A PHD inhibitor prevents changes in the phosphoproteome and capillary rarefaction by CsA: treatment option for CKD?

Labes et al. analyze the phosphoproteome in a mouse model of chronic cyclosporine A nephrotoxicity and detect significant changes in the angiogenic pathway. Furthermore, they observe reduced hemoglobin levels and capillary rarefaction in the kidney. The authors show that coadministration of the hypoxia-inducible factor prolyl hydroxylase inhibitor daprodustat almost completely prevents changes of the phosphoproteome and capillary rarefaction, suggesting that prolyl hydroxylase domain enzyme inhibitors may preserve microvasculature of the kidney, which is commonly impaired in chronic kidney disease.

Inhibition of oxygen-sensing prolyl hydroxylases increases lipid accumulation in human primary tubular epithelial cells without inducing ER stress.

The role of the hypoxia-inducible transcription factor (HIF) pathway in renal lipid metabolism is largely unknown. As HIF stabilizing prolyl hydroxylase (PHD) inhibitors are currently investigated in clinical trials for the treatment of renal anemia, we studied the effects of genetic deletion and pharmacological inhibition of PHDs on renal lipid metabolism in transgenic mice and human primary tubular epithelial cells (hPTEC). Tubular cell-specific deletion of HIF prolyl hydroxylase 2 (Phd2) increased the size of Oil Red-stained lipid droplets in mice. In hPTEC, the PHD inhibitors (PHDi) DMOG and ICA augmented lipid accumulation, which was visualized by Oil Red staining and assessed by microscopy and an infrared imaging system. PHDi-induced lipid accumulation required the exogenous availability of fatty acids and was observed in both proximal and distal hPTEC. PHDi treatment was not associated with structural features of cytotoxicity in contrast to treatment with the immunosuppressant cyclosporine A (CsA). PHDi and CsA differentially upregulated the expression of the lipid droplet-associated genes PLIN2, PLIN4 and HILPDA. Both PHDi and CsA activated AMP-activated protein kinase (AMPK) indicating the initiation of a metabolic stress response. However, only CsA triggered endoplasmic reticulum (ER) stress as determined by the increased mRNA expression of multiple ER stress markers but CsA-induced ER stress was not linked to lipid accumulation. Our data raise the possibility that PHD inhibition may protect tubular cells from toxic free fatty acids by trapping them as triacylglycerides in lipid droplets. This mechanism might contribute to the renoprotective effects of PHDi in experimental kidney diseases.

Multiple renal cancer susceptibility polymorphisms modulate the HIF pathway.

Un-physiological activation of hypoxia inducible factor (HIF) is an early event in most renal cell cancers (RCC) following inactivation of the von Hippel-Lindau tumor suppressor. Despite intense study, how this impinges on cancer development is incompletely understood. To test for the impact of genetic signals on this pathway, we aligned human RCC-susceptibility polymorphisms with genome-wide assays of HIF-binding and observed highly significant overlap. Allele-specific assays of HIF binding, chromatin conformation and gene expression together with eQTL analyses in human tumors were applied to mechanistic analysis of one such overlapping site at chromosome 12p12.1. This defined a novel stage-specific mechanism in which the risk polymorphism, rs12814794, directly creates a new HIF-binding site that mediates HIF-1α isoform specific upregulation of its target BHLHE41. The alignment of multiple sites in the HIF cis-acting apparatus with RCC-susceptibility polymorphisms strongly supports a causal model in which minor variation in this pathway exerts significant effects on RCC development.

Mononuclear phagocytes orchestrate prolyl hydroxylase inhibition-mediated renoprotection in chronic tubulointerstitial nephritis.

