14]. Despite the existence of a burgeoning literature dealing with more biochemical aspects of renal enlargement [14–16], the molecular mechanisms involved in the regulation of such changes are not formally understood. Especially incomplete is an understanding of the nuclear and cytoplasmic regulation of genes differentiating compensatory renal hypertrophy from true hyperplasia. Recent discoveries in the fields of epithelial growth factors, signal transduction and gene activation, however, have shed some new light on the molecular mechanisms of cellular enlargement. We will focus on these more recent findings.

Autosomal dominant polycystic kidney disease, a leading cause of end-stage renal disease in adults, is characterized by progressive focal cyst formation in the kidney. Embryonic lethality of Pkd1 -targeted mice limits the use of these mice. Here we developed a floxed allele of Pkd1 exons 2–6. Global deletion mutants developed polyhydramnios, hydrops fetalis, polycystic kidney and pancreatic disease. Somatic Pkd1 inactivation in the kidney was achieved by crossing Pkd1 flox mice with transgenic mice expressing Cre controlled by a γ-glutamyltranspeptidase promoter. These mutants developed cysts in both proximal and distal nephron segments and survived for about 4 weeks. Somatic loss of heterozygosity was shown in a reporter mouse strain to cause cystogenesis. Some cysts in young mice are positive for multiple tubular markers and a mesenchymal marker, suggesting a delay in tubular epithelial differentiation. A higher cell proliferation rate was observed in distal nephron segments probably accounting for the faster growth rate of distal cysts. Although we observed an overall increase in apoptosis in cystic kidneys, there was no difference between proximal or distal nephron segments. We also found increased cyclic AMP, aquaporin 2 and vasopressin type 2 receptor mRNA levels, and apical membrane translocation of aquaporin 2 in cystic kidneys, all of which may contribute to the differential cyst growth rate observed. The accelerated polycystic kidney phenotype of these mice provides an excellent model for studying molecular pathways of cystogenesis and to test therapeutic strategies.

Immunolocalization of fibroblast growth factor-1 (FGF-1), its receptor (FGFR-1), and fibroblast-specific protein-1 (FSP-1) in inflammatory renal disease.BackgroundThe fibroblast growth factor (FGF) family has functions in development, cell proliferation, migration, and differentiation. While FGF-2 induces fibrosis, the role of FGF-1 in inflammation and fibrosis is less defined. We examined the expression of FGF-1 and FGF receptor (FGFR-1) to determine if renal diseases with varying etiologies of inflammation, including lupus nephritis (LN), acute interstitial nephritis (AIN) and acute rejection superimposed on chronic allograft nephropathy (CAN), showed varying patterns of expression. We also examined the expression of fibroblast-specific protein-1 (FSP-1), which has been linked to epithelial-mesenchymal transition (EMT) and fibrosis, to determine whether it was linked to potential profibrotic and inflammatory FGF-1 mechanisms.MethodsProliferative LN (PLN) (N = 12), nonproliferative lupus nephritis (NPLN) (N = 5), AIN (N = 6), CAN (N = 4), and normal kidneys (N = 3) were studied. FGF, FGFR-1, and FSP-1 were localized by immunohistochemistry, and intensity scored on a 0 to 3+ scale. Double staining with CD68 and separate immunohistochemical staining for CD4 and CD8 with serial sections analysis were done to identify if T lymphocytes or macrophages showed staining for FGF-1 and FGFR-1 or FSP-1.ResultsIn normal kidneys, FGF-1 was expressed in mesangial cells (0.67 ± 0.58), glomerular endothelial (0.67 ± 0.58), visceral, and parietal epithelial cells (1.67 ± 0.58). FGFR-1 showed a similar pattern of staining but also was expressed in tubular epithelium, and arterial endothelium and smooth muscle. Expression of FGF-1 was increased over normal in glomerular parenchymal cells only in CAN in podocytes (2.30 ± 0.58 vs. 3.00 ± 0.00) (P < 0.05) and parietal epithelial cells (1.67 ± 0.58 vs. 2.25 ± 0.50) (P < 0.05). Infiltrating glomerular and interstitial inflammatory cells in diseased glomeruli also expressed FGF-1 and FGFR-1. Tubular cells expressed slightly increased FGFR-1 in renal diseases vs. normal, whereas tubules remained negative for FGF-1 in diseased kidneys. FSP-1 expression was prominent in the interstitium in all kidneys with interstitial inflammation, and most prominent in CAN. Interstitial FSP-1+ cells were consistent with a myofibroblast-type morphology, and did not stain with CD-68. FSP-1 expression was closely associated with inflammatory cells expressing FGF-1 and FGFR-1. FSP-1 also showed positivity within crescents and occasional podocytes in PLN.ConclusionThe expression of FGF-1 and FGFR-1 in infiltrating lymphocytes and macrophages, and of FGFR-1 in tubules, is supportive, but does not prove causality, of the possibility that FGF-1 might have both autocrine and paracrine functions in renal inflammation. However, the initial stimulus for renal inflammation, whether immune complex, hypersensitivity or rejection, did not alter expression patterns of FGF-1 or its receptor. The colocalization of inflammatory infiltrates with interstitial fibrosis supports the possibility of a contribution of FGF-1 for chemotaxis and associated fibrosis, further supported by interstitial FSP-1 expression closely associated with these inflammatory cells expressing FGF-1 and FGFR-1.

