Primary Human Renal Epithelial Cells Research Articles

Farnesol Inhibits PI3 Kinase Signaling and Inflammatory Gene Expression in Primary Human Renal Epithelial Cells.

Chronic inflammation and elevated cytokine levels are closely associated with the progression of chronic kidney disease (CKD), which is responsible for the manifestation of numerous complications and mortality. In addition to conventional CKD therapies, the possibility of using natural compounds with anti-inflammatory potential has attracted widespread attention in scientific research. This study aimed to study the potential anti-inflammatory effects of a natural oil compound, farnesol, in primary human renal proximal tubule epithelial cell (RPTEC) culture. Farnesol was encapsulated in lipid-based small unilamellar vesicles (SUVs) to overcome its insolubility in cell culture medium. The cell attachment of empty vesicles (SUVs) and farnesol-loaded vesicles (farnesol-SUVs) was examined using BODIPY, a fluorescent dye with hydrophobic properties. Next, we used multiple protein, RNA, and protein phosphorylation arrays to investigate the impact of farnesol on inflammatory signaling in RPTECs. The results indicated that farnesol inhibits TNF-α/IL-1β-induced phosphorylation of the PI3 kinase p85 subunit and subsequent transcriptional activation of the inflammatory genes TNFRSF9, CD27, TNFRSF8, DR6, FAS, IL-7, and CCL2. Therefore, farnesol may be a promising natural compound for treating CKD.

Losartan alleviates renal fibrosis by inhibiting the biomechanical stress-induced epithelial-mesenchymal transition of renal epithelial cells

Angiotensin receptor blockers (ARBs) have been reported to be beneficial of renal fibrosis, but the molecular and cellular mechanisms are still unclear. In this study, we investigated the effectiveness and relevant mechanism of ARBs in alleviating renal fibrosis, especially by focusing on biomechanical stress-induced epithelial to mesenchymal transition (EMT) of renal epithelial cells. Unilateral ureteral obstruction (UUO) renal fibrosis model was established in mice by ligating the left ureter, and then randomly received losartan at a low dose (1 mg/kg) or a regular dose (3 mg/kg) for 2 weeks. Compared to the control, histological analysis showed that losartan treatment at either a low dose or a regular dose effectively attenuated renal fibrosis in the UUO model. To further understand the mechanism, we ex vivo loaded primary human renal epithelial cells to 50 mmHg hydrostatic pressure. Western blot and immunostaining analyses indicated that the loading to 50 mmHg hydrostatic pressure for 24 h significantly upregulated vimentin, β-catenin and α-SMA, but downregulated E-cadherin in renal epithelial cells, suggesting the EMT. The addition of 10 or 100 nM losartan in medium effectively attenuated the EMT of renal epithelial cells induced by 50 mmHg hydrostatic pressure loading. Our in vivo and ex vivo experimental data suggest that losartan treatment, even at a low dose can effectively alleviate renal fibrosis in mouse UUO model, at least partly by inhibiting the biomechanical stress-induced EMT of renal epithelial cells. A low dose of ARBs may repurpose for renal fibrosis treatment.

Pleiotropic Devitalization of Renal Cancer Cells by Non-Invasive Physical Plasma: Characterization of Molecular and Cellular Efficacy.