Prolyl hydroxylase domain enzyme inhibitors (PHDIs) stabilize hypoxia-inducible factors (HIFs), and are protective in models of acute ischemic and inflammatory kidney disease. Whether PHDIs also confer protection in chronic inflammatory kidney disease models remains unknown. Here we investigated long-term effects of PHDI treatment in adenine-induced nephropathy as a model for chronic tubulointerstitial nephritis. After three weeks, renal dysfunction and tubulointerstitial damage, including proximal and distal tubular injury, tubular dilation and renal crystal deposition were significantly attenuated in PHDI-treated (the isoquinoline derivative ICA and Roxadustat) compared to vehicle-treated mice with adenine-induced nephropathy. Crystal-induced renal fibrosis was only partially diminished by treatment with ICA. Renoprotective effects of ICA treatment could not be attributed to changes in adenine metabolism or urinary excretion of the metabolite 2,8-dihydroxyadenine. ICA treatment reduced inflammatory infiltrates of F4/80+ mononuclear phagocytes in the kidneys and supported a regulatory, anti-inflammatory immune response. Furthermore, interstitial deposition of complement C1q was decreased in ICA-treated mice fed an adenine-enriched diet. Tubular cell-specific HIF-1α and myeloid cell-specific HIF-1α and HIF-2α expression were not required for the renoprotective effects of ICA. In contrast, depletion of mononuclear phagocytes with clodronate largely abolished the nephroprotective effects of PHD inhibition. Thus, our findings indicate novel and potent systemic anti-inflammatory properties of PHDIs that confer preservation of kidney function and structure in chronic tubulointerstitial inflammation and might counteract kidney disease progression.

Receptor-independent modulation of TGF-ß-induced pro-fibrotic pathways by relaxin-2 in human primary tubular epithelial cells.

Renal tubular epithelial cells actively contribute to the development of renal fibrosis and may be targeted by anti-fibrotic drugs. Relaxin-2 (RLX2) applied as recombinant protein is suggested to be renoprotective. Therefore, we investigated whether human primary tubular epithelial cells (hPTEC) obtained from various donors were target cells for the anti-fibrotic actions of RLX2. Treatment of hPTEC with RLX2 reduced the TGF-ß1-induced secretion of the pro-fibrotic factor CTGF (connective tissue growth factor) and inhibited fibronectin synthesis and secretion. Furthermore, metalloproteinase MMP2 secretion was increased, with no effect on MMP9. Considerable differences were observed between hPTEC obtained from different donors. Therefore, expression of the relaxin family peptide receptor RXFP1, the major mediator of renal RLX2 effects, was analyzed. A validated antibody detected a double band of 80-90 kDa in cellular homogenates by Western blotting. Expression of the detected protein was not altered by incubation with TGF-ß1 and RLX2-induced modulation of CTGF expression did not correlate with the putative receptor expression. Therefore, relaxin family receptors RXFP1-4 were assessed by RNA-seq analysis. No evidence was found for mRNA expression of any of these receptors in several hPTEC preparations. Lack of RXFP1 mRNA was confirmed by qPCR using mRNA obtained from THP-1 cells as positive control. Our data thus provide evidence for primary renal human tubular epithelial cells as targets for the anti-fibrotic actions of RLX2. However, anti-fibrotic effects were observed at micromolar concentrations of RLX2 and shown to be independent of RXFP1 expression.

Inhibitors of oxygen sensing prolyl hydroxylases regulate nuclear localization of the transcription factors Smad2 and YAP/TAZ involved in CTGF synthesis.

Pharmacological inhibition of oxygen sensing prolyl hydroxylase domain enzymes (PHDs) has been shown to preserve renal structure and function in various models of kidney disease. Since transforming growth factor ß-1 (TGFß-1) is one of the major mediators of kidney injury, we investigated if inhibition of PHDs with subsequent stabilization of hypoxia inducible transcription factors (HIF) might interfere with TGFß-1 signaling with special emphasis on its target gene connective tissue growth factor (CTGF). Overnight incubation of human renal tubular cells, primary cells and cell lines, with the PDH inhibitor DMOG increased Smad3 expression, but barely affected Smad2. Both Smads were translocated into the nucleus upon activation of the cells with TGFß-1. Interestingly, Smad3 nuclear localization was enhanced upon pretreatment of the cells with DMOG for several hours, whereas nuclear Smad2 was reduced. This differential localization was independent of Smad2/3 phosphorylation. Reduced nuclear Smad2 correlated with impaired CTGF secretion in DMOG-treated cells and transient downregulation of Smad2 interfered with TGFß-1-induced CTGF synthesis. Furthermore, YAP was confirmed as indispensable transcription factor involved in CTGF synthesis. Nuclear localization of YAP and TAZ was reduced in DMOG-treated cells. Our data thus provide evidence for DMOG-mediated reduction of CTGF expression by regulating the nuclear localization of the transcription factors Smad2, YAP and TAZ. Prolonged inhibition of PHDs was necessary to achieve alterations in cellular localization suggesting an indirect HIF-mediated effect. This mechanism might be extended to other transcription factors and target genes, and may thus represent a novel mechanism of negative regulation of gene expression by PHD inhibition.

