Primary Renal Tubular Epithelial Cells Research Articles

Spontaneous Extracellular Matrix Accumulation in a Human in vitro Model of Renal Fibrosis Is Mediated by αV Integrins

Background: Tubulointerstitial fibrosis is a key feature of chronic kidney diseases leading to renal failure. It is characterised by the infiltration of fibroblasts and aberrant accumulation of extracellular matrix (ECM) proteins, which are associated with progressive loss of renal function. Integrins play a major role in fibrosis, but the mechanisms through which they do this are not fully understood. Objective: Using a complex cell system, we test the hypothesis that integrins are pro-fibrotic via regulation of functional interactions between tubular epithelial cells and renal fibroblasts. Method: Contact co-culture of human primary renal proximal tubular epithelial cells and renal fibroblasts promoted the spontaneous accumulation of a mature ECM rich in interstitial collagens, which was considerably in excess of that seen in the individual mono-cultures. Both cell types persisted throughout the culture and were capable of expressing multiple ECM components. Results: While ECM accumulation was inhibited by the clinically proven anti-fibrotic, nintedanib, and was partially abrogated by transforming growth factor β neutralisation, its levels did not return to basal, indicating additional pathways were implicated in the pro-ECM response. Application of anti-integrin blocking antibodies and small molecules demonstrated a major role of the αV integrins in the ECM accumulation during fibroblast: epithelial cell interactions. Conclusion: Integrin-mediated pathways can facilitate the spontaneous accumulation of ECM during fibroblast: epithelial cell interactions, and this direct renal co-culture assay system could provide a translational in vitro assay for investigating novel pathways involved in the pro-ECM response and the screening of renal anti-fibrotic agents.

Interaction between bone marrow-derived dendritic cells and miR-21 of tubular renal epithelial cells under hypoxia.

To investigate the intracellular response and role of microRNA 21 in the regulation of dendritic cell maturation and function. Bone marrow-derived DCs (BMDCs) isolated from male C57BL/6J mice and primary renal tubular epithelial cells were used as primary cells to perform this study. Flow cytometry was used to determine BMDCs and analyze the apoptosis effect. Transmission electron microscopy was used for the identification of the diameter of exosomes. Reverse transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting were used to detect the effect after cells were transfected with oligo. ELISA was used to determine the tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), and IL-1beta in DC supernatants. We found that the upregulation of microRNA 21 in dendritic cells induced by physical hypoxia contributed to decreased expressions of CD80 (cluster of differentiation 80), CD86 (cluster of differentiation 86), and MHCII (major histocompatibility complex class II molecules) of dendritic cells and suppressed secretion of inflammatory cytokines and chemokine receptor type 7. Co-culture with tubular epithelial cells or hypoxia-pretreated tubular epithelial cell-derived conditional medium promoted bone marrow-derived dendritic cell maturation. Exosomes purified from the supernatant of cultured marrow-derived dendritic cells showed upregulated microRNA 21 under hypoxia, whereas anti-microRNA 21 treated tubular epithelial cells promoted co-cultured marrow-derived dendritic cell maturation. Both oxygen concentration and tubular epithelial cells participate in regulating dendritic cell maturation, directly or indirectly through the microRNA 21 signal pathway.

Orbital Shear Stress Regulates Differentiation and Barrier Function of Primary Renal Tubular Epithelial Cells.

Primary cells cultured in vitro gradually lose features characteristic of the in vivo phenotype. Culture techniques that help maintain cell-specific phenotype are advantageous for development of tissue engineered and bioartificial organs. Here we evaluated the phenotype of primary human renal tubular epithelial cells subjected to fluid shear stress by culturing the cells on an orbital shaker. Transepithelial electrical resistance (TEER), cell density, and gene and protein expression of proximal tubule-specific functional markers were measured in cells subjected to orbital shear stress. Cells cultured on an orbital shaker had increased TEER, higher cell density, and enhanced tubular epithelial specific gene and protein expression. This is likely due at least in part to the mechanical stress applied to the apical surface of the cells although other factors including increased nutrient and oxygen delivery and improved mixing could also play a role. These results suggest that orbital shaker culture may be a simple approach to augmenting the differentiated phenotype of cultured renal epithelial cells.

