Supplementary Figure S1 from Up-Regulation of Connective Tissue Growth Factor in Endothelial Cells by the Microtubule-Destabilizing Agent Combretastatin A-4

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.

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.

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.

Non-professional phagocytosis by cancer cells has been described for decades. Recently, non-professional phagocytosis by normal tissue cells has been reported, which prompted us to take a closer look at this phenomenon. Non-professional phagocytosis was studied by staining cultured cells with live-cell staining dyes or by staining paraffin-embedded tissues by immunohistochemistry. Here, we report that each of 21 normal tissue cell lines from seven different organs was capable of phagocytosis, including ex vivo cell cultures examined before the 3rd passage as well as the primary and virus-transformed cell lines. We extended our analysis to an in vivo setting, and we found the occurrence of non-professional phagocytosis in healthy skin biopsies immediately after resection. Using dystrophin immunohistochemistry for membrane staining, human post-infarction myocardial tissue was assessed. We found prominent signs of non-professional phagocytosis at the transition zone of healthy and infarcted myocardia. Taken together, our findings suggest that non-professional phagocytosis is a general feature of normal tissue cells.

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.

Acute kidney injury (AKI) is a common and potentially lethal complication in the hospitalized patients, with hypoxic injury being as a major cause. The loss of renal tubular epithelial cells (TEC), one of the AKI hallmarks, is potentially followed by tubular regeneration process orchestrated by the remaining uninjured TECs that undergo proliferation and migration. In this study, we used human primary TEC to investigate the initiation of tubular cell migration and associated cytoskeletal alterations in response to pharmacological HIF stabilization which resembles the pathophysiology of hypoxia. Tubular cells have been shown to migrate as cohorts in a wound healing assay. Importantly, cells of distal tubular origin moved faster than those of proximal origin. HIF stabilization impaired TEC migration, which was confirmed by live single cell tracking. HIF stabilization significantly reduced tubular cell migration velocity and promoted cell spreading. In contrast to the control conditions, HIF stabilization induced actin filaments rearrangement and cell adhesion molecules including paxillin and focal adhesion kinase. Condensed bundling of keratin fibers was also observed, while the expression of different types of keratins, phosphorylation of keratin 18, and the microtubule structure were not altered. In summary, HIF stabilization reduced the ability of renal tubular cells to migrate and led to cytoskeleton reorganization. Our data suggested an important involvement of HIF stabilization during the epithelial migration underlying the mechanism of renal regeneration in response to AKI.

Changes in cell morphology that involve alterations of the actin cytoskeleton are a hallmark of diseased renal tubular epithelial cells. While the impact of actin remodeling on gene expression has been analyzed in many model systems based on cell lines, this study investigated human primary tubular epithelial cells isolated from healthy parts of tumor nephrectomies.Latrunculin B (LatB) and cytochalasin D (CytoD) were used to modulate G-actin levels in a receptor-independent manner. Both compounds (at 0.5μM) profoundly altered F-actin structures in a Rho kinase-dependent manner, but only CytoD strongly induced the pro-fibrotic factor CTGF (connective tissue growth factor). CTGF induction was dependent on YAP as shown by transient downregulation experiments. However, CytoD did not alter the nuclear localization of either YAP or TAZ, whereas LatB reduced nuclear levels particularly of TAZ. CytoD modified MKL1, a coactivator of serum response factor (SRF) regulating CTGF induction, and promoted its nuclear localization.TGFβ-1 is one of the major factors involved in tubulointerstitial disease and an inducer of CTGF. Preincubation with CytoD but not LatB synergistically enhanced the TGFβ-1-stimulated synthesis of CTGF, both in cells cultured on plastic dishes as well as in polarized epithelial cells. CytoD had no direct effect on the phosphorylation of Smad2/3, but facilitated their phosphorylation and thus activation by TGFβ-1.Our present findings provide evidence that morphological alterations have a strong impact on cellular signaling of one of the major pro-fibrotic factors, TGFβ-1. However, our data also indicate that changes in cell morphology per se cannot predict those interactions which are critically dependent on molecular fine tuning of actin reorganization.

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.