P2Y2R is a direct target of HIF-1α and mediates secretion-dependent cyst growth of renal cyst-forming epithelial cells.

Polycystic kidney diseases are characterized by numerous renal cysts that continuously enlarge resulting in compression of intact nephrons and tissue hypoxia. Recently, we have shown that hypoxia-inducible factor (HIF)-1α promotes secretion-dependent cyst expansion, presumably by transcriptional regulation of proteins that are involved in calcium-activated chloride secretion. Here, we report that HIF-1α directly activates expression of the purinergic receptor P2Y2R in human primary renal tubular cells. In addition, we found that P2Y2R is highly expressed in cyst-lining cells of human ADPKD kidneys as well as PKD1 orthologous mouse kidneys. Knockdown of P2Y2R in renal collecting duct cells inhibited calcium-dependent chloride secretion in Ussing chamber analyses. In line with these findings, knockdown of P2Y2R retarded cyst expansion in vitro and prevented ATP- and HIF-1α-dependent cyst growth. In conclusion, P2Y2R mediates ATP-dependent cyst growth and is transcriptionally regulated by HIF-1α. These findings provide further mechanistic evidence on how hypoxia promotes cyst growth.

HIF-1α activation results in actin cytoskeleton reorganization and modulation of Rac-1 signaling in endothelial cells.

BACKGROUND: Hypoxia is a major driving force in vascularization and vascular remodeling. Pharmacological inhibition of prolyl hydroxylases (PHDs) leads to an oxygen-independent and long-lasting activation of hypoxia-inducible factors (HIFs). Whereas effects of HIF-stabilization on transcriptional responses have been thoroughly investigated in endothelial cells, the molecular details of cytoskeletal changes elicited by PHD-inhibition remain largely unknown. To investigate this important aspect of PHD-inhibition, we used a spheroid-on-matrix cell culture model. RESULTS: Microvascular endothelial cells (glEND.2) were organized into spheroids. Migration of cells from the spheroids was quantified and analyzed by immunocytochemistry. The PHD inhibitor dimethyloxalyl glycine (DMOG) induced F-actin stress fiber formation in migrating cells, but only weakly affected microvascular endothelial cells firmly attached in a monolayer. Compared to control spheroids, the residual spheroids were larger upon PHD inhibition and contained more cells with tight VE-cadherin positive cell-cell contacts. Morphological alterations were dependent on stabilization of HIF-1α and not HIF-2α as shown in cells with stable knockdown of HIF-α isoforms. DMOG-treated endothelial cells exhibited a reduction of immunoreactive Rac-1 at the migrating front, concomitant with a diminished Rac-1 activity, whereas total Rac-1 protein remained unchanged. Two chemically distinct Rac-1 inhibitors mimicked the effects of DMOG in terms of F-actin fiber formation and orientation, as well as stabilization of residual spheroids. Furthermore, phosphorylation of p21-activated kinase PAK downstream of Rac-1 was reduced by DMOG in a HIF-1α-dependent manner. Stabilization of cell-cell contacts associated with decreased Rac-1 activity was also confirmed in human umbilical vein endothelial cells. CONCLUSIONS: Our data demonstrates that PHD inhibition induces HIF-1α-dependent cytoskeletal remodeling in endothelial cells, which is mediated essentially by a reduction in Rac-1 signaling.

Reconfiguration and loss of peritubular capillaries in chronic kidney disease.

