Tubular Cell Phenotype Research Articles

GPR97 deficiency suppresses Wnt/β-catenin signaling in hypertensive nephropathy.

Accumulating evidence shows that renal fibrosis plays a key role in the development of hypertensive nephropathy (HTN). Therefore, a better understanding of the underlying mechanism of renal fibrosis regulation in HTN would be critical for designing rational strategies for therapeutic interventions. In this study, we revealed that GPR97, a novel identified adhesion G coupled receptor, plays an important role in the regulation of Wnt/β-catenin signaling, which is the crucial driver of renal fibrosis in HTN. First, we identified that the expression of GPR97 correlated with the β-catenin expression in renal biopsy from patients with HTN. Moreover, we found that GPR97 deficiency inhibited Wnt/β-catenin signaling in mice with HTN, as evidenced by the reduction of β-catenin expression and downstream target proteins, including MMP7 and Fibronectin. Mechanistically, we found that GPR97 could directly bind with Wnt1 in cultured tubular cells and TGF-β1 treatment enhanced the binding ability of GPR97 and Wnt1. In addition, the gene silencing of GPR97 could decrease the Wnt1-induced fibrotic phenotype of tubular cells and inflammatory responses, suggesting that the binding of GPR97 and Wnt1 promoted Wnt/β-catenin signaling. Collectively, our studies reveal that GPR97 is a regulator of Wnt/β-catenin signaling in HTN, and targeting GPR97 may be a novel therapeutic strategy for HTN treatment.

The Scattered Tubular Cell Phenotype Is Induced in Cultured Primary Human Tubular Cells and Correlates With Loss of HNF4A Expression

The Scattered Tubular Cell Phenotype Is Induced in Cultured Primary Human Tubular Cells and Correlates With Loss of HNF4A Expression

Kidney microRNA Expression Pattern in Type 2 Diabetic Nephropathy in BTBR Ob/Ob Mice.

Diabetic nephropathy (DN) is the main leading cause of chronic kidney disease worldwide. Although remarkable therapeutic advances have been made during the last few years, there still exists a high residual risk of disease progression to end-stage renal failure. To further understand the pathogenesis of tissue injury in this disease, by means of the Next-Generation Sequencing, we have studied the microRNA (miRNA) differential expression pattern in kidneys of Black and Tan Brachyury (BTBR) ob/ob (leptin deficiency mutation) mouse. This experimental model of type 2 diabetes and obesity recapitulates the key histopathological features described in advanced human DN and therefore can provide potential useful translational information. The miRNA-seq analysis, performed in the renal cortex of 22-week-old BTBR ob/ob mice, pointed out a set of 99 miRNAs significantly increased compared to non-diabetic, non-obese control mice of the same age, whereas no miRNAs were significantly decreased. Among them, miR-802, miR-34a, miR-132, miR-101a, and mir-379 were the most upregulated ones in diabetic kidneys. The in silico prediction of potential targets for the 99 miRNAs highlighted inflammatory and immune processes, as the most relevant pathways, emphasizing the importance of inflammation in the pathogenesis of kidney damage associated to diabetes. Other identified top canonical pathways were adipogenesis (related with ectopic fatty accumulation), necroptosis (an inflammatory and regulated form of cell death), and epithelial-to-mesenchymal transition, the latter supporting the importance of tubular cell phenotype changes in the pathogenesis of DN. These findings could facilitate a better understanding of this complex disease and potentially open new avenues for the design of novel therapeutic approaches to DN.

Decoy receptor 2 mediation of the senescent phenotype of tubular cells by interacting with peroxiredoxin 1 presents a novel mechanism of renal fibrosis in diabetic nephropathy

