Renal fibroblasts and myofibroblasts in chronic kidney disease.
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Chronic Disease↗
Biomedical subjects
Frank Strutz
Publications and source records attributed to Frank Strutz.
Renal fibrosis and the origin of the renal fibroblast.
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Animals↗
Decrease in expression of bone morphogenetic proteins 4 and 5 in synovial tissue of patients with osteoarthritis and rheumatoid arthritis.
Bone morphogenetic proteins (BMPs) have been identified as important morphogens with pleiotropic functions in regulating the development, homeostasis and repair of various tissues. The aim of this study was to characterize the expression of BMPs in synovial tissues under normal and arthritic conditions. Synovial tissue from normal donors (ND) and from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) were analyzed for BMP expression by using microarray hybridization. Differential expression of BMP-4 and BMP-5 was validated by semiquantitative RT-PCR, in situ hybridization and immunohistochemistry. Activity of arthritis was determined by routine parameters for systemic inflammation, by histological scoring of synovitis and by semiquantitative RT-PCR of IL-1beta, TNF-alpha, stromelysin and collagenase I in synovial tissue. Expression of BMP-4 and BMP-5 mRNA was found to be significantly decreased in synovial tissue of patients with RA in comparison with ND by microarray analysis (p < 0.0083 and p < 0.0091). Validation by PCR confirmed these data in RA (p < 0.002) and also revealed a significant decrease in BMP-4 and BMP-5 expression in OA compared with ND (p < 0.015). Furthermore, histomorphological distribution of both morphogens as determined by in situ hybridization and immunohistochemistry showed a dominance in the lining layer of normal tissues, whereas chronically inflamed tissue from patients with RA revealed BMP expression mainly scattered across deeper layers. In OA, these changes were less pronounced with variable distribution of BMPs in the lining and sublining layer. BMP-4 and BMP-5 are expressed in normal synovial tissue and were found decreased in OA and RA. This may suggest a role of distinct BMPs in joint homeostasis that is disturbed in inflammatory and degenerative joint diseases. In comparison with previous reports, these data underline the complex impact of these factors on homeostasis and remodeling in joint physiology and pathology.
Adult↗
The antifibrotic effects of relaxin in human renal fibroblasts are mediated in part by inhibition of the Smad2 pathway.
BACKGROUND: The peptide hormone relaxin has been demonstrated to exert antifibrotic effects in renal and extrarenal tissues. The aims of this study were to identify potential anti-fibrotic effects of relaxin on human renal fibroblasts in vitro and to analyze their mechanisms. METHODS: All experiments were performed in established renal fibroblast cell lines and in primary cortical fibroblasts. Effects of relaxin were analyzed on cell proliferation, apoptosis, activation of renal fibroblasts, synthesis and secretion of collagen type I and fibronectin, as well as on the secretion of matrix metalloproteinases (MMPs). Effects on transforming growth factor-beta1 (TGF-beta1) receptor binding were analyzed by flow cytometry and on TGF-beta1 signal transduction by immunoblot analyses for Smad4 and 7, translocation from cytosol to nucleus for Smad2 and 3 as well as for phosphorylated and unphosphorylated forms of p38, c-Jun NH2 terminal kinase (JNK) and extracellular-regulated protein kinase (ERK). Finally, specific siRNAs for Smad2 and 3 were applied to assess the signal transduction pathway. RESULTS: After stimulation with relaxin, tyrosine phosphorylation of a 220 kD protein was demonstrated, indicating interaction with the receptor. Relaxin had only modest inhibitory effects on cell proliferation, and no effects on apoptosis. Conversely, relaxin exerted robust effects on TGF-beta1-induced fibroblast to myofibroblast transformation as well as on matrix synthesis and secretion even at the smallest dose tested. The secretion of MMP-2 and MMP-9 was induced noticeably by all investigated relaxin concentrations. TGF-beta1 receptor binding was not influenced by relaxin; however, it prevented Smad2 phosphorylation, translocation to nucleus, and complex formation between Smad2 and 3 indicating a possible interaction with TGF-beta1 signaling. These findings were corroborated by studies using siRNAs to Smad2 and 3 where siRNA to Smad2 but not to Smad3 inhibited the TGF-beta1 induction of fibronectin synthesis. There was no influence of relaxin on intracellular Smad3, Smad4, and Smad7 translocation or phosphorylation of mitogen-activated protein (MAP) kinases. CONCLUSION: Relaxin is a potent inhibitor of TGF-beta1-induced extracellular matrix (ECM) synthesis and secretion as well as fibroblast activation. Furthermore, it induces ECM degradation by induction of MMP-2 and MMP-9. These effects are mediated, at least in part, by inhibition of TGF-beta1 signaling.