Renal cell carcinoma (RCC) is the third most common urological tumor and has an extremely poor prognosis after metastasis has occurred. Therapeutic options are highly restricted, primarily due to resistance to classical chemotherapeutics. The development of new, innovative therapeutic procedures is thus of great urgency. In the present study, the influence of non-invasive physical plasma (NIPP) on malignant and non-malignant renal cells is characterized. The biological efficacy of NIPP has been demonstrated in malignant renal cell lines (786-O, Caki-1) and non-malignant primary human renal epithelial cells (HREpC). The cell responses that were experimentally examined were cell growth (cell number determination, calculation of growth rate and doubling time), cell motility (scratch assay, invasiveness assay), membrane integrity (uptake of fluorescent dye, ATP release), and induction of apoptosis (TUNEL assay, caspase-3/7 assay, comet assay). A single NIPP treatment of the malignant cells significantly inhibited cell proliferation, invasiveness, and metastasis. This treatment has been attributed to the disruption of membrane functionality and the induction of apoptotic mechanisms. Comparison of NIPP sensitivity of malignant 786-O and Caki-1 cells with non-malignant HREpC cells showed significant differences. Our results suggest that renal cancer cells are significantly more sensitive to NIPP than non-malignant renal cells. Treatment with NIPP could represent a promising innovative option for the therapy of RCC and might supplement established treatment procedures. Of high clinical relevance would be the chemo-sensitizing properties of NIPP, which could potentially allow a combination of NIPP treatment with low-dose chemotherapy.

POS-476 OPTIMIZATION OF SDS-BASED KIDNEY DECELLULARIZATION RESULTS IN ECM SCAFFOLDS APPLICABLE FOR 3D RECELLULARIZATION

Kidney decellularization is used to obtain extracellular matrix (ECM) scaffolds as a platform for 3D culturing and kidney tissue regeneration. ECM contains biological molecules such as growth factors necessary for cell survival, proliferation, and maturation during organogenesis. While decellularization allows for the complete removal of cells, it also causes the removal of specific growth factors from the ECM, which could have deleterious effects on cell survival, proliferation, and differentiation during recellularization. We hypothesize that using a mild decellularization protocol which employs the use of sodium dodecyl sulphate (SDS) below its critical micelle concentration (CMC) of 0.2% in water at room temperature will allow for the greater retention of bioactive molecules which will improve recellularization efficiencies. Porcine kidney cortex was cut into 1mm x 6mm slices and decellularized using 0.05%, 0.075%, or 0.1% SDS in a soak and agitation protocol for 24 hours at room temperature. Decellularization was determined through histological and immunofluorescent staining, and DNA quantification. The retention of ECM proteins and biomolecules was determined by histological and immunofluorescent staining, mass spectrometry, and protein array analysis. Four different cell lines were used for recellularization; human-induced pluripotent stem cells (hiPSC), mesoderm derived from hiPSC, intermediate mesoderm derived from hiPSC, and primary human renal epithelial cells. ECM scaffolds were dried on Transwell inserts and cells were pipetted on the surface of the scaffold. ECM-cell constructs were grown for two weeks in culture at air-liquid interface. Recellularization efficiency, cell organization, and differentiation were determined using whole mount staining. Complete decellularization was achieved when SDS was used in concentrations below its CMC. Decellularizing with 0.05% SDS resulted in the greatest retention of glycosaminoglycan chains (GAGs), revealing that GAGs are still significantly removed from the ECM when using SDS below its CMC. Upon protein quantification, scaffolds decellularized with higher SDS concentrations were more enriched with matrisome proteins compared to scaffolds decellularized with lower SDS concentrations, which correlated to a decrease in growth factors and cytokines present on the scaffolds as determined by protein array. Following 14-day recellularization, hiPSCs and kidney progenitor cells proliferated on the ECM and expressed markers such as LIM1, WT1, PBX1 and CD31. Primary renal epithelial cells maintained a mature phenotype, expressing LTL, cytokeratin, and KSP. Herein, we describe a novel protocol for the decellularization of kidney cortex scaffolds that allows for greater retention of biomolecules. This retention of growth factors and cytokines aids in the proliferation and directed differentiation of hiPS and kidney progenitor cells toward mature renal lineages. Thus, we determined that our ECM scaffolds are able to support the growth of both hiPS and organ-matched cells. This can be applied to other organ decellularization/ recellularization protocols, as well as for the generation of kidney tissue for further in vitro studies.

PI3K-Akt-mTOR inhibition by GNE-477 inhibits renal cell carcinoma cell growth in vitro and in vivo.