Receptor-independent modulation of TGF-β-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-β1-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 80-90kDa in cellular homogenates by Western blotting. Expression of the detected protein was not altered by incubation with TGF-β1 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.

HBx gene transfection affects the cycle of primary renal tubular epithelial cells through regulating cyclin expression

Hepatitis B virus X protein (HBx) has been previously demonstrated to be associated with the regulation of cell proliferation; however, the exact mechanisms underlying this effect remain unclear. The present study aimed to investigate the regulatory mechanism of HBx on the cycle progression of primary renal tubular epithelial cells. Primary renal tubular epithelial cells of Sprague Dawley (SD) rats were separated and cultured. The morphology of cultured cells was characterized by immunohistochemical analysis and the results demonstrated that primary renal tubular epithelial cells with the expected morphology and distribution were successfully separated and cultured from SD rats. HBx gene pcDNA3.1/myc vector and empty vector were constructed and transfected into cells as HBx and empty groups, respectively. Following transfection, the mRNA and protein levels of HBx, cyclin A, cyclin D1 and cyclin E in cells were determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The results demonstrated that following HBx gene transfection, the mRNA and protein levels of HBx, cyclin A, cyclin D1 and cyclin E in cells were significantly upregulated, compared with the empty control group (P<0.05). Furthermore, cell apoptosis and the cell cycle were evaluated by Annexin V-fluorescein isothiocyanate/propidium iodide staining and flow cytometry. HBx gene transfection significantly inhibited the cell apoptosis (P<0.05), promoted cell cycle progression from the G1 to S phase and arrested the cell cycle in the S phase. Therefore, the results of the present study indicated that HBx gene transfection may regulate the apoptosis and cell cycle of primary renal tubular epithelial cells by affecting the expression of cyclins. The results of the present study may improve the understanding of pathogenesis associated with HBV-associated glomerulonephritis, and may also provide insight and theoretical support for the future design and development of drugs for the treatment of hepatitis B virus.

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

Mitochondrial DNA is Released in Urine of SIRS Patients With Acute Kidney Injury and Correlates With Severity of Renal Dysfunction.

Systemic inflammatory response syndrome (SIRS) is characterized by the activation of the innate immune system resulting in stimulation of inflammatory responses, coagulation, and platelet activation that may contribute to complication such as the development of acute kidney injury (AKI). AKI importantly worsens the outcome of SIRS, implying the existence of a detrimental cross talk via systemic messages. Mitochondria are a source of damage-associated molecular patterns (DAMPs) and are thought to form a molecular link between tissue injury and stimulation of innate immunity. The role of mitochondrial DNA (mtDNA) in the cross talk between the onset of SIRS and subsequent development of AKI is unknown. Hence, we performed a case control study in critically ill patients with SIRS diagnosed with or without AKI, in which we determined mtDNA levels in plasma and urine, and correlated these to markers of renal impairment, inflammation, coagulation, and platelet activation. In addition, we exposed mice, primary renal tubular epithelial cells (TECs), and platelets to mtDNA or purified mitochondrial ligands, and measured their response to elucidate underlying pathophysiological mechanisms. Our data reveal that increased systemic mtDNA levels in SIRS patients do not correlate with systemic inflammation and renal disease activity. Moreover, AKI does not have an additional effect on circulating mtDNA levels. In contrast, we found that urinary mtDNA levels correlate with an elevated albumin creatinine ratio (ACR) as well as with increased urinary markers of inflammation, coagulation, and platelet activation. Both renal TECs and platelets respond to mtDNA and mtDNA ligands, leading to increased expression of, respectively, inflammatory cytokines and P-selectin. Moreover, activation of platelets results in mtDNA release. Together, these data suggest that circulating mtDNA is probably not important in the detrimental cross talk between SIRS and AKI, whereas renal mtDNA accumulation may be related to intrarenal inflammation, coagulation processes, and renal dysfunction in the pathophysiology of SIRS.

An immunofluorescence assay for extracellular matrix components highlights the role of epithelial cells in producing a stable, fibrillar extracellular matrix.