Apoptosis↗
Tubular cell apoptosis and cidofovir-induced acute renal failure.
Cidofovir is an antiviral drug with activity against a wide array of DNA viruses including poxvirus. The therapeutic use of cidofovir is marred by a dose-limiting side effect, nephrotoxicity, leading to proximal tubular cell injury and acute renal failure. Treatment with cidofovir requires the routine use of prophylactic measures. A correct knowledge of the cellular and molecular mechanisms of cidofovir toxicity may lead to the development of alternative prophylactic strategies. We recently cared for a patient with irreversible acute renal failure due to cidofovir. Renal biopsy showed tubular cell apoptosis. Cidofovir induced apoptosis in primary cultures of human proximal tubular cells in a temporal (peak apoptosis at 7 days) and concentration (10-40 microg/ml) pattern consistent with that of clinical toxicity. Apoptosis was identified by the presence of hypodiploid cells, by the exposure of annexin V binding sites and by morphological features and was associated with the appearance of active caspase-3 fragments. Cell death was specific as it was also present in a human proximal tubular epithelial cell line (HK-2), but not in a human kidney fibroblast cell line, and was prevented by probenecid. An inhibitor of caspase-3 (DEVD) prevented cidofovir apoptosis. The survival factors present in serum, insulin-like growth factor-1 and hepatocyte growth factor, were also protective. The present data suggest that apoptosis induction is a mechanism contributing to cidofovir nephrotoxicity. The prophylactic administration of factors with survival activity for tubular epithelium should be further explored in cidofovir renal injury.
Acute Kidney Injury↗
Transforming growth factor-beta1 stimulates collagen matrix remodeling through increased adhesive and contractive potential by human renal fibroblasts.
Renal tubulointerstitial fibrosis is the common final pathway leading to end-stage renal failure. Tubulointerstitial fibrosis is characterized by fibroblast proliferation and excessive matrix accumulation. Transforming growth factor-beta1 (TGF-beta1) has been implicated in the development of renal fibrosis accompanied by alpha-smooth muscle actin (alpha-SMA) expression in renal fibroblasts. To investigate the molecular and cellular mechanisms involved in tubulointerstitial fibrosis, we examined the effect of TGF-beta1 on collagen type I (collagen) gel contraction, an in vitro model of scar collagen remodeling. TGF-beta1 enhanced collagen gel contraction by human renal fibroblasts in a dose- and time-dependent manner. Function-blocking anti-alpha1 or anti-alpha2 integrin subunit antibodies significantly suppressed TGF-beta1-stimulated collagen gel contraction. Scanning electron microscopy showed that TGF-beta1 enhanced the formation of the collagen fibrils by cell attachment to collagen via alpha1beta1 and alpha2beta1 integrins. Flow cytometry and cell adhesion analyses revealed that the stimulation of renal fibroblasts with TGF-beta1 enhanced cell adhesion to collagen via the increased expression of alpha1 and alpha2 integrin subunits within collagen gels. Fibroblast migration to collagen was not up-regulated by TGF-beta1. Furthermore, TGF-beta1 increased the expression of a putative contractile protein, alpha-SMA, by human renal fibroblasts in collagen gels. These results suggest that TGF-beta1 stimulates fibroblast-collagen matrix remodeling by increasing both integrin-mediated cell attachment to collagen and alpha-SMA expression, thereby contributing to pathological tubulointerstitial collagen matrix reorganization in renal fibrosis.