Connective tissue growth factor (CTGF, also named CCN2) plays an important role in the development of tubulointerstitial fibrosis, which most critically determines the progression to end-stage renal failure in autosomal-dominant polycystic kidney disease (ADPKD), the most common genetically caused renal disease. We determined CTGF expression in a well-characterized animal model of human ADPKD, the PKD/Mhm (cy/+) rat. Kidneys of 12 weeks old (cy/+) as well as (+/+) non-affected rats were analyzed for CTGF RNA and protein expression by RT-PCR, Northern and Western blot analyses, in situ hybridization, and IHC. Besides the established expression of CTGF in glomerular cells in kidneys of wild-type (+/+) animals, in (cy/+) rats, CTGF mRNA and protein were robustly expressed in interstitial, stellate-shaped cells, located in a scattered pattern underlying the cystic epithelium and in focal areas of advanced tubulointerstitial remodeling. Renal CTGF mRNA and protein expression levels were significantly higher in (cy/+) rats compared with their (+/+) littermates. Detection of CTGF expression in cells adjacent to cystic epithelium and in areas of marked fibrosis suggests a role in the local response to cyst development and indicates that CTGF may be a relevant factor contributing to tubulointerstitial fibrosis in polycystic kidney disease.

Megakaryoblastic Leukemia 1 and 2 (MKL1/2) are transcriptional coactivators of Serum Response Factor (SRF) with an essential role for hepatocellular carcinoma (HCC) growth and oncogene-induced senescence. In this report, we identified myoferlin as a novel MKL/SRF target gene by gene expression profiling and verification in vivo in HCC xenografts. Myoferlin was overexpressed in human and murine HCCs triggered by conditional expression of constitutively active SRF-VP16 protein in hepatocytes. Furthermore, myoferlin was required for HCC cell invasion, proliferation and anchorage-independent cell growth. We provide evidence that myoferlin is a crucial gene target of MKL1/2 mediating its effect on oncogene-induced senescence by modulating the activation state of the EGFR and downstream MAPK and p16-/Rb pathways. Depletion of myoferlin in tumour cells from SRF-VP16-derived murine HCCs induced a senescence phenotype. These findings identify MKL1/2 and myoferlin as novel therapeutic targets to treat human HCC by a senescence-inducing strategy.

Abstract Superparamagnetic iron oxide nanoparticles (SPIONs) are in use for many clinical diagnostic and experimental therapeutic applications, for example, for targeted drug delivery. To analyze the cellular responses to mitoxantrone-carrying SPIONs (SPION-MTO), and to the drug released from SPIONs, we used an in vitro system that allows comparison of primary human cells with different endocytotic capacities, namely, epithelial cells from proximal and distal parts of the nephron. SPIONs were selectively and rapidly internalized by proximal tubular cells with high endocytotic potential, but not by distal tubular cells. Uptake did not affect cell viability or morphology. In both cell types, free MTO (10–100 nM) induced double-strand DNA breaks and senescence, cell hypertrophy and reduced cell proliferation. However, cadherin-mediated cell–cell adhesion, cytoskeletal structures or polarity of the cells were not affected. Interestingly, a comparable response was also observed upon treatment with SPION-MTO and was independent of uptake of the particles. The effect of SPION-MTO on cells which did not internalize particles was primarily related to the release of MTO from drug-coated particles upon incubation in serum-containing cell growth medium. In conclusion, we show that whereas the uptake of SPIONs does not affect cellular functions or viability, the toxicity of drug-loaded SPIONs depends essentially on the type of drug bound to nanoparticles. Due to the relatively low systemic toxicity of MTO, the effects of MTO-SPIONs on human tubular cells were moderate, but they may become clinically relevant when more nephrotoxic drugs are bound to SPIONs.

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.

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 β-1 (TGFβ-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 TGFβ-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 TGFβ-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 TGFβ-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.

BACKGROUND: Tissue injuries and pathological changes are often associated with changes in cell adhesion and reorganization of cytoskeletal structures. This is of particular relevance for renal epithelial cells, which form a highly regulated barrier along the nephron. Here, we investigated the effect of the calcium chelator EGTA on morphological alterations and changes in pro-fibrotic gene expression in proximal renal tubular epithelial cells. METHODS: Epithelial cells were exposed to EGTA alone or in combination with TGF -1, followed by the analysis of N-cadherin and paxillin localization and cytoskeletal rearrangement. The expression of connective tissue growth factor (CTGF), a pro-fibrotic protein regulated by alterations of cell morphology, was investigated. RESULTS: Even low concentrations of EGTA, which did not lead to the loss of cell-cell interactions or cell detachment, were sufficient to reduce N-cadherin binding, to rearrange focal adhesion, to induce CTGF expression, and to strongly enhance TGF -1-mediated signaling. These data indicate a substantial crosstalk between the changes in cell cytoskeletal structures at the cell-cell adhesions and the signaling of soluble mediators. CONCLUSIONS: By linking cell-cell and cell-matrix interactions with the regulation of pro-fibrotic signaling, our studies provide deeper insights into the physiological mechanisms underlying the natural regeneration and repair processes in the kidney.