Mutant prenyltransferase-like mitochondrial protein (PLMP) and mitochondrial abnormalities in kd/kd mice. Background Mice that are homozygous for the kidney disease ( kd ) mutation are apparently healthy for the first 8 weeks of life, but spontaneously develop a severe form of interstitial nephritis that progresses to end-stage renal disease (ESRD) by 4 to 8 months of age. By testing for linkage to microsatellite markers, we previously localized the kd gene to a YAC/BAC contig. Methods The sequence of the entire critical region was examined, and candidate genes were identified. These candidate genes were sequenced in both mutant ( kd/kd ) mice and normal controls. The phenotype was further characterized by immunohistochemistry and electron microscopy. Transgenic mice were constructed that carried the wild-type allele of the prime candidate gene, and this transgene was transferred to a kd/kd background by breeding. Results We have obtained evidence that kd is a mutant allele of a novel gene for a prenyltransferase-like mitochondrial protein (PLMP). This gene is alternatively spliced, with the larger gene product having one domain that resembles transprenyltransferase and another that is similar to geranylgeranyl pyrophosphate synthase. The smaller gene product includes only the first domain. An antiserum to PLMP localizes to mitochondria, and ultrastructural defects are present in the mitochondria of renal tubular epithelial cells, and to a lesser extent, hepatocytes and heart cells from kd/kd mice. In a line of kd/kd mice that carried the wild-type PLMP allele as a transgene, only 1 out of 13 animals expressed the disease by 120 days of age. Conclusion The kd allele codes for a novel protein that localizes to the mitochondria, and the kd/kd mouse has dysmorphic mitochondria in the renal tubular epithelial cells. This mouse is therefore a unique animal model for studying mechanisms that lead to tubulointerstitial nephritis.