Actinin↗
Activation of toll-like receptor-9 induces progression of renal disease in MRL-Fas(lpr) mice.
How bacterial or viral infections trigger flares of autoimmunity is poorly understood. As toll-like receptor (TLR)-9 activation by exogenous or endogenous CpG-DNA may contribute to disease activity of systemic lupus erythematosus, we examined the effects of CpG-oligodeoxynucleotides (ODN) or DNA derived from Escherichia coli (E. coli) on the course of nephritis in MRL(lpr/lpr) mice. In kidneys of these mice, TLR9 localized to glomerular, tubulointerstitial, and perivascular infiltrates. After intraperitoneal injection labeled CpG-ODN localized to glomerular and interstitial macrophages and dendritic cells in nephritic kidneys of MRL(lpr/lpr) mice but not in healthy MRL controls. Furthermore, murine J774 macrophages and splenocytes from MRL(lpr/lpr) mice, but not tubular epithelial cells, renal fibroblasts, or mesangial cells, expressed TLR9 and up-regulated CCL5/RANTES mRNA upon stimulation with CpG-ODN in vitro. In vivo both E. coli DNA and CpG-ODN increased serum DNA autoantibodies of the IgG2a isotype in MRL(lpr/lpr) mice. This was associated with progression of mild to crescentic glomerulonephritis, interstitial fibrosis, and heavy proteinuria. CpG-ODN increased renal CCL2/MCP-1 and CCL5/RANTES expression associated with increased glomerular and interstitial leukocyte recruitment. In contrast control GpC-ODN had no effect. We conclude that TLR9 activation triggers disease activity of systemic autoimmunity, for example, lupus nephritis, and that adaptive and innate immune mechanisms contribute to the CpG-DNA-induced progression of lupus nephritis.
Animals↗
Impact of chronic LDL-apheresis treatment on Achilles tendon affection in patients with severe familial hypercholesterolemia: a clinical and ultrasonographic 3-year follow-up study.
OBJECTIVE: Pain of Achilles tendon (AT) is a common symptom in patients with severe familial hypercholesterolemia (FH) and often associated with AT xanthomas. It is unknown if these changes are potentially reversible during lipid lowering treatment. LDL-apheresis (LA) represents the most effective lipid lowering regimen. Aim of this investigation was to determine clinical and ultrasonographic reduction of AT xanthomas in patients with severe FH undergoing regular LA. METHODS: At baseline, patient history of 22 patients with FH undergoing LA was obtained and their 44 ATs were evaluated clinically and with ultrasound for the presence of xanthomas. Three years later, both examinations of ATs could be repeated at follow-up visits in 16 patients. AT thickness and changes in echo structure were assessed at both points of time and compared to each other as well as to a healthy control group (n = 21). ROC analysis was performed to identify the optimal cut-off in AT thickness between healthy and affected ATs. RESULTS: Twelve of 22 FH patients suffered from AT pain at least once during their life-time. At baseline, AT thickness was significantly increased compared to the healthy control group (mean sagittal diameter 10.1 +/- 3.6 mm). At follow-up, AT thickness was significantly reduced to 8.2 +/- 3.3 mm (mean) under LA, whereas changes in echo structure were less distinct between both visits. Conversely, in the control group, mean AT thickness was 5.2 +/- 0.6 mm. The optimal cut-off between healthy and affected ATs was determined to be 6mm. CONCLUSIONS: Ultrasonographic changes of the AT (thickening and changes in echo structure) are frequent in patients with severe FH, even if xanthomas are not clinically evident. LA treatment has the capability to reduce AT xanthomas and thickness. Ultrasound may give information about diagnosis and follow-up of AT affection in patients with FH.