Primary cilia are antenna-like structures projected from the apical surface of various mammalian cells including renal tubular cells. Functional or structural defects of the cilium lead to systemic disorders comprising polycystic kidneys as a key feature. Here we show that anoctamin 6 (ANO6), a member of the anoctamin chloride channel family, is localized in the primary cilium of renal epithelial cells in vitro and in vivo . ANO6 was not essential for cilia formation and had no effect on in vitro cyst expansion. However, knockdown of ANO6 impaired cyst lumen formation of MDCK cells in three-dimensional culture. In the absence of ANO6, apoptosis was reduced and epithelial cells were incompletely removed from the center of cell aggregates, which form in the early phase of cystogenesis. In line with these data, we show that ANO6 is highly expressed in apoptotic cyst epithelial cells of human polycystic kidneys. These data identify ANO6 as a cilium-associated protein and suggest its functional relevance in cyst formation.

Morphological alterations of cells can lead to modulation of gene expression. An essential link is the MKL1-dependent activation of serum response factor (SRF), which translates changes in the ratio of G- and F-actin into mRNA transcription. SRF activation is only partially characterized in non-transformed epithelial cells. Therefore, the impact of GTPases of the Rho family and changes in F-actin structures were analyzed in renal proximal tubular epithelial cells. Activation of SRF signaling was compared to the regulation of a known MKL1/SRF target gene, connective tissue growth factor (CTGF). In the human proximal tubular cell line HKC-8 overexpression of two actin mutants either favoring or preventing the formation of F-actin fibers regulated SRF-mediated transcription as well as CTGF expression. Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants. To elucidate the functional role of Rho kinases as downstream mediators of RhoA, pharmacological inhibition and genetic inhibition by transient siRNA knock down were compared. Upon stimulation with lysophosphatidic acid (LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression. Together with the partial inhibition of CTGF expression by the pharmacological inhibitors Y27432 and H1154, Rho kinases seem to be less important in mediating RhoA signaling related to CTGF expression in HKC-8 epithelial cells. Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h. Similarly, human primary cells of proximal but not of distal tubular origin showed inhibitory as well as stimulatory effects of Rac1 inhibition. Thus, RhoA signaling activates MKL1-SRF-mediated CTGF expression in proximal tubular cells, whereas Rac1 signaling is more complex with adaptive cellular responses.

Src kinases are regulators of the expression of connective tissue growth factor (CTGF/CCN2), which plays a role in fibrotic injuries. The aim of the present study was to evaluate the potential of SU6656, a dual inhibitor of Src family and Aurora kinases, to interfere with the synthesis of this pro-fibrotic factor. SU6656 impaired TGF-β-mediated upregulation of CTGF mRNA and protein in proximal epithelial HKC-8 cells, and also reduced CTGF expression in cells exposed to autocrine growth factors. In association with the inhibition of Src family kinases and diminished focal adhesion kinase activity, adherence of the cells was reduced. Furthermore, SU6656 interfered with Aurora kinase activity resulting in inhibition of cell division and formation multilobular nuclei after 24h. Comparable alterations were observed in primary tubular cells. When cell division was inhibited by SU6656 or ZM447439, a specific inhibitor of Aurora kinases, CTGF levels were back to control or even increased after 48h. The activity of RhoA-Rho kinase and ERK signaling was analyzed to delineate the signaling pathways responsible for the biphasic regulation of CTGF. While Rho kinase was not significantly altered by SU6656, ERK activity was inhibited in the early phase and increased after 24-48h. ERK activity correlated with secreted CTGF. As ZM447439 increased ERK activity only after 48h, cellular reorganization is likely responsible for triggering the ERK-dependent upregulation of CTGF. Taken together, in non-transformed epithelial cells, SU6656 modulates the expression of the pro-fibrotic factor CTGF in a time-dependent manner by inhibition of Src kinases and Aurora kinases.

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.