Previous studies have identified regions within alpha3(IV) collagen in human antiglomerular basement membrane (anti-GBM) disease, however, information pertaining to the nature of the pathogenic human autoantibodies has been limited by a lack of a relevant disease model. Availability of engineered mice that produce antibodies (that is, XenoMouse II strains) provides an ideal opportunity to examine the human antibody response.XenoMouse II mice that produce human IgG2 (gamma2kappa) in response to antigenic challenge were immunized with various forms of alpha3(IV)NC1 GBM collagen, including native bovine alpha3(IV) NCl collagen, E. coli expressed r alpha3(IV)NCl, and mammalian fetal kidney 293 cell expressed r alpha3(IV)NC1 preparations. The mice were evaluated for autoantibody (Ab) production and nephritis.All immunized XenoMouse II animals produced human anti-GBM Ab associated with proliferative glomerulonephritis, linear IgG deposits along the murine GBM and tubular basement membrane (TBM), C3 deposits (weaker). A fully human mAb (Ig gamma2kappa), produced from a mouse immunized with native bovine alpha3(IV)NCl collagen produced basement membrane deposits, nephritis and proteinuria on transfer to normal XenoMouse II. Furthermore, monoclonal antibodies (mAb) shared idiotypic properties with polyclonal autoantibodies derived from patients with anti-GBM disease, supporting a structural relationship among the antibodies.The results further support the importance of alpha3(IV)NCl collagen in the pathogenesis of anti-GBM disease. Moreover, to our knowledge this is the first demonstration that experimentally induced, pathogenic human autoantibodies result in disease. This new model of anti-GBM disease, therefore, provides the means and unique reagents to both decipher the molecular basis of the human anti-GBM autoantibody response and the opportunity to test specific therapies aimed at modulation of either B cells producing human autoantibodies or the human pathogenic antibodies themselves, in vivo, prior to trial in patients with the spontaneous form of the disease.

Heterogeneous T cell receptor Vβ gene repertoire in murine interstitial nephritis. Anti-tubular basement membrane disease (αTBM) produces T cell-mediated interstitial nephritis in SJL/J mice following immunization with heterologous renal tubular antigen. Initial mononuclear infiltrates appear in vivo after six to eight weeks, with subsequent progression to renal fibrosis and endstage kidney disease. Cultured lymph node derived nephritogenic T cells from these mice react to a small epitopic region of the 3M-1 target antigen and share a common amino acid motif in their Vβ CDR3 regions. We now have used RT-PCR to further characterize the renal expression of T cell receptor (TcR) Vβ gene repertoires during the course of this disease. Individual kidneys with focal mononuclear infiltrates characteristic of early αTBM disease express up to three different TcR Vβ genes; however, the same Vβ genes are not found in all kidneys at the same early stage of injury. DNA sequencing of the Vβ RT-PCR products reveals a heterogeneous population of VDJ recombinations and deduced CDR3 amino acid sequences. Our studies do not support TcR Vβ region gene restriction in histologically-detectable αTBM disease, but are more consistent with a dynamic, organ-specific autoimmune disease, directed at multiple autoantigenic epitopes.