Functional and structural alterations of peritubular capillaries (PTCs) are a major determinant of chronic kidney disease (CKD). Using a software-based algorithm for semiautomatic segmentation and morphometric quantification, this study analyzes alterations of PTC shape associated with chronic tubulointerstitial injury in three mouse models and in human biopsies. In normal kidney tissue PTC shape was predominantly elongated, whereas the majority of PTCs associated with chronic tubulointerstitial injury had a rounder shape. This was reflected by significantly reduced PTC luminal area, perimeter and diameters as well as by significantly increased circularity and roundness. These morphological alterations were consistent in all mouse models and human kidney biopsies. The mean circularity of PTCs correlated significantly with categorized glomerular filtration rates and the degree of interstitial fibrosis and tubular atrophy (IFTA) and classified the presence of CKD or IFTA. 3D reconstruction of renal capillaries revealed not only a significant reduction, but more importantly a substantial simplification and reconfiguration of the renal microvasculature in mice with chronic tubulointerstitial injury. Computational modelling predicted that round PTCs can deliver oxygen more homogeneously to the surrounding tissue. Our findings indicate that alterations of PTC shape represent a common and uniform reaction to chronic tubulointerstitial injury independent of the underlying kidney disease.

Vectorial secretion of CTGF as a cell-type specific response to LPA and TGF-ß in human tubular epithelial cells.

BACKGROUND: Increased expression of the pro-fibrotic protein connective tissue growth factor (CTGF) has been detected in injured kidneys and elevated urinary levels of CTGF are discussed as prognostic marker of chronic kidney disease. There is evidence that epithelial cells lining the renal tubular system contribute to uptake and secretion of CTGF. However, the role of different types of tubular epithelial cells in these processes so far has not been addressed in primary cultures of human cells. RESULTS: Tubular epithelial cells of proximal and distal origin were isolated from human kidneys and cultured as polarized cells in insert wells. The pro-fibrotic stimuli lysophosphatidic acid (LPA) and transforming growth factor ß (TGF-ß) were used to induce CTGF secretion.LPA activated CTGF secretion in proximal tubular cells when applied from either the apical or the basolateral side as shown by immunocytochemistry. CTGF was secreted exclusively to the apical side. Signaling pathways activated by LPA included MAP kinase and Rho kinase signaling. TGF-ß applied from either side also stimulated CTGF secretion primarily to the apical side with little basolateral release.Interestingly, TGF-ß activation induced different signaling pathways depending on the side of TGF-ß application. Smad signaling was almost exclusively activated from the basolateral side most prominently in cells of distal origin. Only part of these cells also synthesized CTGF indicating that Smad activation alone was not sufficient for CTGF induction. MAP kinases were involved in apical TGF-ß-mediated activation of CTGF synthesis in proximal cells and a subset of epithelial cells of distal origin. This subpopulation of distal tubular cells was also able to internalize recombinant apical CTGF, in addition to proximal cells which were the main cells to take up exogenous CTGF. CONCLUSIONS: Analysis of polarized human primary renal epithelial cells in a transwell system shows that vectorial secretion of the pro-fibrotic protein CTGF depends on the cell type, the stimulus and the signaling pathway activated. In all conditions, CTGF was secreted mainly to the apical side upon TGF-ß and LPA treatment and therefore, likely contributes to increased urinary CTGF levels in vivo. Moreover, CTGF secreted basolaterally may be active as paracrine pro-fibrotic mediator.

A complex microdeletion 17q12 phenotype in a patient with recurrent de novo membranous nephropathy.

BACKGROUND: Microdeletions on chromosome 17q12 cause of diverse spectrum of disorders and have only recently been identified as a rare cause of Mayer-Rokitansky-Kuester-Hauser-Syndrome (MRKH), which is characterized by uterus aplasia ± partial/complete vaginal aplasia in females with a regular karyotype. For the first time we report about a patient with a 17q12 microdeletion who is affected by MRKH in combination with a vascular and soft tissue disorder. Repeatedly she suffered from kidney transplant failure caused by consuming membranous nephropathy. CASE PRESENTATION: A 38-year-old female patient had been diagnosed with right kidney aplasia, left kidney dysplasia and significantly impaired renal function during infancy. Aged 16 she had to start hemodialysis. Three years later she received her first kidney transplant. Only then she was diagnosed with MRKH. The kidney transplant was lost due to consuming nephrotic syndrome caused by de novo membranous nephropathy, as was a second kidney transplant years later. In addition, a hyperelasticity syndrome affects the patient with congenital joint laxity, kyphoscoliosis, bilateral hip dysplasia, persistent hypermobility of both elbows, knees and hips. Her clinical picture resembles a combination of traits of a hypermobile and a vascular form of Ehlers-Danlos-Syndrome, but no mutations in the COL3A1 gene was underlying. Instead, array-based comparative genomic hybridisation (CGH) detected a heterozygous 1.43 Mb deletion on chromosome 17q12 encompassing the two renal developmental genes HNF1ß and LHX1. CONCLUSIONS: Deletions of HNF1ß have recently drawn significant attention in pediatric nephrology as an important cause of prenatally hyperechogenic kidneys, renal aplasia and renal hypodysplasia. In contrast, membranous nephropathy represents an often-unaccounted cause of nephrotic syndrome in the adult population. A causative connection between theses two conditions has never been postulated, but is suggestive enough in this case to hypothesize it.