Achilles Tendon↗
CCR1 blockade reduces interstitial inflammation and fibrosis in mice with glomerulosclerosis and nephrotic syndrome.
BACKGROUND: CC chemokines mediate leukocyte infiltration into inflamed tissue. We have recently shown that blockade of the CC chemokine receptor CCR1 reduces interstitial inflammation and fibrosis in murine obstructive nephropathy. However, it is not known whether CCR 1 blockade is protective in progressive renal injury associated with severe proteinuria. We therefore studied the effect of the small-molecule CCR1 antagonist BX471 in a murine model of adriamycin-induced focal segmental glomerulosclerosis (FSGS) with nephrotic syndrome and progressive interstitial inflammation and fibrosis. METHODS: Adriamycin nephropathy with persistent proteinuria was induced in male BALB/c mice by two intravenous injections of adriamycin (13 mg/kg) at day 0 and 14. BX471 treatment was started at day 14 when proteinuria and interstitial inflammation had developed. At 6 weeks, renal histology was studied by morphometry and immunohistochemistry. RESULTS: At week 6, adriamycin-treated mice showed FSGS, associated with tubulointerstitial injury consisting of tubular dilation and atrophy, interstitial leukocyte infiltration, and fibrosis. The mRNA expression of CCR1 and CC chemokines, including the CCR1 ligands CCL3 (MIP-1alpha) and CCL5 (RANTES), was up-regulated in diseased kidneys, with a prominent interstitial expression of CCL5. Compared to vehicle-treated controls BX471 significantly reduced the amount of macrophages and T lymphocytes in interstitial lesions by 51% and 22%, respectively. Markers of renal fibrosis such as interstitial fibroblasts (48%) and interstitial volume (23%) were significantly reduced by BX471 treatment. In contrast, the extent of proteinuria and glomerular sclerosis was not affected by BX471 treatment. CONCLUSION: Blockade of CCR1 substantially reduced interstitial leukocyte accumulation and the subsequent renal fibrosis in a murine model of nephrotic syndrome and FSGS. These findings support a role for CCR1 in interstitial leukocyte recruitment and suggest that CCR1 blockade might be a new therapeutic strategy in progressive nephropathies such as FSGS.
Animals↗
Bone morphogenic protein-7 inhibits progression of chronic renal fibrosis associated with two genetic mouse models.
Tubulointerstitial fibrosis is a hallmark feature of chronic renal injury. Specific therapies to control the progression of renal fibrosis toward end-stage renal failure are limited. Previous studies have demonstrated that expression of endogenous bone morphogenic protein-7 (BMP-7) is reduced in the kidneys of several inducible mouse models of acute and chronic renal disease and that administration of exogenous recombinant human BMP-7 (rhBMP-7) has a beneficial effect on kidney function. Here we report that treatment with rhBMP-7 leads to improved renal function, histology, and survival in mice deficient in the alpha3-chain of type IV collagen and MRL/MpJlpr/lpr lupus mice, two genetic models for chronic renal injury and fibrosis. Such therapeutic benefit is also associated with a significant decrease in the expression of profibrotic molecules, such as type I collagen and fibronectin, in renal fibroblasts. Additionally, rhBMP-7 induces expression of active matrix metalloproteinase-2, which is potentially important for removal of fibrotic matrix. Collectively, these studies provide further evidence for rhBMP-7 as an important bone-associated protein with protective function against renal pathology.
Animals↗
Direct contact between human peripheral blood mononuclear cells and renal fibroblasts facilitates the expression of monocyte chemoattractant protein-1.
