Epithelial-mesenchymal transition (EMT) in kidney fibrosis: fact or fantasy?

Epithelial-mesenchymal transition (EMT) has become widely accepted as a mechanism by which injured renal tubular cells transform into mesenchymal cells that contribute to the development of fibrosis in chronic renal failure. However, an increasing number of studies raise doubts about the existence of this process in vivo. Herein, we review and summarize both sides of this debate, but it is our view that unequivocal evidence supporting EMT as an in vivo process in kidney fibrosis is lacking.

Pulse mTOR inhibitor treatment effectively controls cyst growth but leads to severe parenchymal and glomerular hypertrophy in rat polycystic kidney disease.

The efficacy of mammalian target of rapamycin (mTOR) inhibitors is currently tested in patients affected by autosomal dominant polycystic kidney disease. Treatment with mTOR inhibitors has been associated with numerous side effects. However, the renal-specific effect of mTOR inhibitor treatment cessation in polycystic kidney disease is currently unknown. Therefore, we compared pulse and continuous everolimus treatment in Han:SPRD rats. Four-week-old male heterozygous polycystic and wild-type rats were administered everolimus or vehicle by gavage feeding for 5 wk, followed by 7 wk without treatment, or continuously for 12 wk. Cessation of everolimus did not result in the appearance of renal cysts up to 7 wk postwithdrawal despite the reemergence of S6 kinase activity coupled with an overall increase in cell proliferation. Pulse everolimus treatment resulted in striking noncystic renal parenchymal enlargement and glomerular hypertrophy that was not associated with compromised kidney function. Both treatment regimens ameliorated kidney function, preserved the glomerular-tubular connection, and reduced proteinuria. Pulse treatment at an early age delays cyst development but leads to striking glomerular and parenchymal hypertrophy. Our data might have an impact when long-term treatment using mTOR inhibitors in patients with autosomal dominant polycystic kidney disease is being considered.

The renal cortical interstitium: morphological and functional aspects.

The renal interstitial compartment, situated between basement membranes of epithelia and vessels, contains two contiguous cellular networks. One network is formed by interstitial fibroblasts, the second one by dendritic cells. Both are in intimate contact with each other. Fibroblasts are interconnected by junctions and connected to basement membranes of vessels and tubules by focal adhesions. Fibroblasts constitute the "skeleton" of the kidney. In the renal cortex, fibroblasts produce erythropoietin and are distinguished from other interstitial cells by their prominent F-actin cytoskeleton, abundance of rough endoplasmic reticulum, and by ecto-5'-nucleotidase expression in their plasma membrane. The resident dendritic cells belong to the mononuclear phagocyte system and fulfil a sentinel function. They are characterized by their expression of MHC class II and CD11c. The central situation of fibroblasts suggests that signals from tubules, vessels, and inflammatory cells converge in fibroblasts and elicit an integrated response. Following tubular damage and inflammatory signals fibroblasts proliferate, change to the myofibroblast phenotype and increase their collagen production, potentially resulting in renal fibrosis. The acquisition of a profibrotic phenotype by fibroblasts in renal diseases is generally considered a main causal event in the progression of chronic renal failure. However, it might also be seen as a repair process.

Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat.

Tubulo-interstitial fibrosis is a constant feature of chronic renal failure and it is suspected to contribute importantly to the deterioration of renal function. In the fibrotic kidney there exists, besides normal fibroblasts, a large population of myofibroblasts, which are supposedly responsible for the increased production of intercellular matrix. It has been proposed that myofibroblasts in chronic renal failure originate from the transformation of tubular cells via epithelial-mesenchymal transition (EMT) or from infiltration by bone marrow-derived precursors. Little attention has been paid to the possibility of a transformation of resident fibroblasts into myofibroblasts in renal fibrosis. Therefore we examined the fate of resident fibroblasts in the initial phase of renal fibrosis in the classical model of unilateral ureter obstruction (UUO) in the rat. Rats were perfusion-fixed on days 1, 2, 3 and 4 after ligature of the right ureter. Starting from 1 day of UUO an increasing expression of alpha-smooth muscle actin (alphaSMA) in resident fibroblasts was revealed by immunofluorescence and confirmed by the observation of bundles of microfilaments and webs of intermediate filaments in the electron microscope. Inversely, there was a decreased expression of 5'-nucleotidase (5'NT), a marker of renal cortical fibroblasts. The RER became more voluminous, suggesting an increased synthesis of matrix. Intercellular junctions, a characteristic feature of myofibroblasts, became more frequent. The mitotic activity in fibroblasts was strongly increased. Renal tubules underwent severe regressive changes but the cells retained their epithelial characteristics and there was no sign of EMT. In conclusion, after ureter ligature, resident peritubular fibroblasts proliferated and they showed progressive alterations, suggesting a transformation in myofibroblasts. Thus the resident fibroblasts likely play a central role in fibrosis in that model.