Sustained activation of PI3K-Akt-mTOR cascade is important for renal cell carcinoma (RCC) cell progression. GNE-477 is a novel and efficacious PI3K-mTOR dual inhibitor. The current study tested its anti-RCC cell activity. In the primary cultured human RCC cells, GNE-477 potently inhibited cell growth, viability and proliferation, as well as cell cycle progression, migration and invasion. Furthermore, it induced robust apoptosis activation in primary RCC cells, but being non-cytotoxic to HK-2 epithelial cells and primary human renal epithelial cells. In the primary RCC cells GNE-477 inactivated PI3K-Akt-mTOR cascade by blocking phosphorylation of p85, Akt1, p70S6K1 and S6. Restoring Akt-mTOR activation by a constitutively-active Akt1 reversed GNE-477-induced anti-RCC cell activity. In nude mice intraperitoneal injection of GNE-477 potently suppressed RCC xenograft tumor growth. Collectively, targeting PI3K-Akt-mTOR cascade by GNE-477 inhibits RCC cell growth in vitro and in vivo.

Active mixture of serum-circulating small molecules selectively inhibits proliferation and triggers apoptosis in cancer cells via induction of ER stress

Genetic and epigenetic regulation as well as immune surveillance are known defense mechanisms to protect organisms from developing cancer. Based on experimental evidence, we proposed that small metabolically active molecules accumulating in cancer cells may play a role in an alternative antitumor surveillance system. Previously, we reported that treatment with a mixture of experimentally selected small molecules, usually found in the serum (defined ‘active mixture’, AM), selectively induces apoptosis in cancer cells and significantly inhibits tumor formation in vivo. In this study, we show that the AM elicits gene expression changes characteristic of endoplasmic reticulum (ER) stress in HeLa, MCF-7, PC-3 and Caco-2 cancer cells, but not in primary human renal epithelial cells. The activation of the ER stress pathway was confirmed by the upregulation of ATF3, ATF4, CHAC1, DDIT3 and GDF15 proteins. Mechanistically, our investigation revealed that eIF2α, PERK and IRE1α are phosphorylated upon treatment with the AM, linking the induction of ER stress to the antiproliferative and proapoptotic effects of the AM previously demonstrated. Inhibition of ER stress in combination with BBC3 and PMAIP1 knockdown completely abrogated the effect of the AM. Moreover, we also demonstrated that the AM induces mIR-3189-3p, which in turn enhances the expression of ATF3 and DDIT3, thus representing a possible new feedback mechanism in the regulation of ATF3 and DDIT3 during ER stress. Our results highlight small molecules as attractive anticancer agents and warrant further evaluation of the AM in cancer therapy, either alone or in combination with other ER stress inducing agents.

Direct reprogramming to human nephron progenitor-like cells using inducible piggyBac transposon expression of SNAI2-EYA1-SIX1

All nephrons in the mammalian kidney arise from a transient nephron progenitor population that is lost close to the time of birth. The generation of new nephron progenitors and their maintenance in culture are central to the success of kidney regenerative strategies. Using a lentiviral screening approach, we previously generated a human induced nephron progenitor-like state invitro using a pool of six transcription factors. Here, we sought to develop a more efficient approach for direct reprogramming of human cells that could be applied invivo. PiggyBac transposons are a non-viral integrating gene delivery system that is suitable for invivo use and allows for simultaneous delivery of multiple genes. Using an inducible piggyBac transposon system, we optimized a protocol for the direct reprogramming of HK2 cells to induced nephron progenitor-like cells with expression of only 3 transcription factors (SNAI2, EYA1, and SIX1). Culture in conditions supportive of the nephron progenitor state further increased the expression of nephron progenitor genes. The refined protocol was then applied to primary human renal epithelial cells, which integrated into developing nephron structures invitro and invivo. Such inducible reprogramming to nephron progenitor-like cells could facilitate direct cellular reprogramming for kidney regeneration.