ABSTRACTActivated fibroblasts are considered major drivers of fibrotic disease progression through the production of excessive extracellular matrix (ECM) in response to signals from damaged epithelial and inflammatory cells. Nevertheless, epithelial cells are capable of expressing components of the ECM, cross-linking enzymes that increase its stability and are sensitive to factors involved in the early stages of fibrosis. We therefore wanted to test the hypothesis that epithelial cells can deposit ECM in response to stimulation in a comparable manner to fibroblasts. We performed immunofluorescence analysis of components of stable, mature extracellular matrix produced by primary human renal proximal tubular epithelial cells and renal fibroblasts in response to cytokine stimulation. Whilst fibroblasts produced a higher basal level of extracellular matrix components, epithelial cells were able to deposit significant levels of fibronectin, collagen I, III and IV in response to cytokine stimulation. In response to hypoxia, epithelial cells showed an increase in collagen IV deposition but not in response to the acute stress stimuli aristolochic acid or hydrogen peroxide. When epithelial cells were in co-culture with fibroblasts we observed significant increases in the level of matrix deposition which could be reduced by transforming growth factor beta (TGF-β) blockade. Our results highlight the role of epithelial cells acting as efficient producers of stable extracellular matrix which could contribute to renal tubule thickening in fibrosis.

A differential expression of uncoupling protein-2 associates with renal damage in stroke-resistant spontaneously hypertensive rat/stroke-prone spontaneously hypertensive rat-derived stroke congenic lines.

Uncoupling protein-2 (UCP2), a mitochondrial anion transporter involved in mitochondrial uncoupling, limiting reactive oxygen species formation, is significantly downregulated in kidneys of high-salt-fed stroke-prone spontaneously hypertensive rat (SHRSP), where it associates with increased renal damage occurrence. We aimed at establishing whether UCP2 differential expression associates with renal damage in two stroke-resistant spontaneously hypertensive rat (SHRSR)/SHRSP-derived stroke congenic lines. For this purpose, SHRSR, SHRSP, and two reciprocal stroke congenic lines carrying the (D1Rat134-Mt1pa) segment of chromosome 1 were fed with Japanese style diet for 8 weeks. At 4, 6, and 8 weeks of Japanese diet, kidneys were removed and analyzed for UCP2 gene and protein expression [UCP2 maps within (D1Rat134-Mt1pa)]; nuclear factor kappa-light-chain-enhancer of activated B cells protein expression; oxidized total protein levels; mitochondrial function; gene expression of cubulin, megalin, and nephrin. At 6 and 8 weeks of Japanese diet, histological damage and percentage of high molecular weight urinary proteins excretion were assessed. Introgression of UCP2 in the SHRSP configuration within the SHRSR genome led to UCP2 downregulation upon Japanese diet, as compared with the SHRSR, with significantly reduced ATP levels, increased rate of inflammation, oxidative stress, renal damage, and excretion of high molecular weight proteins. The opposite phenomena were observed in the reciprocal congenic line, compared with the SHRSP. In vitro, high-NaCl medium led to UCP2 downregulation, increased apoptosis/necrosis, and reduced viability in primary renal proximal tubular epithelial cells isolated from SHRSP. Exposure of the SHRSP/proximal tubular epithelial cells to recombinant UCP2 rescued the high-salt-dependent deleterious effects. A differential UCP2 expression associates with different degree of renal damage upon Japanese diet in two SHRSR/SHRSP-derived stroke congenic lines through modulation of mitochondrial function, inflammation, and oxidative stress.

Caspase-11 promotes cisplatin-induced renal tubular apoptosis through a caspase-3-dependent pathway.