Proliferation capacity of the renal proximal tubule involves the bulk of differentiated epithelial cells.

We investigated the proliferative capacity of renal proximal tubular cells in healthy rats. Previously, we observed that tubular cells originate from differentiated cells. We now found 1) by application of bromo-deoxyuridine (BrdU) for 14 days and costaining for BrdU, and the G(1)-phase marker cyclin D1 that the bulk of cells in the S3 segment of juvenile rats were involved in proliferation; 2) that although the proliferation rate was about 10-fold higher in juvenile rats compared with adult rats, roughly 40% of S3 cells were in G(1) in both groups; 3) that after a strong mitotic stimulus (lead acetate), proliferation was similar in juveniles and adults; 4) that there was a high incidence of cyclin D1-positive cells also in the healthy human kidney; and 5) by labeling dividing cells with BrdU for 2 days before the application of lead acetate and subsequent costaining for BrdU and cell cycle markers, that, although a strong mitotic stimulus does not abolish the period of quiescence following division, it shortens it markedly. Thus the capacity of the proximal tubule to rapidly recruit cells into division relies on a large reserve pool of cells in G(1) and on the shortening of the obligatory period of quiescence that follows division.

Sirolimus ameliorates the enhanced expression of metalloproteinases in a rat model of autosomal dominant polycystic kidney disease.

BACKGROUND: Remodelling of matrix and tubular basement membranes (TBM) is a characteristic of polycystic kidney disease. We hypothesized that matrix and TBM degradation by metalloproteinases (MMPs) could promote cyst formation. We therefore investigated the renal expression of MMPs in the Han:SPRD rat model of autosomal dominant polycystic kidney disease (ADPKD) and examined the effect of sirolimus treatment on MMPs. METHODS: 5-week-old male heterozygous (Cy/+) and wild-type normal (+/+) rats were treated with sirolimus (2 mg/kg/day) through drinking water for 3 months. RESULTS: The mRNA and protein levels of MMP-2 and MMP-14 were markedly increased in the kidneys of heterozygous Cy/+ animals compared to wild-type +/+ as shown by RT-PCR and Western blot analyses for MMP-2 and MMP-14, and by zymography for MMP-2. Strong MMP-2 expression was detected by immunoperoxidase staining in cystic epithelial cells that also displayed an altered, thickened TBM. Tissue inhibitor of metalloproteinases-2 (TIMP-2) expression was not changed in Cy/+ kidneys. Sirolimus treatment leads to decreased protein expression of MMP-2 and MMP-14 in Cy/+, whereas MMP-2 and MMP-14 mRNA levels and TIMP-2 protein levels were not affected by sirolimus. CONCLUSION: In summary, in kidneys of the Han:SPRD rat model of ADPKD, there is a marked upregulation of MMP-2 and MMP-14. Sirolimus treatment was associated with a marked improvement of MMP-2 and MMP-14 overexpression, and this correlated also with less matrix and TBM alterations and milder cystic disease.

Mitotic activation of Akt signalling pathway in Han:SPRD rats with polycystic kidney disease.