Premature senescence of renal tubular epithelial cell (RTEC), which is involved in kidney fibrosis, is a key event in the progression of diabetic nephropathy. However, the underlying mechanism remains unclear. Here we investigated the role and mechanism of decoy receptor 2 (DcR2) in kidney fibrosis and the senescent phenotype of RTEC. DcR2 was specifically expressed in senescent RTEC and associated with kidney fibrosis in patients with diabetic nephropathy and mice with streptozotocin-induced with diabetic nephropathy. Knockdown of DcR2 decreased the expression of α-smooth muscle actin, collagen I, fibronectin and serum creatinine levels in streptozotocin-induced mice. DcR2 knockdown also inhibited the expression of senescent markers p16, p21, senescence-associated beta-galactosidase and senescence-associated heterochromatic foci and promoted the secretion of a senescence-associated secretory phenotype including IL-6, TGF-β1, and matrix metalloproteinase 2 invitro and invivo. However, DcR2 overexpression showed the opposite effects. Quantitative proteomics and validation studies revealed that DcR2 interacted with peroxiredoxin 1 (PRDX1), which regulated the cell cycle and senescence. Knockdown of PRDX1 upregulated p16 and cyclin D1 while downregulating cyclin-dependent kinase 6 expression invitro, resulting in RTEC senescence. Furthermore, PRDX1 knockdown promoted DcR2-induced p16, cyclin D1, IL-6, and TGF-β1 expression, whereas PRDX1 overexpression led to the opposite results. Subsequently, DcR2 regulated PRDX1 phosphorylation, which could be inhibited by the specific tyrosine kinase inhibitor genistein. Thus, DcR2 mediated the senescent phenotype of RTEC and kidney fibrosis by interacting with PRDX1. Hence, DcR2 may act as a potential therapeutic target for the amelioration of diabetic nephropathy progression.

Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells

The tight junctional pore-forming protein claudin-2 (CLDN-2) mediates paracellular Na+ and water transport in leaky epithelia and alters cancer cell proliferation. Previously, we reported that tumor necrosis factor-α time-dependently alters CLDN-2 expression in tubular epithelial cells. Here, we found a similar expression pattern in a mouse kidney injury model (unilateral ureteral obstruction), consisting of an initial increase followed by a drop in CLDN-2 protein expression. CLDN-2 silencing in LLC-PK1 tubular cells induced activation and phosphorylation of guanine nucleotide exchange factor H1 (GEF-H1), leading to Ras homolog family member A (RHOA) activation. Silencing of other claudins had no such effects, and re-expression of an siRNA-resistant CLDN-2 prevented RHOA activation, indicating specific effects of CLDN-2 on RHOA. Moreover, kidneys from CLDN-2 knockout mice had elevated levels of active RHOA. Of note, CLDN-2 silencing reduced LLC-PK1 cell proliferation and elevated expression of cyclin-dependent kinase inhibitor P27 (P27KIP1) in a GEF-H1/RHOA-dependent manner. P27KIP1 silencing abrogated the effects of CLDN-2 depletion on proliferation. CLDN-2 loss also activated myocardin-related transcription factor (MRTF), a fibrogenic RHOA effector, and elevated expression of connective tissue growth factor and smooth muscle actin. Finally, CLDN-2 down-regulation contributed to RHOA activation and smooth muscle actin expression induced by prolonged tumor necrosis factor-α treatment, because they were mitigated by re-expression of CLDN-2. Our results indicate that CLDN-2 suppresses GEF-H1/RHOA. CLDN-2 down-regulation, for example, by inflammation, can reduce proliferation and promote MRTF activation through RHOA. These findings suggest that the initial CLDN-2 elevation might aid epithelial regeneration, and CLDN-2 loss could contribute to fibrotic reprogramming.

Mechanisms of Bone Morphogenetic Protein-7 Protective Effects Against Cold Ischemia-Induced Renal Injury in Rats

Deceased donor kidneys are exposed to cold ischemic insult which makes them particularly susceptible to the effects of cold ischemic injury during hypothermic preservation resulting in high rates of delayed graft function. Bone morphogenetic protein-7 (BMP-7) is a valuable reagent in the field of tissue regeneration and preservation under ischemic conditions. Following these insights, we investigated the effect of recombinant human BMP-7 (rhBMP-7) on graft preservation during cold ischemia.The study was conducted on an experimental model of kidney cold ischemia in rats. Kidneys were perfused with University of Wisconsin (UW) saline solution, rhBMP-7, or rhBMP-7 + UW, and exposed to cold ischemia for 6, 12, and 24 hours.In tubular epithelial cells of kidneys perfused with rhBMP-7 and rhBMP-7+UW solution, the expression of BMP-7 and E-cadherin was observed after 24 hours of cold ischemia. In kidneys not perfused with rhBMP-7, high expression of transforming growth factor-β and α-smooth muscle actin was found. Also, in kidneys perfused with rhBMP-7 solution, statistically higher levels of Smad1, Smad5, and Smad8 messenger RNA expressions were proven.BMP-7 maintains the morphology of kidney tissue better than UW solution during 24 hours of cold ischemia. BMP-7 prevents epithelial to mesenchymal transformation and consequently maintains epithelial phenotype of tubular cells.