FoxO proteins mediate hypoxic induction of connective tissue growth factor in endothelial cells.

Hypoxia, a driving force in neovascularization, promotes alterations in gene expression mediated by hypoxia-inducible factor (HIF)-1alpha. Connective tissue growth factor (CTGF, CCN2) is a modulator of endothelial cell growth and migration, but its regulation by hypoxia is poorly understood. Therefore, we analyzed signaling pathways involved in the regulation of CTGF by hypoxia in endothelial cells. Exposure to low oxygen tension or treatment with the hypoxia-mimetic dimethyloxalyl glycine (DMOG) stabilized HIF-1alpha and up-regulated CTGF in human umbilical vein endothelial cells and in a murine microvascular endothelial cell line. Induction of CTGF correlated with a HIF-dependent increase in protein and mRNA levels, and nuclear accumulation of the transcription factor FoxO3a. By contrast, gene expression and cellular localization of FoxO1 were not significantly altered by hypoxia. Expression of CTGF was strongly reduced by siRNA silencing of FoxO1 or FoxO3a. Furthermore, nuclear exclusion of FoxO1/3a transcription factors by inhibition of serine/threonine protein phosphatases by okadaic acid inhibited CTGF expression, providing evidence for both FoxO proteins as regulators of CTGF expression. The DMOG-stimulated induction of CTGF was further increased when endothelial cells were co-incubated with transforming growth factor-beta, an activator of Smad signaling. Activation of RhoA-Rho kinase signaling by the microtubule-disrupting drug combretastatin A4 also enhanced the DMOG-induced CTGF expression, thus placing CTGF induction by hypoxia in a network of interacting signaling pathways. Our findings provide evidence that FoxO1, hypoxia-stimulated expression of FoxO3a and its nuclear accumulation are required for the induction of CTGF by hypoxia in endothelial cells.

The lysyl oxidases LOX and LOXL2 are necessary and sufficient to repress E-cadherin in hypoxia: insights into cellular transformation processes mediated by HIF-1.

Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.

Carotid plaque vulnerability: a positive feedback between hemodynamic and biochemical mechanisms.

BACKGROUND AND PURPOSE: Rupture of atherosclerotic plaques is one of the main causes of ischemic strokes. The aim of this study was to investigate carotid plaque vulnerability markers in relation to blood flow direction and the mechanisms leading to plaque rupture at the upstream side of carotid stenoses. METHODS: Frequency and location of rupture, endothelial erosion, neovascularization, and hemorrhage were determined in longitudinal sections of 80 human carotid specimens. Plaques were immunohistochemically analyzed for markers of vulnerability. Plaque geometry was measured to reconstruct shape profiles of ruptured versus stable plaques and to perform computational fluid dynamics analyses. RESULTS: In 86% of ruptured plaques, rupture was observed upstream. In this region, neovascularization and hemorrhage were increased, along with increased immunoreactivity of vascular endothelial and connective tissue growth factor, whereas endothelial erosion was more frequent downstream. Proteolytic enzymes, mast cell chymase and cathepsin L, and the proapoptotic protein Bax showed significantly higher expression upstream as compared with the downstream shoulder of atherosclerotic lesions. Comparison of geometric profiles for ruptured and stable plaques showed increased longitudinal asymmetry of fibrous cap and lipid core thickness in ruptured plaques. The specific geometry of plaques ruptured upstream induced increased levels of shear stress and increased pressure drop between the upstream and the downstream plaque shoulders. CONCLUSIONS: Vulnerability of the upstream plaque region is associated with enhanced neovascularization, hemorrhage, and cap thinning induced by proteolytic and proapoptotic mechanisms. These processes are reflected in structural plaque characteristics, analyses of which could improve the efficacy of vascular diagnostics and prevention.