AIM: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by an imbalance between tubular epithelial cell proliferation and apoptosis. We have previously shown that the mammalian target of rapamycin (mTOR) signalling pathway is aberrantly activated in the cystic kidneys of Han:SPRD rats with ADPKD. Because the Akt kinase is an upstream regulator of mTOR, we hypothesized that the activity of Akt could be enhanced in the kidneys of Han:SPRD rats. METHODS: Reverse transcription-polymerase chain reaction, western blot, enzyme-linked immunosorbent assay and immunohistochemistry were used to analyse Akt expression in rat polycystic kidneys. RESULTS: Wild-type (+/+) and heterozygous (Cy/+) Han:SPRD rats showed constitutive expression of Akt-1, -2 and -3 mRNA by reverse transcription-polymerase chain reaction analysis with no significant difference between Cy/+ and +/+ kidneys. Western blotting and enzyme-linked immunosorbent assay showed a significant increase in phosphorylated Akt in Cy/+ compared with +/+ kidneys. The pattern of immunoreactivity for phosphorylated Akt in kidney sections was the same in +/+ and in Cy/+ rats, with very low levels in interphase cells, but extremely bright signals in mitotic cells, beginning with the onset of the prophase. The in vivo incorporation of bromo-deoxyuridine revealed approximately a ninefold higher rate of proliferation in Cy/+ cyst epithelia compared with normal tubule epithelia in +/+ rats, while the expression of the cell cycle marker Ki67 revealed approximately a sixfold higher rate of proliferation. In summary, enhanced phosphorylation of Akt can be demonstrated in Cy/+ kidneys which correlates with a markedly elevated proliferation rate of epithelial cells in cysts. Mitotic but not resting cells display strong phosphorylation of Akt. CONCLUSION: Because Akt is a proximal target of mTOR, its inhibition with specific antagonists could be useful to prevent or halt cystogenesis in ADPKD.

Everolimus retards cyst growth and preserves kidney function in a rodent model for polycystic kidney disease.

BACKGROUND/AIMS: Rapamycin inhibits cyst growth in polycystic kidney disease by targeting the mammalian target of rapamycin (mTOR). To determine if this is a class effect of the mTOR inhibitors, we examined the effect of everolimus, the analogue of rapamycin, on disease progression in the Han:SPRD rat model of polycystic kidney disease. METHODS: Four-week-old male heterozygous cystic (Cy/+) and wild-type normal (+/+) Han:SPRD rats were administered everolimus or vehicle (3 mg/kg/day) by gavage for 5 weeks. Kidney function and whole-blood trough levels of everolimus were monitored. After treatment kidney weight and cyst volume density were assessed. Tubule epithelial cell proliferation was assessed by BrdU staining. RESULTS: Everolimus trough levels between 5 and 7 microg/l were sufficient to significantly reduce kidney and cyst volume density by approximately 50 and 40%, respectively. The steady decrease of kidney function in Cy/+ rats was reduced by 30% compared with vehicle-treated Cy/+ rats. Everolimus treatment markedly reduced the number of 5-bromo-2-deoxyuridine-labeled nuclei in cyst epithelia. Body weight gain and kidney function were impaired in everolimus-treated wild-type rats. CONCLUSION: Moderate dosage of everolimus inhibits cystogenesis in Han:SPRD rats. The inhibitory effect of everolimus appears to represent a class effect of mTOR inhibitors.

Abrogation of protein uptake through megalin-deficient proximal tubules does not safeguard against tubulointerstitial injury.

Sustained proteinuria and tubulointerstitial damage have been closely linked with progressive renal failure. Upon excess protein endocytosis, tubular epithelial cells are thought to produce mediators that promote inflammation, tubular degeneration, and fibrosis. This concept was tested in a transgenic mouse model with megalin deficiency. Application of an anti-glomerular basement membrane serum to transgenic megalin-deficient mice [Cre(+)/GN] and megalin-positive littermates [Cre(-)/GN] produced the typical glomerulonephritis (GN) with heavy proteinuria in both groups. Tubulointerstitial damages correlated closely with glomerular damages in pooled Cre(+)/GN and Cre(-)/GN mice. Owing to a mosaic pattern of megalin expression in the mutant mice, Cre(+)/GN kidneys permitted side-by-side analysis of megalin-deficient and megalin-positive tubules in the same kidney. Protein endocytosis was found only in megalin-positive cells. TGF-beta, intercellular adhesion molecule, vascular cellular adhesion molecule, endothelin-1, and cell proliferation were high in megalin-positive cells, whereas apoptosis, heat-shock protein 25, and osteopontin were enhanced in megalin-deficient cells. No fibrotic changes were associated with either phenotype. Tubular degeneration with interstitial inflammation was found only in nephrons with extensive crescentic lesions at the glomerulotubular junction. In sum, enhanced protein endocytosis indeed led to an upregulation of profibrotic mediators in a megalin-dependent way; however, there was no evidence that endocytosis played a pathogenetic role in the development of the tubulointerstitial disease.