Tubular cell phenotype in HIV-associated nephropathy: Role of phospholipid lysophosphatidic acid

Collapsing glomerulopathy and microcysts are characteristic histological features of HIV-associated nephropathy (HIVAN). We have previously reported the role of epithelial mesenchymal transition (EMT) in the development of glomerular and tubular cell phenotypes in HIVAN. Since persistent tubular cell activation of NFκB has been reported in HIVAN, we now hypothesize that HIV may be contributing to tubular cell phenotype via lysophosphatidic acid (LPA) mediated downstream signaling. Interestingly, LPA and its receptors have also been implicated in the tubular interstitial cell fibrosis (TIF) and cyst formation in autosomal dominant polycystic kidney disease (PKD). Primary human proximal tubular cells (HRPTCs) were transduced with either empty vector (EV/HRPTCs), HIV (HIV/HRPTCs) or treated with LPA (LPA/HRPTC). Immunoelectrophoresis of HIV/HRPTCs and LPA/HRPTCs displayed enhanced expression of pro-fibrotic markers: a) fibronectin (2.25 fold), b) connective tissue growth factor (CTGF; 4.8 fold), c) α-smooth muscle actin (α-SMA; 12 fold), and d) collagen I (5.7 fold). HIV enhanced tubular cell phosphorylation of ILK-1, FAK, PI3K, Akt, ERKs and P38 MAPK. HIV increased tubular cell transcriptional binding activity of NF-κB; whereas, a LPA biosynthesis inhibitor (AACOCF3), a DAG kinase inhibitor, a LPA receptor blocker (Ki16425), a NF-κB inhibitor (PDTC) and NFκB-siRNA not only displayed downregulation of a NFκB activity but also showed attenuated expression of profibrotic/EMT genes in HIV milieu. These findings suggest that LPA could be contributing to HIV-induced tubular cell phenotype via NFκB activation in HIVAN.

Differential impact of glucose levels and advanced glycation end-products on tubular cell viability and pro-inflammatory/profibrotic functions

High glucose (HG) or synthetic advanced glycation end-products (AGE) conditions are generally used to mimic diabetes in cellular models. Both models have shown an increase of apoptosis, oxidative stress and pro-inflammatory cytokine production in tubular cells. However, the impact of the two conditions combined has rarely been studied. In addition, the impact of glucose level variation due to cellular consumption is not clearly characterized in such experiments. Therefore, the aim of this study was to compare the effect of HG and AGE separately and of both on tubular cell phenotype changes in the HK2 cell line. Moreover, glucose consumption was monitored every hour to maintain the glucose level by supplementation throughout the experiments. We thus observed a significant decrease of apoptosis and H2O2 production in the HK2 cell. HG or AGE treatment induced an increase of total and mitochondrial apoptosis as well as TGF-β release compared to control conditions; however, AGE or HG led to apoptosis preferentially involving the mitochondria pathway. No cumulative effect of HG and AGE treatment was observed on apoptosis. However, a pretreatment with RAGE antibodies partially abolished the apoptotic effect of HG and completely abolished the apoptotic effect of AGE. In conclusion, tubular cells are sensitive to the lack of glucose as well as to the HG and AGE treatments, the AGE effect being more deleterious than the HG effect. Absence of a potential synergistic effect of HG and AGE could indicate that they act through a common pathway, possibly via the activation of the RAGE receptors.

A microRNA-30e/mitochondrial uncoupling protein 2 axis mediates TGF-β1-induced tubular epithelial cell extracellular matrix production and kidney fibrosis

Mitochondria dysfunction has been reported in various kidney diseases but how it leads to kidney fibrosis and how this is regulated is unknown. Here we found that mitochondrial uncoupling protein 2 (UCP2) was induced in kidney tubular epithelial cells after unilateral ureteral obstruction in mice and that mice with ablated UCP2 resisted obstruction-induced kidney fibrosis. We tested this association further in cultured NRK-52E cells and found that TGF-β1 remarkably induced UCP2 expression. Knockdown of UCP2 largely abolished the effect of TGF-β1, whereas overexpression of UCP2 promoted tubular cell phenotype changes. Analysis using a UCP2 mRNA-3′-untranslated region luciferase construct showed that UCP2 mRNA is a direct target of miR-30e. MiR-30e was downregulated in tubular cells from fibrotic kidneys and TGF-β1-treated NRK-52E cells. A miR-30e mimic significantly inhibited TGF-β1-induced tubular-cell epithelial–mesenchymal transition, whereas a miR-30e inhibitor imitated TGF-β1 effects. Finally, genipin, an aglycone UCP2 inhibitor, significantly ameliorated kidney fibrosis in mice. Thus, the miR-30e/UCP2 axis has an important role in mediating TGF-β1-induced epithelial–mesenchymal transition and kidney fibrosis. Targeting this pathway may shed new light for the future of fibrotic kidney disease therapy.