The normal architecture and biologic function of the kidney depends on an integrated network of cellular and extracellular matrix interactions. The extracellular matrix, in addition to its role in the organization of renal tissue, also provides structural dimensions for fluid and protein movement through which physiologic systems operate to modify the filtration of ions and macromolecules, or to alter the size, proliferation and biological expressions of renal cells [1], Thus, the renal extracellular milieu constitutes the primary communication and signal processing pathway for the constituents of surrounding tissue. Although poorly characterized and poorly understood, recent advances in molecular and cellular biology have provided a variety of new experimental approaches from which to further analyze this subject matter [2–5].Several highly dynamic processes are thought to modulate the integrity of the extracellular matrix as well as its associated cellular components. Among these processes are cell-cell contact [6] and cell-matrix adhesion [7] mediated, in both spatial and temporal circumstances, by an increasingly abundant set of matrix-relevant cytokines. These interactive events, and their regulatory cascade, provide for the elegant remodelling of structural tissues within developing kidney, their homeostatic maintenance and turnover in adult life, as well as their obliteration during prolonged inflammation when regenerative processes are no longer capable of recreating naive tissues.Any alteration in the composition and structure of the extracellular matrix is, therefore, likely to have important consequences for the function of the nephron, and kidney, in general [8]. Such changes may accompany renal injury, or appear as sequelae of a variety of etiologic events, including deviation from the genetic wild-type, new metabolic disturbances, altered immunological responses, or the use of pharmaceuticals.Perhaps the most commonly recognized disturbance in the topography of extracellular matrix resulting from prolonged injury is fibrosis (Table 1). This represents the major lesion of end-stage renal disease, and is characterized by the elaboration of a variety of autocrine and paracrine factors affecting several cellular components, their cellular proliferation and migration, and their net contribution to the deposition of matrix components, particularly the interstitial collagens.The homeostatic role of the fibrogenic process is difficult to evaluate because it provides structural success mixed with architectural failure. On one hand, it represents a compensatory mechanism initiated to maintain the structural integrity of tissue, to contain the deleterious consequences of inflammatory reactions, and to recruit and stimulate cells necessary for the repair of damaged tissue and subsequent restoration of function. Fibrogenesis, however, can also reflect the relentless sequelae of chronic inflammatory events. The resultant disruption and destruction of tissue architecture, and the subsequent loss of function at both local and distant sites within the kidney can have widespread clinical implications. Progressive fibrogenesis, therefore, can be viewed as a permissive extension of normal restorative processes which have deviated to remodel tissue in an aberrant fashion.Oddly enough, despite their significant clinical impact, fibrogenic mechanisms operating locally in the kidney are not particularly well understood at the present time. On a comparative basis, a more informative literature exists for other organ systems, particularly the liver, lung, and skin [9–11] where several experimental paradigms have been extensively exploited. There is a need for a more thorough comprehension of the regulatory mechanisms governing fibrogenesis within the nephron mass. In particular it may be necessary to identify and characterize the fibrogenic response both from the perspective of the tubulointerstitium and the glomerulus, where major focus has been traditional. The tubulointerstitium takes on significance in these matters, not only because of its more extensive spatial involvement, but also because of a close correlation between the compromise of renal function and the alteration in the tubulointerstitial substance [12]. In the following review the general mechanisms of fibrogenesis will be discussed and an attempt to identify those components and factors which especially participate in tubulointerstitial fibrogenesis will be made.

candidategeneandthetheorythatsympatheticoveractivityisan underestimated contributor to ESRD.Although considerable further evidence would need to fol-low to verify the putative causal chain, ideally early develop-ments would be that others would reproduce the findings inlarger populations, and longitudinal studies in patient groupsor animal models would demonstrate that sympathetic over-activity (and lower catestatin) precedes progressive decline torenal failure. Eventually, then, the value of these CHGA hap-lotypes and catestatin as markers and perhaps the benefits ofmedication that is capable of limiting the distorted sympa-thetic outflow, perhaps limiting certain adrenergic effects,might then be worth assessment in those who are at high riskfor ESRD.DISCLOSURES

An otherwise healthy, 27-year-old white female went to her local physician complaining of 2 weeks of fever, malaise, and poor appetite. She was taking no medication and had no history of substance abuse. Physical examination revealed pallor and a temperature of 101°F orally; the blood pressure was 110/70 mm Hg. Her eye grounds were normal, no lymphadenopathy was present, the chest was clear to auscultation and percussion, and she had no extra heart sounds. An abdominal examination was within normal limits, pelvic examination revealed no adnexal masses or tenderness, and a general neurologic examination was within normal limits. Urinalysis revealed a pH of 5.5, with +1 protein, a trace of blood, and no glucose. Microscopic examination revealed 10 to 12 white blood cells/high-powered field, 10 to 15 red blood cells/high-powered field, occasional epithelial cells, and 2 white blood cell casts; a stain for eosinophils was negative. Routine screening tests revealed a hemoglobin of 11.4 g/dl; the white blood cell count was 11,500 mm3, with 85% polymorphonuclear leukocytes, 5% bands, 1% eosinophils, and 9% monocytes. Sodium was 134 mEq/liter; potassium, 4.8 mEq/liter; bicarbonate, 25 mEq/liter; and chloride, 103 mEq/liter. The BUN was 34 mg/dl, and the creatinine was 2.1 mg/dl. Glucose was 114 mg/dl. A 24-hour urine collection revealed 1 g of creatinine, 800 mg of protein, and a creatinine clearance of 33 mI/mm. A plain chest film was normal, and serologic studies were unrevealing. The patient was admitted to the hospital, where a renal biopsy was performed subsequent to a diagnostic ultrasound examination, blood cultures, and a urine culture, The cultures were negative and the renal sonogram demonstrated two kidneys approximately 13.5 cm in length