Proximal tubular epithelial cells are generated by division of differentiated cells in the healthy kidney.

We searched for evidence for a contribution of stem cells in growth of the proximal S3 segments of healthy rats. According to the stem cell model, stem cells are undifferentiated and slow cycling; the bulk of cycling cells are transit amplifying, rapidly cycling cells. We show the following. 1) By continuous application of a thymidine analog (ThA) for 7 days, S3 proximal epithelial cells in healthy kidneys display a high-cycling rate. 2) Slow-cycling cells, identified by lack of ThA uptake during 14 days of continuous ThA application up to death and by expression of the cell cycle protein Ki67 at death, have the same degree of differentiation as quiescent cells. 3) To detect rapidly cycling cells, rats were killed at various time points after injection of a ThA. Double immunofluorescence for ThA and a cell cycle marker was performed, with colocalization indicating successive divisions. During one week after division, daughter cells display a very low proliferation rate, indicating the absence of rapidly cycling cells. 4) Labeling with cyclin D1 showed that this low proliferation rate is due to cycle arrest. 5) More than 50% of the S3 cells entered the cell cycle 36 h after a potent proliferative stimulus (lead acetate injection). We conclude that generation of new cells in the proximal tubule relies on division of differentiated, normally slow-cycling cells. These may rapidly enter the cycle under an adequate stimulus.

Immunolocalization of phospho-S6 kinases: a new way to detect mitosis in tissue sections and in cell culture.

During a study on the mTor pathway in the rat kidney we observed a striking increase of the phosphorylation of the S6 kinase in mitosis. In cryostat sections of perfusion-fixed tissue mitotic cells appeared as bright spots in immunofluorescence using an antibody specific for the phosphorylation site Thr421/Ser424. They were easily spotted in overviews with the objective 4x and 10x. Immunofluorescence was weak during the interphase. During the prophase it increased in both the nucleus and the cytoplasm and it remained bright during the subsequent phases of mitosis. All mitotic cells which were found in tubules and in the interstitium of the kidney using a chromatin stain displayed the bright immunofluorescence for phospho-S6 kinase. The same phenomenon was observed in rat liver and in mouse kidney as well as in a human cell line. Provided a rapid fixation, mitotic cells could be identified with the immunoperoxidase technique in paraffin sections of immersion-fixed tissue. This is the first report of phosphorylation of S6 kinase during mitosis in vivo. Thus, immunohistochemistry with anti-phospho-S6 kinase (Thr421/Ser424) appears to provide a convenient way to detect mitotic cells at low magnification.

Inhibition of mTOR with sirolimus slows disease progression in Han:SPRD rats with autosomal dominant polycystic kidney disease (ADPKD).