Matrix-independent stimulation of human tubular epithelial cell migration by Rho kinase inhibitors.

Proximal tubular epithelial cells differ from other epithelial cells in the expression of N-cadherin as major adherens junction protein instead of E-cadherin. Migration of proximal epithelial cells (HKC-8) was analyzed by scratch wounding and by a barrier assay, which allowed determination of migration velocity on different extracellular matrices. Migration velocity was about threefold higher on fibronectin compared to collagen IV. The differential migration velocity was reflected by the orientation of F-actin stress fibers. TGF-beta activated secretion of fibronectin and thus increased migration on collagen IV, but did not further promote migration on fibronectin. Pharmacological inhibition of Rho kinases (ROCKs) by Y-27632, hydroxyfasudil and H-1152, or siRNA against ROCKs significantly increased migration velocity independently of the extracellular matrix. Cells at the migration front showed long filopodia, which could not be mimicked by overexpression of consitutively active Cdc42, indicative of a more complex regulation of F-actin structures. N-cadherin was reorganized from tight zipper-like structures into loosened cell-cell contacts upon incubation with Y-27632, but HKC-8 cells still migrated as cohort. Migration through single cell pores in a modified Boyden chamber assay was also stimulated by ROCK inhibitors. ROCK inhibitors enhanced migration of primary cultures of renal tubular cells which consisted of proximal and distal tubular cells expressing N-cadherin and E-cadherin, respectively. There was no indication of a switch in cadherin expression in these cells or a preferential migration of N-cadherin expressing cells. Pharmacologic inhibition of ROCKs may thus favor repair processes in renal tubules by increasing the migratory capacity of tubular epithelial cells.

Lack of α8 integrin leads to morphological changes in renal mesangial cells, but not in vascular smooth muscle cells.

BACKGROUND: Extracellular matrix receptors of the integrin family are known to regulate cell adhesion, shape and functions. The α8 integrin chain is expressed in glomerular mesangial cells and in vascular smooth muscle cells. Mice deficient for α8 integrin have structural alterations in glomeruli but not in renal arteries. For this reason we hypothesized that mesangial cells and vascular smooth muscle cells differ in their respective capacity to compensate for the lack of α8 integrin. RESULTS: Wild type and α8 integrin-deficient mesangial cells varied markedly in cell morphology and expression or localization of cytoskeletal molecules. In α8 integrin-deficient mesangial cells α-smooth muscle actin and CTGF were downregulated. In contrast, there were no comparable differences between α8 integrin-deficient and wild type vascular smooth muscle cells. Expression patterns of integrins were altered in α8 integrin-deficient mesangial cells compared to wild type mesangial cells, displaying a prominent overexpression of α2 and α6 integrins, while expression patterns of the these integrins were not different between wild type and α8 integrin-deficient vascular smooth muscle cells, respectively. Cell proliferation was augmented in α8 integrin-deficient mesangial cells, but not in vascular smooth muscle cells, compared to wild type cells. CONCLUSIONS: Our findings suggest that α8 integrin deficiency has differential effects in mesangial cells and vascular smooth muscle cells. While the phenotype of vascular smooth muscle cells lacking α8 integrin is not altered, mesangial cells lacking α8 integrin differ considerably from wild type mesangial cells which might be a consequence of compensatory changes in the expression patterns of other integrins. This could result in glomerular changes in α8 integrin-deficient mice, while the vasculature is not affected in these mice.

Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia.