The effects of daily cyclophosphamide administration on the development and extent of primary experimental interstitial nephritis in rats. We examined the effects of daily cyclophosphamide administration on the development and extent of tubulointerstitial nephritis produced in rats injected with tubular basement membranes in adjuvant. 15 mg/kg/day of cyclophophamide completely blocked the development of interstitial lesions, while 2 mg/kg/day enhanced the degree of interstitial injury. When cyclophosphamide in the higher dose was started early in disease, 12 days after immunization, protection from progression was also observed as well as significant reductive improvement. If cyclophosphamide was administered late in disease, 21 days after immunization, no further progression was demonstrable, but substantial injury remained. In the latter two experiments, the beneficial effects of cyclophosphamide could not be explained by a reduction in anti-tubular basement membrane antibodies bound to the kidney. In groups of immunized rats that were tested, however, cyclophosphamide was able to non-specifically impair the delayed-type hypersensitivity response to tubular antigen and PPD. We conclude, therefore, that cyclophosphamide, in high but not low dosage, if given before damage is extensive and prolonged, may successfully inhibit the cellular immune response producing primary interstitial nephritis.

Effects of cyclosporin A on the development of immune-mediated interstitial nephritis. We examined the effect of daily cyclosporin A administration on the development and extent of tubulointerstitial nephritis produced in rats immunized with tubular basement membranes in adjuvant. Six mg/kg/day of cyclosporin A, given from the time of immunization, completely blocked the development of interstitial lesions and renal insufficiency. The administration of cyclosporin A after the development of interstitial nephritis also arrested the progression of the histological lesions. Both T cell-mediated and humoral immunity were markedly reduced by the administration of cyclosporin A, as evidenced by the near absence of delayed-type hypersensitivity responses and by the reduced production of anti-tubular basement membrane antibodies. Cell admixture experiments indicated that impairment of the delayed-type hypersensitivity response to tubular antigen was probably not the result of active suppression, and suggested that the protective effect of cyclosporin A might be secondary to its direct inhibitory action on the activation of nephritogenic T and B cells. Finally, the treatment of control rats with cyclosporin A, in the doses used, did not produce any detectable kidney damage nor did it impair renal function. We conclude that cyclosporin A can be an effective prophylactic and therapeutic agent in autoimmune interstitial nephritis in rats.

This overview has examined some of the current experimental options available for the study of cellular immunity in the immunopathogenesis of renal disease. T cell immunity, where it has been examined, seems to have a particularly pivotal role in orchestrating and regulating functional patterns of renal injury. The use of the research methods presented here for the study of cell-mediated interactional events in kidney disease, however, has lagged behind similar efforts in other organ systems. We hope, therefore, this report will serve to stimulate and strengthen further interest in the cell biology of the nephritogenic immune response.

Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation. Background Epithelial-mesenchymal transformation (EMT) plays an important role in embryonic development and tumorigenesis and has been described in organ remodeling during fibrogenesis. In the kidney, EMT can be induced efficiently in cultured proximal tubular epithelium by coincubation of transforming growth factor (TGF)-β1 and epidermal growth factor (EGF). Recently, we also have observed overexpression of basic fibroblast growth factor-2 (FGF-2) protein and mRNA in human kidneys with marked interstitial fibrosis. The aims of the present study were to compare the effects of FGF-2 as a facilitator of EMT in tubular epithelial cells with EGF and TGF-β1. We analyzed the morphogenic effects of the three cytokines on four different aspects of EMT: cell motility, expression and regulation of cellular markers, synthesis and secretion of extracellular matrix (ECM) proteins as well as matrix degradation. Methods Cell motility was studied by a migration assay and cell differentiation markers were analyzed by immunofluorescence and immunoblots. In addition, regulation of the epithelial adhesion molecule E-cadherin and fibroblast-specific protein 1 (FSP1) were analyzed by luciferase reporter constructs and stable transfections. ELISAs for collagen types I and IV and fibronectin were used for ECM synthesis, and zymograms were utilized for analysis of matrix degradation. Results FGF-2 induced cell motility across a tubular basement membrane in two tubular cell lines. All three cytokines induced the expression of vimentin and FSP1, but only FGF-2 and TGF-β1 reduced cytokeratin expression by immunofluorescence. These effects were most demonstrable in the distal tubular epithelial cell line and were confirmed by immunoblot analyses. Expression of E-cadherin was reduced by 61.5 ± 3.3% and expression of cytokeratin by 91 ± 0.5% by TGF-β1 plus FGF-2. Conversely, the mesenchymal markers α-smooth muscle actin (SMA) and FSP1 were induced with FGF-2 by 2.2 ± 0.1-fold and 6.8 ± 0.9-fold, respectively. Interestingly, de novo expression of the mesenchymal marker OB-cadherin was induced only by FGF-2 and EGF but not by TGF-β1. All three cytokines stimulated FSP1 and decreased E-cadherin promoter activity. FGF-2 also induced intracellular fibronectin synthesis but not secretion, the latter of which was stimulated exclusively by TGF-β1. Finally, zymographic analyses demonstrated that FGF-2 induced MMP-2 activity by 2.6 ± 0.5-fold and MMP-9 activity by 2.4 ± 0.1-fold, providing a mechanism for basement membrane disintegration and migratory access of transforming epithelium to the interstitium. Conclusions FGF-2 makes an important contribution to the mechanisms of EMT by stimulating microenvironmental proteases essential for disaggregation of organ-based epithelial units. Furthermore, the expression of epithelial and mesenchymal marker proteins seems to be affected at the promoter level.

Analysis of peritoneal macrophages in continuous ambulatory dialysis patients. Peritoneal macrophages (PMC) from patients undergoing continuous ambulatory peritoneal dialysis (CAPD) were compared to peritoneal macrophages from healthy volunteers and to peripheral blood monocytes (PBM) from CAPD patients, hemodialysis patients, and healthy volunteers. PMC from CAPD patients had morphology similar to PMC and PBM from healthy volunteers. HLA-DR antigen and Fc receptors were present on the cell surface. These monocytes had similar sequential morphologic changes in long-term culture compared to PBM from healthy volunteers. Phagocytosis, hydrogen peroxide generation and bactericidal activity were the same in PMC from CAPD patients as in PBM from healthy volunteers. Chemotaxis and eicosanoid precursor uptake studies suggest that PMC from CAPD patients may be relatively immature bone-marrow-derived cells. Although these cells function well as phagocytes, further study is warranted to define their immune competence, many components of which develop during differentiation into mature macrophages and may therefore be deficient in patients undergoing CAPD.Analyse des macrophages péritonéaux chez des malades en dialyse péritonéale continue ambulatoire. Des macrophages péritonéaux (PMC) provenant de malades en dialyse péritonéale continue ambulatoire (CAPD) ont été comparés à des macrophages péritonéaux de volontaires sains et à des monocytes sanguins périphériques (PBM) provenant de malades en CAPD, d'hémodialysés, et de volontaires sains. Les PMC provenant des malades en CAPD avaient une morphologie identique aux PMC et aux PBM des volontaires sains. L'antigène HLA-DR et les récepteurs Fc étaient présents à la surface cellulaire. Ces monocytes subissaient les mêmes changements morphologiques séquentiels en culture à long terme que les PBM des volontaires sains. La phagocytose, la génération de peroxyde d'hydrogène et l'activité bactéricide étaient les mêmes pour les PMC de malades en CAPD que pour les PBM des volontaires sains. Des études de chémotaxie et de captation du précurseur des eicosanoïdes suggèrent que les PMC des malades en CAPD pourraient être des cellules relativement immatures dérivées de la moelle osseuse. Quoique ces cellules fonctionnent bien en tant que phagocytes, une étude supplémentaire est nécessaire pour définir leur compétence immunitaire, dont plusieurs constituants se développent pendant la différenciation en macrophages matures, et pourraient donc être déficients chez les malades en CAPD.