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by dysregulated tubular epithelial cell growth, resulting in the formation of multiple renal cysts and progressive renal failure. To date, there is no effective treatment for ADPKD. The mammalian target of rapamycin (mTOR) is an atypical protein kinase and a central controller of cell growth and proliferation. We examined the effect of the mTOR inhibitor sirolimus (rapamycin) on renal functional loss and cyst progression in the Han:SPRD rat model of ADPKD. METHODS: Five-week-old male heterozygous cystic (Cy/+) and wild-type normal (+/+) rats were administered sirolimus (2 mg/kg/day) orally through the drinking water for 3 months. The renal function was monitored throughout the treatment phase, and rats were sacrificed thereafter. Kidneys were analysed histomorphometrically, and for the expression and phosphorylation of S6K, a well-characterized target of mTOR in the regulation of cell growth. RESULTS: The steady increase in BUN and creatinine in Cy/+ rats was reduced by 39 and 34%, respectively with sirolimus after 3 months treatment. Kidney weight and 2-kidney/total body weight (2K/TBW) ratios were reduced by 34 and 26% in sirolimus-treated Cy/+ rats. Cyst volume density was also reduced by 18%. Of importance, Cy/+ rats displayed enhanced levels of total and phosphorylated S6K. Sirolimus effectively reduced total and phosphorylated levels of S6K. CONCLUSION: We conclude that oral sirolimus markedly delays the loss of renal function and retards cyst development in Han:SPRD rats with ADPKD. Our data also suggest that activation of the S6K signalling pathway plays an important role in the pathogenesis of PKD. Sirolimus could be a useful drug to retard progressive renal failure in patients with ADPKD.

Tubular cell proliferation in the healthy rat kidney.

We searched for morphological evidence to support the hypothesis that stem cells are responsible for renal tubular cell proliferation. The rationale of the study was that if proliferation relies on progenitors, mitotically active cells should be less differentiated than their neighbors. As the retention of the thymidine analog BrdU has been the only approach employed to identify stem cells in the kidney up to now we additionally characterized BrdU-retaining cells. Rat kidneys were fixed by perfusion. Cycling cells identified by mitotic figures or the expression of the proliferating cell nuclear antigen (PCNA) were examined by light microscopy and electron microscopy as well as immunofluorescence for four differentiation markers. Newborn rats were injected with BrdU in order to detect label-retaining cells. After a period of 8, 14 and 35 weeks the kidneys were examined for BrdU by immunofluorescence and the four differentiation markers mentioned above. All cycling cells showed the same degree of differentiation compared to non-cycling cells. Most of the detected label-retaining cells were differentiated. We conclude that cycling cells in tubules of the healthy kidney are differentiated and that the retention of label is not a criterion to identify stem cells in renal tubules.

Characterization of renal interstitial fibroblast-specific protein 1/S100A4-positive cells in healthy and inflamed rodent kidneys.

Fibrosis is considered as a central factor in the loss of renal function in chronic kidney diseases. The origin of fibroblasts and myofibroblasts that accumulate in the interstitium of the diseased kidney is still a matter of debate. It has been shown that accumulation of myofibroblasts in inflamed and fibrotic kidneys is associated with upregulation of fibroblast-specific protein 1 (FSP1, S100A4), not only in the renal interstitium but also in the injured renal epithelia. The tubular expression of FSP1 has been taken as evidence of myofibroblast formation by epithelial-mesenchymal transition (EMT). The identity of FSP1/S100A4 cells has not been defined in detail. We originally intended to use FSP1/S100A4 as a marker of putative EMT in a model of distal tubular injury. However, since the immunoreactivity of FSP1 did not seem to fit with the distribution and shape of fibroblasts or myofibroblasts, we undertook the characterization of FSP1/S100A4-expressing cells in the interstitium of rodent kidneys. We performed immunolabeling for FSP1/S100A4 on thin cryostat sections of perfusion-fixed rat and mouse kidneys with peritubular inflammation, induced by thiazides and glomerulonephritis, respectively, in combination with ecto-5'-nucleotidase (5'NT), recognizing local cortical peritubular fibroblasts, with CD45, MHC class II, CD3, CD4 and Thy 1, recognizing mononuclear cells, with alpha smooth muscle actin (alphaSMA), as marker for myofibroblasts, and vimentin for intracellular intermediate filaments in cells of mesenchymal origin. In the healthy interstitium of rodents the rare FSP1/S100A4+ cells consistently co-expressed CD45 or lymphocyte surface molecules. Around the injured distal tubules of rats treated for 3-4 days with thiazides, FSP1+/S100A4+, 5'NT+, alphaSMA+, CD45+ and MHC class II+ cells accumulated. FSP1+/S100A4+ cells consistently co-expressed CD45. In the inflamed regions, alphaSMA was co-expressed by 5'NT+ cells. In glomerulonephritic mice, FSP1+/S100A4+ cells co-expressed Thy 1, CD4 or CD3. Thus, in the inflamed interstitium around distal tubules of rats and of glomerulonephritic mice, the majority of FSP1+ cells express markers of mononuclear cells. Consequently, the usefulness of FSP1/S100A4 as a tool for detection of (myo)fibroblasts in inflamed kidneys and of EMT in vivo is put into question. In the given rat model the consistent co-expression of alphaSMA and 5'NT suggests that myofibroblasts originate from resident peritubular fibroblasts.

