Brigitte Kaissling

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.

Tubular Overexpression of Transforming Growth Factor-β1 Induces Autophagy and Fibrosis but Not Mesenchymal Transition of Renal Epithelial Cells

We recently showed in a tetracycline-controlled transgenic mouse model that overexpression of transforming growth factor (TGF)-beta1 in renal tubules induces widespread peritubular fibrosis and focal degeneration of nephrons. In the present study we have analyzed the mechanisms underlying these phenomena. The initial response to tubular cell-derived TGF-beta1 consisted of a robust proliferation of peritubular cells and deposition of collagen. On sustained expression, nephrons degenerated in a focal pattern. This process started with tubular dedifferentiation and proceeded to total decomposition of tubular cells by autophagy. The final outcome was empty collapsed remnants of tubular basement membrane embedded into a dense collagenous fibrous tissue. The corresponding glomeruli survived as atubular remnants. Thus, TGF-beta1 driven autophagy may represent a novel mechanism of tubular decomposition. The fibrosis seen in between intact tubules and in areas of tubular decomposition resulted from myofibroblasts that were derived from local fibroblasts. No evidence was found for a transition of tubular cells into myofibroblasts. Neither tracing of injured tubules in electron micrographs nor genetic tagging of tubular epithelial cells revealed cells transgressing the tubular basement membrane. In conclusion, overexpression of TGF-beta1 in renal tubules in vivo induces interstitial proliferation, tubular autophagy, and fibrosis, but not epithelial-to-mesenchymal transition.

Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content.

Renal proximal tubular response to acute administration of a low Pi diet is characterized by a rapid adaptive increase in apical brush border membrane (BBM) Na-Pi cotransport activity and Na-Pi cotransporter protein abundance, independent of a change in Na-Pi cotransporter mRNA levels (Levi, M., M. Lötscher, V. Sorribas, M. Custer, M. Arar, B. Kaissling, H. Murer, and J. Biber. 1994. Am. J. Physiol. 267: F900-F908). The purposes of the present study were to determine if the acute adaptive response occurs independent of de novo protein synthesis, and if microtubules play a role in the rapid upregulation of the Na-Pi cotransporters at the apical BBM. We found that inhibition of transcription by actinomycin D and translation by cycloheximide did not prevent the rapid adaptive response. In addition, in spite of a 3.3-fold increase in apical BBM Na-Pi cotransporter protein abundance, there was no change in cortical homogenate Na-Pi cotransporter protein abundance. Pretreatment with colchicine, which resulted in almost complete disruption of the microtubular network, abolished the adaptive increases in BBM Na-Pi cotransport activity and Na-Pi cotransporter protein abundance. In contrast, colchicine had no effect on the rapid downregulation of Na-Pi cotransport in response to acute administration of a high Pi diet. We conclude that the rapid adaptive increase in renal proximal tubular apical BBM Na-Pi cotransport activity and Na-Pi cotransporter abundance is independent of de novo protein synthesis, and is mediated by microtubule-dependent translocation of presynthesized Na-Pi cotransporter protein to the apical BBM.

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.

In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature

We describe a new methodology, based on terminal perfusion of rodents with a reactive ester derivative of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ extracts can be purified on streptavidin resin in the presence of strong detergents, digested on the resin and subjected to liquid chromatography–tandem mass spectrometry for identification. In the present study, in vivo biotinylation procedure led to the identification of hundreds of proteins in different mouse organs, including some showing a restricted pattern of expression in certain body tissues. Furthermore, biotinylation of mice with F9 subcutaneous tumors or orthotopic kidney tumors revealed both quantitative and qualitative differences in the recovery of biotinylated proteins, as compared to normal tissues. This technology is applicable to proteomic investigations of the differential expression of accessible proteins in physiological and pathological processes in animal models, and to human surgical specimens using ex vivo perfusion procedures.

Morphology of interstitial cells in the healthy kidney.

Renal interstitial cells play an important role in renal function and renal diseases. We describe the morphology of renal interstitial cells in the healthy kidney. We distinguish within the renal interstitium (1) renal fibroblasts and (2) cells of the immune system. Fibroblasts are in the majority and constitute the scaffold of the kidney; they are interconnected by junctions, and are attached to tubules and vessels. Although the phenotype of fibroblasts shows some variation depending on their location in the kidney and on their functional stage, their recognition as fibroblasts is possible on account of structural features. Among the cell types of the second group, antigen-presenting dendritic cells are the most abundant in in the peritubular interstitial spaces of healthy kidneys. Their incidence is highest in the inner stripe of the outer medulla. They share some morphological features with fibroblasts but lack others — junctional complexes, morphologically defined connections with tubules and vessels, and the prominent layer of actin filaments under the plasma membrane — that are characteristic for fibroblasts. Dendritic cells in healthy kidneys are morphologically different from macrophages, which are characterized by abundant primary and secondary lysosomes. In healthy kidneys macrophages are restricted to the connective tissue of the renal capsule and the pelvic wall, and to the periarterial connective tissue. Lymphocytes are rare in healthy kidneys. The distinction of cell types by morphology is supported by differences of membrane proteins. Among all interstitial cells in the renal cortex, fibroblasts alone exhibit ecto-5′-nucleotidase. Dendritic cells constitutively have a high abundance of MHC class II protein. Both proteins are mutually exclusive. Rat macrophages display the membrane antigen ED 2 and lymphocytes exhibit specific surface antigens, depending on their type and functional stage, e.g., CD4 or CD8.