Bone morphogenetic protein-7 reduces kidney cold ischemic injury by maintaining epithelial phenotype of tubular cells

Deceased donor kidneys are exposed to cold ischemic insult, which makes them particularly susceptible to the effects of cold ischemic injury during hypothermic preservation resulting in high rates of delayed graft function. Although cold storage reduces cellular oxygen demand, ischemia causes the rapid depletion of adenosine triphosphate and accumulation of toxicsubstances leading to cell death. BMP-7 is a valuable reagent in a field of tissue regeneration and preservation under ischemic conditions. Following these insights, we investigated the effect of rhBMP-7 on graft preservation during cold ischemia. The study was conducted on experimental model of kidney cold ischemia in rats. Kidneys were perfused with: saline, University of Wisconsin (UW), rhBMP-7 or rhBMP-7 + UW and exposed to cold ischemia during 6, 12 and 24 hours. Using PCR method was analyzed the expression of mRNA BMP-7, TGF-β1, Smad1, Smad2, Smad3, Smad5 and Smad8. Immunohistochemical analysis was used to show expression and localization of BMP-7, TGF-β1, E-cadherin and α-SMA . In tubular epithelial cells of the kidneys perfused with rhBMP-7 and rhBMP-7+UW solution the expression of BMP-7 and E-cadherin was observed after 24 hours of cold ischemia. In the kidneys not perfused with rhBMP-7 high expression of TGF-β1 and α-SMA was found. Also, in the kidneys perfused with rhBMP-7 solution level of mRNA BMP-7 expression was increased. In the same tissue higher level of mRNA Smad1, Smad5 and Smad8 expression, molecules of intracellular BMP-7 signal pathway, was proved. The levels of mRNA BMP-7, Smad1, Smad5 and Smad8 expression were equally present during whole time of cold ischemia. BMP-7 maintains the morphology of the kidney tissue better than UW solution during 24 hours of cold ischemia. BMP-7 prevents epithelial to mesenchymal transformation and consequently maintains epithelial phenotype of tubular cells.

Tubular shear stress and phenotype of renal proximal tubular cells.

Phenotypic alterations resulting from flow-induced mechanical strains is a growing field of research in many cell types such as vascular endothelial and smooth muscle cells, chondrocytes, and osteocytes. Because renal mass reduction is followed by a dramatic increase in GFR in the remaining nephron, modulation of tubular cell phenotype by flow-induced mechanical strains could be one of the events initiating the deleterious pathways that lead to the destruction of renal parenchyma after renal mass reduction. This study demonstrates that increased flow induced, in vitro and in vivo, a reinforcement of the apical domain of actin cytoskeleton and an inhibition of plasminogen activator expression. These effects of flow on plasminogen activator expression were prevented by blocking the reorganization of actin cytoskeleton and were associated with an increase in a shear-stress responsive element binding activity. These results confirm that tubular flow affects the phenotype of renal epithelial cells and suggest that flow-induced mechanical strains could be one determinant of tubulointerstitial lesions during the progression of renal diseases.

TGF-beta1-mediated alterations of renal proximal tubular epithelial cell phenotype.

The aim of this study was to characterize the mechanism of transforming growth factor (TGF)-beta1-mediated alteration of renal proximal tubular cell phenotype. TGF-beta1 altered cell phenotype, with cells appearing elongated and spindle shaped. This was associated with loss of cell-cell contact and rearrangement of the actin cytoskeleton, increased formation of stress fibers, and focal adhesions. Addition of the tyrosine phosphatase inhibitor sodium orthovanadate also led to rapid but transient loss of cell-cell contact, but it did not lead to a change of phenotype comparable to that seen following addition of TGF-beta1. There was, however, no change in the formation of focal adhesions and no associated reorganization of the Factin cytoskeleton. Disruption of the actin cytoskeleton with cytochalasin D prevented phenotypic alterations following addition of TGF-beta1. Transient transfection with Smad2/4 or Smad3/4 expression vectors did not alter cell phenotype. Previously, we demonstrated beta-catenin translocation to proximal tubule cell nuclei and its association with Smad proteins following addition of TGF-beta1, suggesting the possibility that TGF-beta1 may modulate Wnt signaling. The Wnt-responsive Xtwn-reporter construct was, however, silent in response to TGF-beta1. Similarly, a second Wnt/LEF-1-regulated element, Toplflash, which does not contain Smad binding sites, was insensitive to TGF-beta1 signaling. In contrast, phenotypic changes in response to TGF-beta1 were abrogated by inhibitors of the RhoA downstream target ROCK, which also prevented loss of cell-cell contact and adherens junction disassembly.