Suppressed T-cell activation by IFN-gamma-induced expression of PD-L1 on renal tubular epithelial cells.

BACKGROUND: The interaction of the T-cell molecule PD-1 (programmed death-1) with its ligands PD-L1 and PD-L2 represents a known mechanism of T-cell inhibition. PD-1 is homologous to CD28 while the PD-1 ligands share homology with the B7 family of co-stimulatory molecules. METHODS: We have studied surface expression and transcript levels of PD-L1 and PD-L2 on murine renal tubular epithelial cells (TEC) by flow cytometric analysis and reverse transcription-polymerase chain reaction. Western blot analysis was used to confirm protein expression of PD-L1. We also tested the functional role of PD-L1 and PD-1 in antigen presentation. Furthermore, we stained mouse kidney transplants with rejection for the expression of the PD-1 ligands. RESULTS: We found that PD-L1 but not PD-L2 was weakly expressed on unstimulated TEC. Upon stimulation with IFN-gamma, a dose-dependent upregulation of PD-L1 expression was observed. Blockade of the PD-L1/PD-1 pathway with monoclonal antibodies in antigen presentation assays uncovered an inhibitory role of this ligand system in Th1 and Th2 cell activation. Staining for PD-L1 was strong in proximal and distal tubules in mouse kidney transplants with rejection, whereas staining of normal kidneys and syngenic mouse kidney transplants did not reveal PD-L1 expression. PD-L2 was not observed in normal or rejected mouse kidneys. CONCLUSIONS: These data demonstrate that PD-L1 is an inducible renal tubular epithelial antigen that negatively regulates T-cell responses elicited by IFN-gamma-stimulated TEC. We speculate that the PD-1/PD-L1 pathway may play a role in protecting the epithelium from immune-mediated tubulointerstitial injury.

Alteration of podocytes in a murine model of crescentic glomerulonephritis.

Recent observations suggest a central role of podocytes in crescent formation. In experimental glomerulonephritis podocytes disrupt the parietal epithelial layer and attach on its basement membrane, thus forming bridges between the tuft and Bowman's capsule, and they are a major constituent of crescents. In order to explain these findings we hypothesize that inflammation triggers motility in podocytes. In the present study we asked whether podocytes display alterations which suggest a migratory behavior in glomerulonephritis. Glomerulonephritis was induced in mice by injection of a rabbit serum against the glomerular basement membrane. The kidneys were perfusion-fixed 6 days later and examined by light and electron microscopy as well as by immunohistochemistry. In glomerulonephritis the apical cytoplasm of podocytes displayed numerous actin-containing microprotrusions. Cortactin, a protein involved in the regulation of actin polymerization, was predominantly expressed in foot processes of podocytes in control mice. It was redistributed to the cell body in glomerulonephritis. In untreated mice betal-integrin was restricted to the foot processes. In glomerulonephritis it was additionally found in the cytoplasm and in the apical cell membrane. Recycling of integrins is a crucial event in initiation of cell migration. ICAM-1 and CD44, the ligation of which induces migratory behaviors, were absent from healthy podocytes but expressed by some podocytes in glomerulonephritis. Thus, in glomerulonephritis podocytes display some characteristic features of migrating cells. This might explain their ability to break through the parietal epithelium and to become a constituent of early crescents.

Histopathology of humorally mediated anti-glomerular basement membrane (GBM) glomerulonephritis in mice.