LncRNA THOR promotes human renal cell carcinoma cell growth

BackgroundRecent studies have characterized a novel but extremely conserved long non-coding RNA (LncRNA) THOR. THOR directly associates with insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) to promote mRNA stabilization of key pro-cancerous genes. ResultsHere, we show that THOR is expressed in human renal cell carcinoma (RCC) tissues and established/primary human RCC cells. It was not detected in normal renal tissues nor in HK-2 and primary human renal epithelial cells. THOR silencing (by targeted siRNAs) or CRISPR/Cas9 knockout inhibited RCC cell growth, viability and proliferation in vitro. Reversely, forced over-expression of THOR promoted RCC cell survival and proliferation. IGF2BP1-regulated genes, including IGF2, GLI1 and Myc, were downregulated by THOR silencing or knockout, but they were upregulated after THOR over-expression. In vivo, THOR-knockout 786-O tumors grew significantly slower than the control tumors in nude mice. ConclusionTHOR expression promotes RCC cell growth in vitro and in vivo. THOR could be a novel and important therapeutic target for human RCC.

A modular microfluidic bioreactor with improved throughput for evaluation of polarized renal epithelial cells.

Most current microfluidic cell culture systems are integrated single use devices. This can limit throughput and experimental design options, particularly for epithelial cells, which require significant time in culture to obtain a fully differentiated phenotype. In addition, epithelial cells require a porous growth substrate in order to fully polarize their distinct apical and basolateral membranes. We have developed a modular microfluidic system using commercially available porous culture inserts to evaluate polarized epithelial cells under physiologically relevant fluid flow conditions. The cell-support for the bioreactor is a commercially available microporous membrane that is ready to use in a 6-well format, allowing for cells to be seeded in advance in replicates and evaluated for polarization and barrier function prior to experimentation. The reusable modular system can be easily assembled and disassembled using these mature cells, thus improving experimental throughput and minimizing fabrication requirements. The bioreactor consists of an apical microfluidic flow path and a static basolateral chamber that is easily accessible from the outside of the device. The basolateral chamber acts as a reservoir for transport across the cell layer. We evaluated the effect of initiation of apical shear flow on short-term intracellular signaling and mRNA expression using primary human renal epithelial cells (HRECs). Ten min and 5 h after initiation of apical fluid flow over a stable monolayer of HRECs, cells demonstrated increased phosphorylation of extracellular signal-related kinase and increased expression of interleukin 6 (IL-6) mRNA, respectively. This bioreactor design provides a modular platform with rapid experimental turn-around time to study various epithelial cell functions under physiologically meaningful flow conditions.

Synergistic induction of CTGF by cytochalasin D and TGFβ-1 in primary human renal epithelial cells: Role of transcriptional regulators MKL1, YAP/TAZ and Smad2/3

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.

Biocompatibility, uptake and endocytosis pathways of polystyrene nanoparticles in primary human renal epithelial cells

Recent years have witnessed an unprecedented growth in the number of applications – such as drug delivery, nutraceuticals and production of improved biocompatible materials – in the areas of nanoscience and nanotechnology. Engineered nanoparticles (NPs) are an important tool for the development of quite a few of these applications. Despite intense research activity, mechanisms regulating the uptake of NPs into cells are not completely defined, being the phenomenon dramatically influenced by physico-chemical properties of NPs and cell-specific differences. Since the cellular uptake of NPs is a prerequisite for their use in nanomedicine, the definition of their internalization pathway is crucial. For this reason, we used 44nm polystyrene NPs as a model to analyze the uptake and endocytosis pathways in primary human renal cortical epithelial (HRCE) cells, which play a key role in the clearance of drugs. NPs were found not to affect the viability and cell cycle progression of HRCE cells. Distinct internalization pathways were analyzed by the use of drugs known to inhibit specific endocytosis routes. Analyses, performed by confocal microscopy in combination with quantitative spectrofluorimetric assays, indicated that NPs enter HRCE cells through multiple mechanisms, either energy-dependent (endocytosis) or energy-independent.