Human Goodpasture anti-α3(IV)NC1 autoantibodies share structural determinants. To examine the structural relationship among autoantibodies produced by individuals with anti-GBM antibody-mediated disease, a polyclonal anti-idiotype directed against human anti-α3(IV)NC1 antibodies was produced and then used to study autoantibodies from other patients. For this purpose, anti-α3(IV)NC1 antibodies (anti-GBM), derived from a single patient (LL) with high titer and typical anti-GBM antibody specificity, were isolated using recombinant α3(IV)NC1-sepharose affinity chromatography. Following hyperimmunization of rabbits with anti-GBM IgG, irrelevant rabbit anti-human IgG antibodies were removed from the antiserum using a human IgG-sepharose column. The rabbit anti-α3(IV)NC1 antibodies (anti-Id GBM) effluent bound to human anti-GBM antibodies, but it did not bind to either normal human IgG or recombinant α3(IV)NC1 protein. The Id-anti-Id interaction was conformationally dependent on intact heavy and light chains of the anti-α3(IV)NC1 antibodies (ELISA and Western blotting). A competitive immunoassay was developed to evaluate structural and potential genetic relationships among anti-α3(IV)NC1 antibodies from different patients. All patients tested (9 of 9) had a substantial fraction (producing > 50% inhibition) of anti-GBM antibodies expressing Id-GBM. The results indicate that shared determinants are expressed by anti-GBM antibodies from different individuals, and they raise the possibility that common genetic elements are used to encode them. These regions are potential targets for design of reagents to regulate autoreactive B cells and/or interfere with pathogenic antibody-GBM interactions, in individuals with anti-GBM antibody mediated diseases.

Identification of promoter activity and differential expression of transcripts encoding the murine stromelysin-1 gene in renal cells. Stromelysin-1, matrix metalloproteinase-3 (MMP-3), is an important endopeptidase selectively expressed by somatic cells in organ tissues. The renal tubulointerstitium, for example, comprises tubular epithelium and interstitial fibroblasts forming the principal mass of the kidney. We observed that mRNA encoding stromelysin-1 is detectable in murine renal fibroblasts, but not in proximal tubular epithelium. Transcripts measured by RNase protection assay in renal fibroblasts increase following exposure to phorbol ester, and thereafter, activated stromelysin-1 protein can be detected in culture media by Western blotting. A 6.4Kb genomic clone containing the putative stromelysin-1 promoter was isolated and a relevant 2.1Kb PstI restriction fragment including 2.1Kb of the immediate 5′-flanking region was sequenced on both strands. Two transcriptional start sites were identified by primer extension; the major start site corresponded to a previously established position in the rat promoter, and a second undescribed minor transcriptional start site was located 16bp upstream of the primary site. A HiNF-A chromatin-activating element at −106bp was found in the early promoter region of pR336 and an active AP-1 site at −72bp with an Ets/PEA-3 motif at −203bp was suggested by transient transfection of luciferase minigenes into renal fibroblasts responsive to phorbol ester. This Ets element was identical to a site in the early promoter of the fibroblast-specific gene FSP1. A baseline enhancement in activity of pR336 in fibroblasts was further observed with the addition of 5′ flanking sequence out to −1980bp. This additional region of flanking sequence contains two modular regions: one of multiple PEA-3 elements between −684bp and −1955bp and a second region between −1929bp and −1980bps containing a second AP-1 site at −1929 bp, a MBF-1/MEP-1 metal binding site, and a PPAR peroxisome proliferator element at −1950bp. Our findings implicate a gene structure with expected activity in a mesenchymal phenotype. The PKC-dependent regulation of the stromelysin-1 gene supports the notion that it may be modulated during inflammation or tissue remodeling.

