Michel Le Hir

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.

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.

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 1xa0day of UUO an increasing expression of alpha-smooth muscle actin (αSMA) 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.

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 proteinxa01 (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 classxa0II, CD3, CD4 and Thyxa01, recognizing mononuclear cells, with alpha smooth muscle actin (αSMA), 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+, αSMA+, CD45+ and MHC classxa0II+ cells accumulated. FSP1+/S100A4+ cells consistently co-expressed CD45. In the inflamed regions, αSMA was co-expressed by 5′NT+ cells. In glomerulonephritic mice, FSP1+/S100A4+ cells co-expressed Thyxa01, 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 αSMA and 5′NT suggests that myofibroblasts originate from resident peritubular fibroblasts.

Distal tubular segments of the rabbit kidney after adaptation to altered Na- and K-intake

SummaryThe baso-lateral cell-membrane area in kidney tubules appears to be associated with the capacity for electrolyte transport; in the rabbit, it decreases from the distal convoluted tubule (DCT-cells) over the connecting tubule (CNT-cells) to the cortical collecting duct (principal cells).Adaptation to low Na-, high K-intake changes this pattern: CNT-cells at the beginning of the connecting tubule have the highest membrane area, which decreases along the segment, but remains two-fold higher than in controls. Principal cells have a four-fold higher membrane area than in controls. Simultaneous treatment with the antimineralocorticoid canrenoate-K inhibits the structural changes in CNT-cells only in end-portions of the connecting tubule and in principal cells.After prolonged high Na-, low K-intake DCT-cells display a two-fold higher membrane area than controls, while CNT-cells and principal cells are not affected. Simultaneous treatment with DOCA does not affect the DCT-cells but provokes a moderate increase in membrane area in CNT-cells, and a 5.5-fold increase in principal cells.The data provide evidence that DCT-, CNT- and principal cells are functionally different cell types. The baso-lateral cell-membrane area, associated with electrolyte-transport capacity, appears to be influenced in DCT-cells mainly by Na-intake, in CNT-cells mainly by K-intake and in part also by mineralocorticoids, and in principal cells mainly by mineralocorticoids.

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 µg/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.

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 35xa0weeks 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.

Sodium gradient-energized concentrative transport of adenosine in renal brush border vesicles

The uptake of adenosine in brush border vesicles of the proximal tubule of the rat kidney has been studied with a filtration technique. The initial rate of uptake was almost 6 times greater in the presence of NaCl than in the presence of KCl. The stimulatory effect of Na+ was strictly dependent on a gradient of Na+ (out>in). The time course of uptake showed an overshoot with a maximum at 20 s with a gradient of NaCl, but not with KCl. Inosine and 5′-AMP were produced from adenosine within the vesicles. In the presence of an inhibitor or adenosine deaminase adenosine was not significantly metabolized during the first 20 s of uptake. Thus, kinetic parameters of transport could be studied in the absence of interferences with metabolism. AKm of 1.1 μM and aVmax of 232 pmol · min−1 · mg protein−1 were calculated for the Na+ gradient-dependent transport. The dependency on a Na+ gradient, the capacity for uphill transport and the high affinity for adenosine situate this transport system apart from the mechanisms of transport of nucleosides described so far. It may be relevant in regard to the role of adenosine in the regulation of glomerular filtration.

Differential effects of TNF and LTalpha in the host defense against M. bovis BCG

Signaling via TNF receptor type 1 (TNFR1) was shown to be crucial in host defense against the intracellular pathogens L.u2009monocytogenes, M.u2009tuberculosis and M.u2009bovis. Toinvestigate the function of TNF and LTαu2009 in host defense against M.u2009bovis, mice double deficient for TNF and LTαu2009 (TNFu2009/u2009LTαu2009–u2009/u2009–), TNFu2009/u2009LTαu2009–u2009/u2009– mice complemented with a murine LTαu2009 transgene (TNF–u2009/u2009–) and LTαu2009–u2009/u2009– mice were infected with BCG and the ensuing pathology was investigated. Control mice showed a normal host defense with early clearance of bacteria. The granulomatous reaction in the liver was accompanied by recruitment of activated macrophages characterized by their acid phosphatase positivity and differentiation into epithelioid cells as well as a coordinated expression of proinflammatory transcripts. In contrast, TNFu2009/u2009LTαu2009–u2009/u2009– mice showed no comparable recruitment of activated macrophages in the liver. Furthermore, these mice showed extensive necrotic pulmonary lesions with massive growth of acid fast bacilli. Reintroduction of LTαu2009 as a transgene into TNFu2009/u2009LTαu2009–u2009/u2009– mice prolonged survival but did not restore resistance to BCG. This, at least partially protective role of LTαu2009 was further supported by data demonstrating that LTαu2009‐deficient mice as well were susceptible to BCG infection. In contrast to the deleterious effect of TNFu2009/u2009LTαu2009 deficiency in BCG infection, BCG‐infected TNFu2009/u2009LTαu2009–u2009/u2009– mice were tolerant to LPS‐induced shock. These results demonstrate that TNF as well as LTαu2009 are involved in murine host defense against BCG and that absence of TNFu2009/u2009LTαu2009 protects BCG‐infected mice from LPS mediated shock.