BACKGROUND: From a diagnostic point of view it would be important to learn more about the relationship between the immune responses underlying glomerulonephritis and the patterns of glomerular lesions. A murine model of anti-GBM glomerulonephritis in which inflammation is driven by delayed-type hypersensitivity (DTH) has been studied extensively. The aim of this study was to uncover histological features that might be specific for anti-GBM glomerulonephritis driven by a humoral immune response. METHODS: BALB/c mice were immunized with rabbit IgG in incomplete Freund's adjuvant. Six days later, on day 0, they received rabbit anti-GBM serum intravenously. Proteinuria was assessed with dipsticks. Mice were killed on days 4, 8 or 14. Kidneys from days 4 and 8 were processed for immunofluorescence and histology. On day 14 mice were perfusion-fixed for electron microscopy. RESULTS: Proteinuria started on day 3. Autologous IgG and of C3 were found along the GBM. There was only slight infiltration with macrophages and no measurable infiltration by CD4 T cells, indicating the virtual absence of DTH. Besides infiltration with neutrophils there were little histological alterations on day 4. On day 8 many loops were hyalinized. On day 14, cellular crescents were found in 23% of glomeruli. Subendothelial spaces contained hyaline material, cells and fibrin. Podocytes displayed effacement of foot processes and apical microprotrusions. Podocyte bridges were common. These alterations were identical to those reported in the standard model that produces a DTH-like inflammation. CONCLUSION: The qualitative pattern of histological damage in a murine model of anti-GBM glomerulonephritis does not depend on the underlying immunological process.

Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.

Cellular crescents are a defining histologic finding in many forms of inflammatory glomerulonephritis. Despite numerous studies, the origin of glomerular crescents remains unresolved. A genetic cell lineage-mapping study with a novel transgenic mouse model was performed to investigate whether visceral glomerular epithelial cells, termed podocytes, are precursors of cells that populate cellular crescents. The podocyte-specific 2.5P-Cre mouse line was crossed with the ROSA26 reporter line, resulting in irreversible constitutive expression of beta-galactosidase in doubly transgenic 2.5P-Cre/ROSA26 mice. In these mice, crescentic glomerulonephritis was induced with a previously described rabbit anti-glomerular basement membrane antiserum nephritis approach. Interestingly, beta-galactosidase-positive cells derived from podocytes adhered to the parietal basement membrane and populated glomerular crescents during the early phases of cellular crescent formation, accounting for at least one-fourth of the total cell mass. In cellular crescents, the proliferation marker Ki-67 was expressed in beta-galactosidase-positive and beta-galactosidase-negative cells, indicating that both cell types contributed to the formation of cellular crescents through proliferation in situ. Podocyte-specific antigens, including WT-1, synaptopodin, nephrin, and podocin, were not expressed by any cells in glomerular crescents, suggesting that podocytes underwent profound phenotypic changes in this nephritis model.

A novel mechanism of nephron loss in a murine model of crescentic glomerulonephritis.

BACKGROUND: Nephron loss is a major determinant of renal failure in glomerular diseases. The prevalent concept stresses the role of the toxicity of filtered proteins and/or of interstitial inflammation in tubular degeneration. However, whether that concept is compatible with the actual histopathological features of nephron loss has not been investigated specifically. METHODS: We investigated the morphological aspects of tubular degeneration in crescentic glomerulonephritis in mice. Glomerulonephritis was induced by intravenous injection of anti-glomerular basement membrane antiserum in presensitized mice. Kidneys were fixed by perfusion and examined by light- and electron microscopy and by immunohistochemistry. RESULTS: Tubular degeneration started with cellular hypotrophy in the proximal tubule. Hypotrophy appeared to follow obstruction of the initial proximal tubule by a cellular crescent. Whereas induction of intercellular adhesion molecule-1 (ICAM-1) was diffuse in glomerulonephritic mice, expression of CD44 and vascular cell adhesion molecule-1 (VCAM-1) appeared to be restricted to degenerating tubules. Interstitial inflammation developed in the vicinity of degenerating tubules. Inflammatory infiltration of tubules themselves was observed only in late stages of tubular degeneration. CONCLUSION: In a similar manner as described earlier for focal segmental glomerulosclerosis, in crescentic glomerulonephritis nephron loss can be initiated by the progression of a glomerular lesion into the proximal tubule. Interstitial inflammation might be rather a consequence than the cause of tubular degeneration.