Biosynthetic and proliferative characteristics of tubulointerstitial fibroblasts probed with paracrine cytokines. Fibroblasts in parenchymal organs potentially contribute extracellular matrix to local fibrogenic processes. This contribution, in some circumstances, may be initiated by cytokines disseminated from inflammatory lesions. Different populations of fibroblasts, however, might respond distinctively to this cytokine bath depending on the microenvironment in which they reside. We have begun to explore this issue using syngeneic, low-passage fibroblasts cultured in serum-free media that were derived originally from the dermis (DFBs) and from tubulointerstitium (TFBs) of the kidney. Our findings indicate that, while fibroblasts from each compartment appear similar at the ultrastructural level, there are a variety of functional differences which distinguish their proliferative response, and their collagen secretory response (types I, III, IV, and V) following challenge with various doses of immune-relevant cytokines (TGFβ, EGF, IL-1, IL-2 and γIFN) in culture. DFBs, for example, express more surface EGF receptors than do TFBs, and, as a consequence, exhibit a more robust proliferative response to EGF in serum-free media. Unstimulated DFBs also secrete more collagen types I and III than TFBs, while unstimulated TFBs secrete more types IV and V. The expression of these collagens in TFBs was confirmed by Northern blot hybridization. When these sets of fibroblasts were further stimulated by cytokines, some of the cytokines not only differentially effect the secretion of various species of collagens within the same group of cells, but also between cells from populations which are anatomically distinct. DFBs, furthermore, at mid-level doses of cytokine, demonstrated a general trend towards less secretion of all types of collagen (particularly for TGFβ, EGF, and IL-2), while TFBs seemed less repressive. In TFBs the cytokine-induced responses for collagen types I and III tended to be discordant, and for types I and IV EGF inhibited, while TGFβ stimulated the secretory process. These findings speak collectively for the presence of a functional heterogeneity among organ-based populations of syngeneic fibroblasts in normal tissues.

A monoclonal antibody marker for Alport syndrome identifies the Alport antigen as the α5 chain of type IV collagen. The nephropathy of Alport syndrome is associated with unique abnormalities of glomerular basement membranes and is caused in many families by mutations in the X-chromosomal gene COL4A5, which encodes the α5 chain of type IV collagen. We have previously reported that Alport epidermal and glomerular basement membranes fail to bind a monoclonal antibody, Mab A7, that reacts with normal epidermal and glomerular basement membranes, and that this abnormality is unique to Alport syndrome. The molecule in normal tissues that reacts with Mab A7 was termed the “Alport antigen”. In the present study we used recombinant carboxyterminal noncollagenous (NCI) domains of the α1, α2, α3, α4 and α5 chains of type IV collagen to determine the molecular identity of the Alport antigen. Mab A7 was found to bind specifically to the NCI domain of the α5 chain of type IV collagen, by ELISA and immunoblotting studies. This finding provides a molecular explanation for the utility of Mab A7 as a marker for the Alport basement membrane defect. Mab A7 can identify the Alport basement membrane defect in those patients in whom COL4A5 mutations prevent incorporation of α5(IV) into basement membranes.

We thank Drs Parlongo, Tripepi, and Zoccali for their query regarding the statistics in our paper. We feel pretty comfortable with our original analyses based on the biostatistical advice given during their preparation. Technically speaking, all clinical studies never have enough power. The power of a study, however, is usually of concern when no differences are observed – this was not the case in our study.