Rat Kidney Tubular Epithelial Cells Research Articles

Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation.

It has been shown that transforming growth factor-beta (TGF-beta) is a potent mediator in renal fibrosis and that Smad proteins are critical intracellular mediators in TGF-beta signaling. It is here reported that TGF-beta mediates renal fibrogenesis in tubular epithelial cells (TEC) in association with the activation of Smad2 and that overexpression of Smad7 blocks this fibrotic process. Using a normal rat kidney tubular epithelial cell line (NRK52E), it was determined that TGF-beta1 induces Smad2 phosphorylation and nuclear localization in both a dose- and time-dependent manner. The activation of Smad2 was evident at 5 min (20%), peaked at 15 to 30 min (85%), and declined to baseline levels by 2 h (5 to 10%). This was associated with de novo expression of collagens I, III, and IV and the transformation of TEC into a "myofibroblast" phenotype with de novo expression of alpha-smooth muscle actin (alpha-SMA) and with the loss of E-cadherin (>50%). To investigate a negative regulatory role of Smad7 in renal fibrosis, the Smad 7 gene was stably transfected and its expression was tightly controlled by doxycycline into NRK52E cells. Overexpression of Smad7 induced by doxycycline results in marked inhibition of TGF-beta-induced Smad2 activation (90% downward arrow) with the prevention of collagen synthesis and myofibroblast transformation. Thus, Smad2 activation occurs in the fibrogenic response of TEC to TGF-beta, and this process is blocked by overexpression of Smad7. This indicates that Smad signaling is a key pathway of TGF-beta-mediated renal fibrosis and suggests that treatments targeting the inactivation of Smad2 by overexpression of Smad7 may provide a new therapeutic strategy for renal fibrosis.

Up-regulation of the tumour-associated marker CD44V6 in experimental kidney disease

CD44 is an adhesion molecule involved in a wide range of cell-cell and cell-matrix interactions. The standard form of CD44 (CD44S) is a 85-90-kD glycoprotein, but alternative splicing of RNA encoding 10 variable exons (V1-V10) can give rise to many different CD44 variant protein isoforms of higher molecular weight. CD44 isoforms containing the V6 exon play a crucial role in tumour metastasis and lymphocyte activation. However, the role of CD44V6 in the kidney is unknown. The aim of this study was to examined renal CD44V6 expression in health, disease and in vitro. Immunohistochemistry staining with the V6-specific 1.1ASML antibody identified constitutive CD44V6 expression by occasional cortical tubular epithelial cells and medullary tubules in normal rat kidney. In immune-induced kidney disease (rat anti-glomerular basement membrane glomerulonephritis), there was a marked increase in CD44V6 expression by cortical tubules, particularly in areas of tubulointerstitial damage, which was associated with focal macrophage infiltration. There was also a marked increase in CD44V6 expression by damaged tubules in a model of non-immune kidney disease (unilateral ureteric obstruction). Reverse transcription-polymerase chain reaction revealed a complex pattern of CD44V6-containing mRNA isoforms in normal rat kidney. This pattern of CD44V6 splicing was essentially unaltered in disease. The NRK52E normal rat kidney tubular epithelial cell line expresses both CD44S and CD44V6. Stimulation of NRK52E cells with IL-1 or transforming growth factor-beta 1 induced a two-to-five-fold increase in the expression of both CD44S and CD44V6. Furthermore, triggering of NRK52E cells by antibodies to CD44S or CD44V6, but not isotype control antibodies, induced secretion of monocyte chemoattractant protein-1. In conclusion, this study has identified expression of the tumour-associated marker CD44V6 in tubular epithelial cells in normal and diseased rat kidney, and suggests that signalling through the CD44V6 molecule may participate in the pathogenesis of experimental kidney disease.

Interleukin-1 induces tubular epithelial-myofibroblast transdifferentiation through a transforming growth factor-β1-dependent mechanism in vitro

Interleukin-1 (IL-1) has been shown to exert profibrotic activity in a number of disease models, including crescentic glomerulonephritis and pulmonary fibrosis, but the mechanisms by which this operates are poorly understood. Recent studies have identified a novel mechanism promoting renal fibrosis: tubular epithelial-myofibroblast transdifferentaation (TEMT). The present study examined whether IL-1 can stimulate TEMT in vitro. Cells of the normal rat kidney tubular epithelial cell line (NRK52E) were grown to confluence on collagen-coated plates and cultured for 5 days in the presence 1 to 20 ng/mL of IL-1α. Doses of 10 to 20 ng/mL of IL-1 caused transdifferentiation of NRK52E cells into myofibroblast-like cells. Scanning electron microscopy identified IL-1-induced morphological changes as a loss of apical-basal polarity and microvilli, cell hypertrophy, and the development of an elongated and invasive appearance. Phenotypically, IL-1-induced TEMT was characterized by de novo messenger RNA and protein expression of the mesenchymal marker α-smooth muscle actin, shown by Northern blotting, immunohistochemistry, and Western blotting. This was accompanied by loss of the epithelial marker E-cadherin. The addition of an excess of IL-1-receptor antagonist completely inhibited IL-1-induced TEMT. IL-1 was shown to stimulate the secretion of active transforming growth factor-β1 (TGF-β1) by NRK52E cells. Furthermore, the addition of a neutralizing anti-TGF-β1 antibody inhibited IL-1-induced TEMT. In conclusion, IL-1 is a profibrogenic cytokine capable of inducing TEMT through a TGF-β1-dependent mechanism. This may represent a novel mechanism by which IL-1 induces renal fibrosis in vivo.