Shear-stress-responsive signal transduction mechanisms in renal proximal tubule cells.

Phenotypic alterations resulting from flow-induced mechanical strains is a growing field of research in many cell types such as vascular endothelial and smooth muscle cells, chondrocytes or osteocytes. Although it has been acknowledged for several decades that tubular flow is a main determinant of tubular behavior in terms of vectorial transport of water and solutes, the effect of flow on other characteristics of proximal tubular cell phenotype was ignored until recently. The purpose of the review is to summarize the various effects of shear-stress, recently demonstrated in renal proximal cells. New results demonstrate that tubular flow has pleiotropic effects on proximal tubular cells, affecting, in vitro and in vivo, the organization of the cytoskeleton, the synthesis of extracellular matrix proteases and the activity of specific transcription factors. These results suggest that flow-induced mechanical strains could be one determinant of tubulointerstitial lesions during the progression of renal diseases.

Tubular‐interstitial interactions in proteinuric renal diseases

SUMMARY: Progressive chronic renal disease of all types is characterized by a relatively uniform set of tubular (atrophy, hypertrophy, hyperplasia) and interstitial (inflammatory cell infiltration, fibrosis) pathological changes, the severity of which correlates well with the degree of proteinuria and the decline in glomerular filtration. Complex and redundant pathophysiological events underlie the relationship between proteinuria, tubular injury and interstitial damage, forming potential targets for therapy. The outcome of these events is also determined by additional factors not limited to proteinuric diseases, including tissue hypoxia, complement activation and mediators produced in response to glomerular damage. In response to proteinuria and these additional factors, tubular cells are activated to produce a large number of chemoattractants, proinflammatory and profibrotic cytokines and matrix proteins, which cause, at least in part, interstitial inflammation and fibrosis. In turn, soluble products of interstitial inflammatory cells (predominantly T lymphocytes and macrophages), dendritic cells and fibroblasts are at least partially responsible for altering the phenotype of tubular cells. Abnormal tubular‐interstitial interactions may also depend on direct cell contact rather than soluble mediators, as evidenced by up‐regulation of integrins and cell adhesion molecules. Myofibroblasts, a key cell in the production of interstitial fibrosis, may arise in response to the above mediators by trans‐differentiation of fibroblasts, perivascular cells and tubular cells. In addition, tubular cells have been shown to be capable of the induced expression of the signalling molecules necessary for them to behave as antigen‐presenting cells and thereby potentially initiate interstitial inflammation through the classic interactions of cognate immunity. It is probable that several, but not all, of this bewildering number of pathophysiological events are of prime importance to the development of tubulointerstitial damage in proteinuria. The immediate challenge for investigators is to define the relative importance of these events and, thus, the most appropriate targets for new treatments.

Effects of angiotensin II on proximal tubular cells stably transfected with the c-mas oncogene.

Angiotensin II (ANG II) normally induces cellular hypertrophy in proximal tubular epithelium by engaging receptor systems that use a G-protein-signaling mechanism. The c-mas oncogene also encodes part of a superfamily of vasoactive peptide receptor-like moieties that couple to G proteins. To determine whether the stable expression of the c-mas gene might alter or modify the induction of cellular hypertrophy by ANG II in tubular epithelium, a rat c-mas cDNA was cloned into the pSV2 expression vector for use in cell transfection. Scatchard analysis of ANG II binding revealed no significant differences in ANG II receptor number or in the dissociation constant between pSV2mas-transfected or wild-type MCT cells, but rather an increase in the number of receptors not replaceable by known inhibitors. ANG II also induced proliferation in pSV2mas-transfected MCT cells that was not blocked by conventional inhibitors and increased intracellular levels of inositol trisphosphate. ANG II, furthermore, did not increase de novo protein synthesis in pSV2-transfected MCT cells and failed to lower their intracellular concentration of adenosine 3',5'-cyclic monophosphate, both expected parameters of cellular hypertrophy. Our findings demonstrate that expression of c-mas in tubular epithelium can modulate tubular cell phenotype toward proliferation rather than hypertrophy. This effect is likely mediated by a reshuffling of the heterogeneity of ANG II receptors on the cell surface, or perhaps by the emergence of a new ANG II receptor, followed by alterations in the process of signal transduction.