BACKGROUND: Cell-to-cell interaction is thought to be an important feature of a variety of biological processes. As far as the proinflammatory process is concerned, the interaction between mesangial cells and monocytes/macrophages induces the expression of monocyte chemoattractant protein-1 (MCP-1), and this may play a role in glomerulonephritis. In this study, we investigated whether the cell-to-cell interaction between immune cells and renal fibroblasts induces MCP-1 gene expression, which may be involved in interstitial inflammation in the kidney. METHODS: Human renal fibroblast cell lines, tNKF (from a normal kidney) and tFKIF (from a kidney with fibrosis), and peripheral blood mononuclear cells (PBMC) were used to assess the effect of cell-to-cell contact on the expression of MCP-1 mRNA in the fibroblasts. The expression of the MCP-1 gene in the fibroblasts was also examined after stimulation with tumor necrosis factor-alpha (TNF-alpha) and the culture supernatant from PBMC. RT-PCR was used to detect MCP-1 mRNA expression. Neutralizing antibodies to intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial adhesion molecule-1 (VCAM-1) were used to block the cell-to-cell contact between the fibroblasts and PBMC. RESULTS: TNF-alpha and the culture supernatant from PBMC increased MCP-1 gene expression in tNKF cells. Contact culture with PBMC also significantly increased MCP-1 gene expression in tNKF cells. Although the basal level of MCP-1 mRNA was higher in tFKIF than tNKF cells, tFKIF cells did not respond significantly to any stimulation in this study. Following pretreatment with anti-ICAM-1 antibody, MCP-1 gene expression in tNKF cells was significantly suppressed in contact culture with PBMC. Anti-VCAM-1 antibody treatment had no effects. CONCLUSION: It is suggested that the interaction between renal fibroblasts and PBMC was mediated through direct contact and by secreted humoral factors. ICAM-1 on renal fibroblasts may be involved in the direct cell-to-cell interaction inducing MCP-1 gene expression, which seems to be involved in renal interstitial inflammation.
Antibodies↗
New insights into mechanisms of fibrosis in immune renal injury.
. Renal fibrosis is the final common pathway for many kidney lesions that lead to chronic progressive organ failure. The tubulointerstitial space occupies up to 90% of kidney volume, indicating that pathological changes in that space can not be without functional significance. By analogy to wound healing, renal fibrogenesis can be divided arbitrarily into three phases: induction, inflammatory, and post-inflammatory phases. The latter phase is of particular importance, since its length often exceeds what would be required for healing. The induction phase is characterized by the infiltration of the tubulointerstitial space by mononuclear inflammatory cells. This influx is mediated by proinflammatory cytokines and chemokines often secreted by activated tubular epithelial cells. Subsequently, these infiltrating mononuclear cells stimulate a heterogeneous group of resident fibroblasts and tubular epithelial cells to undergo phenotypic conversion into activated fibroblasts that secrete abundant extracellular matrix. Tubular epithelial cells contribute to this process through epithelial-mesenchymal transition. During the inflammatory phase these activated fibroblasts are stimulated to produce collagenous matrix mainly by cytokines, such as TGF-beta1, EGF, ET-1, and FGF-2, which are secreted by inflammatory and injured somatic cells. Occasionally however, when inflammation subsides, the matrix synthesis in the post-inflammatory phase of renal fibrogenesis continues and may be more dependent on autocrine stimulation from resident renal cells such as remaining tubular epithelium. Eventually, the collagenous matrix of fibrogenesis destroys blood supply and the perimeter of viability for fibroblasts regresses to the point where scars become acellular.
Animals↗
BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury.
Bone morphogenic protein (BMP)-7 is a 35-kDa homodimeric protein and a member of the transforming growth factor (TGF)-beta superfamily. BMP-7 expression is highest in the kidney, and its genetic deletion in mice leads to severe impairment of eye, skeletal and kidney development. Here we report that BMP-7 reverses TGF-beta1-induced epithelial-to-mesenchymal transition (EMT) by reinduction of E-cadherin, a key epithelial cell adhesion molecule. Additionally, we provide molecular evidence for Smad-dependent reversal of TGF-beta1-induced EMT by BMP-7 in renal tubular epithelial cells and mammary ductal epithelial cells. In the kidney, EMT-induced accumulation of myofibroblasts and subsequent tubular atrophy are considered key determinants of renal fibrosis during chronic renal injury. We therefore tested the potential of BMP-7 to reverse TGF-beta1-induced de novo EMT in a mouse model of chronic renal injury. Our results show that systemic administration of recombinant human BMP-7 leads to repair of severely damaged renal tubular epithelial cells, in association with reversal of chronic renal injury. Collectively, these results provide evidence of cross talk between BMP-7 and TGF-beta1 in the regulation of EMT in health and disease.