Polar release of pathogenic Old World hantaviruses from renal tubular epithelial cells.

BackgroundEpithelio- and endotheliotropic viruses often exert polarized entry and release that may be responsible for viral spread and dissemination. Hantaviruses, mostly rodent-borne members of the Bunyaviridae family infect epithelial and endothelial cells of different organs leading to organ dysfunction or even failure. Endothelial and renal epithelial cells belong to the target cells of Old World hantavirus. Therefore, we examined the release of hantaviruses in several renal epithelial cell culture models. We used Vero cells that are commonly used in hantavirus studies and primary human renal epithelial cells (HREpC). In addition, we analyzed MDCKII cells, an epithelial cell line of a dog kidney, which represents a widely accepted in vitro model of polarized monolayers for their permissiveness for hantavirus infection.ResultsVero C1008 and primary HREpCs were grown on porous-support filter inserts for polarization. Monolayers were infected with hantavirus Hantaan (HTNV) and Puumala (PUUV) virus. Supernatants from the apical and basolateral chamber of infected cells were analyzed for the presence of infectious particles by re-infection of Vero cells. Viral antigen and infectious particles of HTNV and PUUV were exclusively detected in supernatants collected from the apical chamber of infected Vero C1008 cells and HREpCs. MDCKII cells were permissive for hantavirus infection and polarized MDCKII cells released infectious hantaviral particles from the apical surface corresponding to the results of Vero and primary human epithelial cells.ConclusionsPathogenic Old World hantaviruses are released from the apical surface of different polarized renal epithelial cells. We characterized MDCKII cells as a suitable polarized cell culture model for hantavirus infection studies.

A microfluidic bioreactor for epithelial cell studies under fluid shear stress

We have developed a multilayer microfluidic bioreactor to evaluate polarized epithelial cell behavior under controlled fluid shear stress. The system consists of two microfluidic chambers separated by a synthetic porous membrane which provides a substrate for cell grown and allows apical to basolateral solute transport. The system has been used to evaluate a number of shear stress‐dependent cellular behaviors in kidney epithelial cells. We found that primary human renal epithelial cells undergo cytoskeletal rearrangement when exposed to shear stress. Endocytosis, metabolism and secretion of albumin were also evaluated using the bioreactor. Fluid shear stress increased the cellular capacity for apical uptake of albumin in immortalized proximal tubule epithelial cells. Following internalization, albumin was metabolized into small molecular weight fragments and excreted into the apical and basolateral compartments. This microfluidic bioreactor is a useful tool for evaluating kidney epithelial cell behavior in response to fluid shear stress and may have broader applications for studying a variety of shear stress sensitive cell types. This work was supported by the Wildwood Foundation and a T32 training grant from the National Institutes of Health (T32‐DK‐007470–25).

Intercellular Communication by Exchange of Cytoplasmic Material via Tunneling Nano-Tube Like Structures in Primary Human Renal Epithelial Cells

Transfer of cellular material via tunneling nanotubes (TNT) was recently discovered as a novel mechanism for intercellular communication. The role of intercellular exchange in communication of renal epithelium is not known. Here we report extensive spontaneous intercellular exchange of cargo vesicles and organelles between primary human proximal tubular epithelial cells (RPTEC). Cells were labeled with two different quantum dot nanocrystals (Qtracker 605 or 525) and intercellular exchange was quantified by high-throughput fluorescence imaging and FACS analysis. In co-culture, a substantial fraction of cells (67.5%) contained both dyes indicating high levels of spontaneous intercellular exchange in RPTEC. The double positive cells could be divided into three categories based on the preponderance of 605 Qtracker (46.30%), 525 Qtracker (48.3%) and approximately equal content of both Qtrackers (4.57%). The transfer of mitochondria between RPTECs was also detected using an organelle specific dye. Inhibition of TNT genesis by actin polymerization inhibitor (Latrunculin B) markedly reduced intercellular exchange (>60%) suggesting that intercellular exchange in RPTEC was in part mediated via TNT-like structures. In contrast, induction of cellular stress by Zeocin treatment increased tube-genesis in RPTEC. Our data indicates an unexpected dynamic of intercellular communication between RPTEC by exchange of cytosolic material, which may play an important role in renal physiology.