Transforming growth factor-β regulates tubular epithelial-myofibroblast transdifferentiation in vitro

We recently found evidence of tubular epithelial-myofibroblast transdifferentiation (TEMT) during the development of tubulointerstitial fibrosis in the rat remnant kidney. This study investigated the mechanisms that induce TEMT in vitro. The normal rat kidney tubular epithelial cell line (NRK52E) was cultured for six days on plastic or collagen type I-coated plates in the presence or absence of recombinant transforming growth factor-beta1 (TGF-beta1). Transdifferentiation of tubular cells into myofibroblasts was assessed by electron microscopy and by expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin. NRK52E cells cultured on plastic or collagen-coated plates showed a classic cobblestone morphology. Culture in 1 ng/ml TGF-beta caused only very minor changes in morphology, but culture in 10 or 50 ng/ml TGF-beta1 caused profound changes. This involved hypertrophy, a loss of apical-basal polarity and microvilli, with cells becoming elongated and invasive, the formation of a new front-end back-end polarity, and the appearance of actin microfilaments and dense bodies. These morphological changes were accompanied by phenotypic changes. Double immunohistochemistry staining showed that the addition of TGF-beta1 to confluent cell cultures caused a loss of the epithelial marker E-cadherin and de novo expression of alpha-SMA. An intermediate stage in transdifferentiation could be seen with hypertrophic cells expressing both E-cadherin and alpha-SMA. De novo alpha-SMA expression was confirmed by Northern blotting, Western blotting, and flow cytometry. In particular, cells with a transformed morphology showed strong alpha-SMA immunostaining of characteristic microfilament structures along the cell axis. There was a dose-dependent increase in the percentage of cells expressing alpha-SMA with increasing concentrations of TGF-beta1, which was completely inhibited by the addition of a neutralizing anti-TGF-beta1 antibody. Compared with growth on plastic, cell culture on collagen-coated plates showed a threefold increase in the percentage of cells expressing alpha-SMA in response to TGF-beta1. TGF-beta1 is a key mediator that regulates, in a dose-dependent fashion, transdifferentiation of tubular epithelial cells into alpha-SMA+ myofibroblasts. This transdifferentiation is markedly enhanced by growth on collagen type I. These findings have identified a novel pathway that may contribute to renal fibrosis associated with overexpression of TGF-beta1 within the diseased kidney.

Immunolocalization of AE2 anion exchanger in rat kidney.

The cellular and subcellular localizations of the AE2 anion exchanger in rat kidney have remained elusive despite detection of moderately abundant AE2 mRNA and AE2 polypeptide in all kidney regions. In this report a simple epitope unmasking technique has allowed the immunolocalization of AE2 antigenic sites in basolateral membranes of several rat kidney tubular epithelial cells. AE2 immunostaining was faint or absent in the glomerulus and proximal tubule, present in descending and ascending thin limbs, and stronger in the medullary thick ascending limb (MTAL). A lower staining intensity was found in cortical thick ascending limbs and even less in the distal convoluted tubule. In contrast, there was an enhanced staining in the macula densa. In principal cells (PC) of the connecting segment, AE2 was undetectable but gradually increased in intensity along the collecting duct, with strongest staining in inner medullary collecting duct (IMCD) PC. A sodium dodecyl sulfate-sensitive AE2-related Golgi epitope was also detected in some interstitial and endothelial cells of the inner medulla and in epithelial cells of IMCD and MTAL. Colchicine treatment of the intact animal altered the distribution of this Golgi-associated epitope but left plasmalemmal AE2 undisturbed. Reverse transcription-polymerase chain reaction detected AE2a, AE2b, and AE2c2 but not AE2cl transcripts in rat kidney mRNA. The results suggest a widespread occurrence of the AE2 protein in several renal epithelial cell types.

FAILURE OF RAT KIDNEY NEPHRON COMPONENTS TO INDUCE ALLOGENEIC LYMPHOCYTES TO PROLIFERATE IN MIXED LYMPHOCYTE KIDNEY CELL CULTURE

Rat kidney glomerular mesangial, glomerular epithelial, and tubular epithelial cells were isolated in virtually pure form and studied in mixed lymphocyte kidney cell culture (MLKC) for their ability to induce allogeneic spleen lymphocyte proliferation. The responses were compared with proliferative responses induced by allogeneic endothelial cells or spleen lymphocytes in the same strain combination. Stimulator cells were treated or left untreated with gamma-interferon, known to increase the major histocompatibility complex class II (and class I) expression of the stimulator cells. The results demonstrate that the nephron components are not able to induce lymphoproliferation in the MLKC. In contrast, the endothelial cells of rat heart were potent inducers of lymphoproliferation in mixed lymphocyte endothelial cell cultures (MLEC), as were allogeneic spleen cells. Although the kidney parenchymal cells have been shown to be immunogenic in vivo, the present finding suggests that they are unable to function as antigen-presenting cells on their own.