Activin Receptors, Type I↗
DNA oligonucleotide microarray technology identifies fisp-12 among other potential fibrogenic genes following murine unilateral ureteral obstruction (UUO): modulation during epithelial-mesenchymal transition.
BACKGROUND: Tubulointerstitial inflammation and fibrosis are pathologic hallmarks of end-stage renal disease (ESRD). Here we have used DNA microarray technology to monitor the transcriptomic responses to murine unilateral ureteral obstruction (UUO) with a view to identifying molecular modulators of tubulointerstitial fibrosis. METHODS: Using Affymetrix Mu74Av2 microarrays, gene expression 4 and 10 days postobstruction was investigated relative to control contralateral kidneys. Candidate profibrogenic genes were further investigated in epithelial cells undergoing epithelial to mesenchymal transition (EMT) in vitro. RESULTS: mRNA levels for 1091 gene/EST sequences, of a total of 12,488 displayed on the microarray, were altered twofold or greater by days 4 and 10 postobstruction compared to contralateral control kidneys. Genes were categorised into functional groups, including modulators of cytoskeletal and extracellular matrix metabolism, cell growth, signalling, and transcription/translational events. Among the potentially profibrogenic genes, whose mRNA levels were increased after UUO, were fibroblast-inducible secreted protein (fisp-12), the murine homologue of connective tissue growth factor (CTGF), collagen XVIIIalpha1, secreted protein acidic and rich in cysteine (SPARC), and src-suppressed C-kinase substrate (SSeCKS). A sustained increase in fisp-12 mRNA level was observed during EMT induced by transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF). CONCLUSION: Altered gene expression in murine UUO has been demonstrated. Increased expression of fisp-12, SPARC, and SSeCKS has been shown in response to TGF-beta1 treatment and during EMT, suggesting that these genes may offer potential therapeutic targets against tubulointerstitial fibrosis.
A Kinase Anchor Proteins↗
Glucose-induced fibronectin and collagen type III expression in renal fibroblasts can occur independent of TGF-beta1.
BACKGROUND: Various renal cell types have been shown to contribute to the excessive matrix deposition observed in diabetic nephropathy. The present study examined the effect of high ambient glucose and transforming growth factor-beta1 (TGF-beta1) on matrix production by human renal fibroblasts. METHODS: Human renal fibroblasts (TK173) were used to examine the effects of high glucose and TGF-beta1 on fibronectin and collagen type III expression. Stable transfectants were generated of TK173 cells expressing a dominant negative TGF-beta type II receptor. Matrix components were measured in enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Fibronectin secretion by renal fibroblasts was increased upon exposure to high glucose, but with delayed kinetics compared to TGF-beta1-induced fibronectin. Exposure to high glucose resulted in an increased secretion of latent TGF-beta1. However, treatment with neutralizing pan-specific anti-TGF-beta antibodies could not attenuate the effects of glucose. Furthermore, collagen type III was up-regulated by high glucose, but not by TGF-beta1. Importantly, fibroblasts expressing a dominant negative TGF-beta type II receptor were defective in TGF-beta1-induced fibronectin production, whereas glucose-induced fibronectin and collagen type III were unaffected. CONCLUSIONS: These data show that in renal fibroblasts exposure to high glucose can increase matrix production independent of endogenous TGF-beta1. Although glucose activation is accompanied by an increased production of latent TGF-beta1, which can have an important role in vivo, the data suggest involvement of alternative growth factors in the mechanism by which hyperglycemic conditions can modulate matrix accumulation in diabetic nephropathy.
Antibodies↗
[Metformin-associated lactic acidosis with acute renal failure in type 2 diabetes mellitus].