Downregulation of α-galactosidase A upregulates CD77: functional impact for Fabry nephropathy

Anderson-Fabry disease, an inherited deficiency in the lysosomal enzyme alpha-galactosidase A, is characterized by the progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. We sought to clarify the pathogenesis of Fabry disease by establishing a cell model of this disorder. The expression of alpha-galactosidase A was transiently silenced by RNA interference in HK2 and primary human renal epithelial cells and stably silenced in HK2 cells by retroviral transfection with small hairpin RNA. All of the silenced cells had histological similarities to cells of patients with Fabry disease. The cells had reduced viability, significant accumulation of intracellular Gb3, and a modest but significant increase in membranous Gb3 expression compared to nonsilenced cells. When silenced HK2 cells were reconstituted with agalsidase-alpha, a protein used for enzyme replacement therapy, they decreased their membranous CD77 expression to levels indistinguishable from those of nonsilenced cells. Because plasma and urinary Gb3 levels are not reliable biomarkers for Fabry disease, our study suggests that membranous CD77 levels mirror Gb3 tissue load and that CD77 expression levels may be used to monitor the efficacy of enzyme replacement therapy.

Internalization of Escherichia coli by human renal epithelial cells is associated with tyrosine phosphorylation of specific host cell proteins

Human renal epithelial cells are capable of internalizing Escherichia coli regardless of whether the bacteria are isolated from individuals with pyelonephritis or from healthy volunteers. In this study, we investigated the role of host cell tyrosine kinase activity in internalization. We found that internalization of both fecal and pyelonephritis isolates is blocked by tyrosine kinase inhibitors. We found increased intensity of two tyrosine-phosphorylated proteins, with relative mobilities of approximately 123,000 and 110,000, in Western blots of extracts from human renal epithelial cells infected with E. coli. The increased intensity of these tyrosine-phosphorylated proteins was observed only in the Triton X-100-insoluble fraction, suggesting that these proteins could be associated with the cytoskeleton. Increased tyrosine phosphorylation of these proteins upon E. coli infection was observed in both transformed and primary human renal epithelial cells and in cells infected with several different strains of E. coli isolated from the feces of healthy individuals or from the blood or urine of patients with pyelonephritis. The increased tyrosine phosphorylation of these proteins required live bacteria and was blocked by tyrosine kinase inhibition but not by protein synthesis inhibitors or cytochalasin D. These experiments establish a strong link between E. coli internalization and host cell signaling through tyrosine kinases in human kidney cells and provide evidence that specific proteins are involved in these processes.

Internalization of Escherichia coli into human kidney epithelial cells: comparison of fecal and pyelonephritis-associated strains.

A gentamicin survival assay, using primary human renal epithelial cells and Escherichia coli strains isolated from the feces of asymptomatic individuals and from the urine or blood of patients with acute pyelonephritis, was used to investigate bacterial internalization as a model for renal parenchymal invasion in pyelonephritis. E. coli strains, regardless of their origin, efficiently entered into human renal epithelial cells, a process inhibited by cytochalasin D. While the percentage of survival of nonhemolytic pyelonephritis isolates did not differ from that of fecal isolates, survival of hemolytic pyelonephritis strains was lower than that of nonhemolytic strains, perhaps as a consequence of the greater cytotoxicity of hemolytic strains. There was no evidence of intracellular multiplication of E. coli. These results demonstrate that human renal epithelial cells are capable of efficient uptake of E. coli regardless of the source of the bacteria.