Tubular epithelial cells secrete connective tissue growth factor (CTGF, CCN2), which contributes to tubulointerstitial fibrosis. However, the molecular regulation of CTGF in human primary tubular epithelial cells (hPTECs) is not well defined. Therefore, CTGF expression was characterized in hPTECs isolated from healthy parts of tumor nephrectomies, with special emphasis on the regulation by transforming growth factor-beta (TGF-beta) and hypoxia, essential factors in the development of fibrosis. CTGF synthesis was strongly dependent on cell density. High CTGF levels were detected in sparse cells, whereas CTGF expression was reduced in confluent cells. Concomitantly, stimulation of CTGF by TGF-beta or the histone deacetylase inhibitor trichostatin was prevented in dense cells. Exposure of hPTECs to low oxygen tension (1% O2) or the hypoxia mimetic dimethyl-oxalylglycine for 24 h reduced CTGF gene expression in most of the 17 preparations analyzed. Preincubation of the cells under hypoxic conditions significantly reduced TGF-beta-mediated upregulation of CTGF. In line with these data, CTGF mRNA was only induced in interstitial cells, but not in tubular cells in kidneys of mice exposed to hypoxia. Longer exposure to hypoxia or TGF-beta (up to 72 h) did not induce hPTECs to adopt a mesenchymal phenotype characterized by upregulation of alpha-smooth muscle actin, downregulation of E-cadherin, or increased sensitivity of the cells in terms of CTGF expression. Sensitivity was restored by inhibition of DNA methylation. Taken together, our data provide evidence that exposure to hypoxia decreased CTGF gene expression. Furthermore, hypoxia per se was not sufficient to induce a mesenchymal phenotype in primary tubular epithelial cells.

Up-regulation of connective tissue growth factor in endothelial cells by the microtubule-destabilizing agent combretastatin A-4.

Incubation of microvascular endothelial cells with combretastatin A-4 phosphate (CA-4P), a microtubule-destabilizing compound that preferentially targets tumor vessels, altered cell morphology and induced scattering of Golgi stacks. Concomitantly, CA-4P up-regulated connective tissue growth factor (CTGF/CCN2), a pleiotropic factor with antiangiogenic properties. In contrast to the effects of other microtubule-targeting agents such as colchicine or nocodazole, up-regulation of CTGF was only detectable in sparse cells, which were not embedded in a cell monolayer. Furthermore, CA-4P induced CTGF expression in endothelial cells, forming tube-like structures on basement membrane gels. Up-regulation of CTGF by CA-4P was dependent on Rho kinase signaling and was increased when p42/44 mitogen-activated protein kinase was inhibited. Additionally, FoxO transcription factors were identified as potent regulators of CTGF expression in endothelial cells. Activation of FoxO transcription factors by inhibition of phosphatidylinositol 3-kinase/AKT signaling resulted in a synergistic increase in CA-4P-mediated CTGF induction. CA-4P-mediated expression of CTGF was thus potentiated by the inhibition of kinase pathways, which are targets of novel antineoplastic drugs. Up-regulation of CTGF by low concentrations of CA-4P may thus occur in newly formed tumor vessels and contribute to the microvessel destabilization and antiangiogenic effects of CA-4P observed in vivo.

Author Correction: Non-professional phagocytosis: a general feature of normal tissue cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Differential regulation of connective tissue growth factor in renal cells by histone deacetylase inhibitors.

Regulation of the profibrotic and angiogenesis modulating cytokine connective tissue growth factor (CTGF) occurs primarily at the transcriptional level. Therefore, we hypothesized that histone deacetylating enzymes (HDAC), which modulate the accessibility of transcriptionally active promoter regions, might play a role in the regulation of CTGF gene expression. We analyzed microvascular endothelial cells, which showed immunoreactivity for acetylated histone in kidney sections, and compared them with renal tubular epithelial cells. Treatment of cultured endothelial cells with different HDAC inhibitors up-regulated CTGF mRNA and protein. Pre-treatment with HDAC inhibitors facilitated induction of CTGF by transforming growth factor-beta (TGF-beta) or lysophosphatidic acid. Transcription factors of the FoxO family were involved in the up-regulation of CTGF as shown at protein level and by reporter gene analyses. In tubular epithelial cells, up-regulation of CTGF was only observed when these cells were cultured as subconfluent cells. Dense cells, which are more likely to resemble tubular cells in vivo, showed no up-regulation upon treatment with HDAC inhibitors and were protected against CTGF induction by TGF-beta. Taken together, our data indicate that the effect of HDAC inhibitors on CTGF expression is largely cell dependent in non-tumour cells. Different cell type-specific transcription factors seem to determine whether CTGF expression is reduced or increased in cells exposed to HDAC inhibitors.