CASE REPORT: An 83-year-old patient was admitted to our hospital because of gastrointestinal symptoms, mental confusion and dysarthria. The patient suffered from type 2 diabetes mellitus and was taking metformin. A mild renal insufficiency was known. On admission, we found impaired consciousness, Kussmaul breathing, a body temperature of 32.1 degrees C, and hemodynamic instability. Laboratory testing revealed lactic acidosis (pH 6.71, base excess--30, standard bicarbonate 4.0 mmol/l, lactate 24.4 mmol/l) and acute renal failure with a creatinine of 10.6 mg/dl and blood urea nitrogen of 134 mg/dl. Electrolytes were not altered; the blood glucose was elevated (147 mg/dl). According to history, physical examination, and laboratory testing the diagnosis metformin-induced lactic acidosis with acute renal failure was made. This diagnosis was confirmed by an elevated level of metformin. As soon as possible a bicarbonate hemodialysis was initiated. After 8 hours of hemodialysis the acid-base metabolism was almost balanced and the vigilance of the patient normalized. No further sessions of hemodialysis were needed and insulin therapy was started. CONCLUSIONS: Metformin-induced lactic acidosis is a common side effect in patients with renal insufficiency. For an early diagnosis, clinical symptoms of intoxication should be well known by physicians and patients. First-line therapy for correction of lactic acidosis and effective elimination of metformin is bicarbonate hemodialysis. Sodium bicarbonate infusions are not able to correct the acid-base metabolism sufficiently. For prevention the renal function should be monitored closely and metaformin therapy should be stopped, if a deterioration of renal function is observed.
Acidosis, Lactic↗
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)-beta1 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-beta1. 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-beta1 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-beta1 plus FGF-2. Conversely, the mesenchymal markers alpha-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-beta1. 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-beta1. 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.
Animals↗
Epithelial-mesenchymal transition of tubular epithelial cells in human renal biopsies.
BACKGROUND: In recent studies performed on cultured cells and experimental nephropathies, it has been hypothesized that tubular epithelial cells (TEC), via epithelial-mesenchymal transformation (EMT), can become collagen-producing cells. According to this theory, they should proceed through several activating steps, such as proliferation and phenotype changes, to eventually synthesize extracellular matrix (ECM). METHODS: To evaluate whether EMT operates in human TECs, 133 renal biopsies of different renal diseases were studied, analyzing by immunohistochemistry and in situ hybridization the possible expression of markers of proliferation (PCNA, Mib-1), cellular phenotype (vimentin, alpha-SMA, cytokeratin, ZO-1) and ECM production (prolyl 4-hydroxylase, HSP47, interstitial collagens). RESULTS: Independently of histological diagnosis, variable degrees of TEC positivity for PCNA (2.7 +/- 2.4 cells/field) and Mib-1 (1.9 +/- 2.3) were present. TECs expressing vimentin (1.4 +/- 4.7) and alpha-smooth muscle actin (alpha-SMA; 0.04 +/- 0.4) also were detected. It was possible to observe loss of epithelial antigens from 8 to 10% of the tubular cross sections. Moreover, TECs were stained by prolyl 4-hydroxylase (3.6 +/- 4.3), heat shock protein-47 (HSP47; 2.9 +/- 5.4), collagen type I (0.2 +/- 2.7) and type III (0.3 +/- 2.0). Collagen types I and III mRNAs were found in 0.8 to 1.4 cells/field. The number of TEC with EMT features were associated with serum creatinine and the degree of interstitial damage (P< or = 0.03), and even considering the 45 cases with mild interstitial lesions, the tubular expression of all markers remained strictly associated with renal function (P< or = 0.01). CONCLUSIONS: Our results suggest that, via transition to a mesenchymal phenotype, TEC can produce ECM proteins in human disease and directly intervene in the fibrotic processes. Moreover, the association of EMT features with serum creatinine supports the value of these markers in the assessment of disease severity.
Actins↗