Rapid downregulation of rat renal Na/Pi cotransporter in response to parathyroid hormone involves microtubule rearrangement

Renal proximal tubule cells express in their apical brush border membrane (BBM) a Na/P(i) cotransporter type IIa that is rapidly downregulated in response to parathyroid hormone (PTH). We used the rat renal Na/P(i) cotransporter type IIa (NaPi-2) as an in vivo model to assess early cellular events in the rapid downregulation of this transporter. When rats were treated with PTH for 15 minutes, NaPi-2 abundance in the BBM was decreased. In parallel, transporter accumulated in intracellular vesicles. Concomitantly, microtubules (MTs) were found to form dense bundles of apical-to-basal orientation. After 60 minutes of PTH action, the cells were vastly depleted of NaPi-2, whereas their microtubular cytoskeleton had returned to its normal appearance. Prevention of MT rearrangement by taxol resulted in accumulation of NaPi-2 in the subapical cell portion after 15 minutes and a strong delay in depletion of intracellular transporter after 60 minutes of PTH action. Furthermore, the subapical accumulation of NaPi-2 was associated with the expansion of dense apical tubules of the subapical endocytic apparatus (SEA). Depolymerization of MTs by colchicine likewise caused a retardation of intracellular NaPi-2 depletion. These results suggest that NaPi-2 is downregulated in response to PTH through a rapid endocytic process in 2 separate steps: (a) internalization of the transporter into the SEA, and (b) its delivery to degradative organelles by a trafficking mechanism whose efficiency depends on a taxol-sensitive rearrangement of MTs.

Organization of the pronephric kidney revealed by large-scale gene expression mapping

BackgroundThe pronephros, the simplest form of a vertebrate excretory organ, has recently become an important model of vertebrate kidney organogenesis. Here, we elucidated the nephron organization of the Xenopus pronephros and determined the similarities in segmentation with the metanephros, the adult kidney of mammals.ResultsWe performed large-scale gene expression mapping of terminal differentiation markers to identify gene expression patterns that define distinct domains of the pronephric kidney. We analyzed the expression of over 240 genes, which included members of the solute carrier, claudin, and aquaporin gene families, as well as selected ion channels. The obtained expression patterns were deposited in the searchable European Renal Genome Project Xenopus Gene Expression Database. We found that 112 genes exhibited highly regionalized expression patterns that were adequate to define the segmental organization of the pronephric nephron. Eight functionally distinct domains were discovered that shared significant analogies in gene expression with the mammalian metanephric nephron. We therefore propose a new nomenclature, which is in line with the mammalian one. The Xenopus pronephric nephron is composed of four basic domains: proximal tubule, intermediate tubule, distal tubule, and connecting tubule. Each tubule may be further subdivided into distinct segments. Finally, we also provide compelling evidence that the expression of key genes underlying inherited renal diseases in humans has been evolutionarily conserved down to the level of the pronephric kidney.ConclusionThe present study validates the Xenopus pronephros as a genuine model that may be used to elucidate the molecular basis of nephron segmentation and human renal disease.

Localization of NaPi-1, a Na/Pi cotransporter, in rabbit kidney proximal tubules

Polyclonal antibodies have been raised against a C-terminal peptide of NaPi-1, a recently cloned Na-Pi cotransport system of rabbit kidney cortex with a predicted (unglycosylated) molecular mass of 52 kDa. By Western blot analysis using brush-border membranes isolated from rabbit kidney cortex, two proteins with apparent molecular masses of 64 kDa and 35 kDa were specifically recognized (peptide protectable) by the antiserum obtained. The 64-kDa protein was found to migrate in parallel with the luminal membrane during separation by free-flow electrophoresis of brush-border and basolateral membranes. In immunofluorescence studies using cryostat sections of rabbit kidney, specific binding of antibodies was observed in proximal tubules (including S1, S2 and S3 segments) of superficial and deep nephrons. Anti-(NaPi-1)-antibody-mediated fluorescence was restricted to the brush border of proximal tubular cells. No specific immunoreaction was observed in other tubular segments. The results suggest that the native NaPi-1-related protein (Na-Pi cotransport system) has an apparent molecular mass of 64 kDa and is uniformly expressed in the apical membrane of proximal tubules of all nephron generations in the rabbit kidney. Immunohistochemical localization of the Na-Pi cotransport system NaPi-1 confirms the segmental localization within the nephron of NaPi-1-related mRNA as revealed by the reverse transcriptase/polymerase chain reaction (see preceding paper).

Effects of increased renal tubular vascular endothelial growth factor (VEGF) on fibrosis, cyst formation, and glomerular disease.

The role of vascular endothelial growth factor (VEGF) in renal fibrosis, tubular cyst formation, and glomerular diseases is incompletely understood. We studied a new conditional transgenic mouse system [Pax8-rtTA/(tetO)(7)VEGF], which allows increased tubular VEGF production in adult mice. The following pathology was observed. The interstitial changes consisted of a ubiquitous proliferation of peritubular capillaries and fibroblasts, followed by deposition of matrix leading to a unique kind of fibrosis, ie, healthy tubules amid a capillary-rich dense fibrotic tissue. In tubular segments with high expression of VEGF, cysts developed that were surrounded by a dense network of peritubular capillaries. The glomerular effects consisted of a proliferative enlargement of glomerular capillaries, followed by mesangial proliferation. This resulted in enlarged glomeruli with loss of the characteristic lobular structure. Capillaries became randomly embedded into mesangial nodules, losing their filtration surface. Serum VEGF levels were increased, whereas endogenous VEGF production by podocytes was down-regulated. Taken together, this study shows that systemic VEGF interferes with the intraglomerular cross-talk between podocytes and the endocapillary compartment. It suppresses VEGF secretion by podocytes but cannot compensate for the deficit. VEGF from podocytes induces a directional effect, attracting the capillaries to the lobular surface, a relevant mechanism to optimize filtration surface. Systemic VEGF lacks this effect, leading to severe deterioration in glomerular architecture, similar to that seen in diabetic nephropathy.