Renal tubular injury is the hallmark of cisplatin-induced nephrotoxicity. Caspase-11, a member of the caspase family, plays an important role in inflammation and cell death. However, its role in cisplatin-induced renal tubular injury remains unclear. In cisplatin-treated mice, caspase-11 expression was significantly elevated and the expression of caspase-11 was mainly located in renal tubule. Inhibition of caspase-11 by small-interference RNA or its inhibitor wedelolactone attenuated cisplatin-induced renal dysfunction and tubular injury. In cultured primary renal tubular epithelial cells, cisplatin significantly promoted the expression and activation of caspase-11. Inhibition of caspase-11 by small-interference RNA reduced cisplatin-induced cell apoptosis. Overexpression of caspase-11 promoted cell apoptosis by activating the caspase-3-related cell apoptosis. Furthermore, coimmunoprecipitation results showed there was a direct interaction between caspase-11 and caspase-3, and the interaction was enhanced by cisplatin. The fluorescence confocal microscopy results showed that caspase-11 and caspase-3 were colocalized in the cytoplasm of renal tubular epithelial cells. These results demonstrate that caspase-11 plays an important role in cisplatin-induced renal tubular injury. Caspase-11 promotes renal epithelial cell apoptosis by activating the caspase-3-dependent apoptotic pathway. Caspase-11 might be a potential target for therapeutic treatment against cisplatin-induced nephrotoxicity.

Preconditioning of primary human renal proximal tubular epithelial cells without tryptophan increases survival under hypoxia by inducing autophagy

Hypoxia plays a significant role in the pathogenesis of acute kidney injury (AKI). Autophagy protects from AKI. Amino acid deprivation induces autophagy. The effect of L-tryptophan depletion on survival and autophagy in cultures of renal proximal tubular epithelial cells (RPTECs) under hypoxia was evaluated. RPTECs were preconditioned in a medium containing or not tryptophan, following culture under hypoxia and treatment with or without the autophagy inhibitor chloroquine. Cell survival was assessed by cell imaging, the level of certain proteins by western blotting and cellular ATP fluorometrically. Preconditioning of RPTECs in a medium without tryptophan activated general control nonderepressible 2 kinase and induced changes that favored autophagy and cell survival under hypoxic conditions. Additionally, it increased cellular ATP, while it inhibited apoptosis. Inhibition of autophagy nullified the induced increase in cellular ATP and cell survival by the absence of tryptophan. The absence of tryptophan increased p53, although its effect on p53's transcriptional targets was heterogeneous. In accordance with the decreased apoptosis, expression of p21 increased, while expression of Bax decreased. The expression of BNIP3L, which may be pro-apoptotic or pro-autophagic, increased. Considering the decreased apoptosis, it is likely that tryptophan depletion enhances autophagy through a p53-mediated increase of BNIP3L. Preconditioning of primary human RPTECs in a medium without tryptophan increases their survival under hypoxia by inducing autophagy. Identifying new molecular mechanisms that protect renal tissue from hypoxia could be proved clinically important in the prevention of AKI.

Expression and functional activity of bitter taste receptors in primary renal tubular epithelial cells and M-1 cells.

The kidney is essential in the maintenance of in vivo homeostasis by body fluid and electrolyte conservation and metabolic waste removal. Previously, we reported the expression of a novel G protein family (Tas2rs), which includes bitter taste receptors, in the kidney tubule system, including the nephrons and the collecting duct system. Bitter taste receptors could affect kidney function via Ca2+ intake. Alkaloids such as phenylthiocarbamide stimulate these receptors and cause an increase in Ca2+ intake. In this study, we determined the expression of bitter taste receptors in the immature kidney and small intestine and in primary renal epithelial cells and M-1 (collecting tubule cell line) cells, by using QPCR and immunostaining. We found no expression of bitter taste receptors in the immature kidney and small intestine several days after birth; the relative abundance of Tas2rs transcripts varied depending on the developmental stage. Tas2rs were expressed in primary renal epithelial cells and M-1 cells. The traditional Chinese medicinal plant extracts phellodendrine and coptisine caused a rapid rise in intracellular Ca2+ concentration, which was inhibited by the phospholipase C (PLC) inhibitor U-73122. Thus, phellodendrine and coptisine could change the physiological status of renal cells in vitro by mediation of bitter taste receptors in a PLC-dependent manner. Our results provide new insights on the expression and role of bitter taste receptors in renal development and function.

Zika virus infects renal proximal tubular epithelial cells with prolonged persistency and cytopathic effects

Zika virus (ZIKV) infection can cause fetal developmental abnormalities and Guillain–Barré syndrome in adults. Although progress has been made in understanding the link between ZIKV infection and microcephaly, the pathology of ZIKV, particularly the viral reservoirs in human, remains poorly understood. Several studies have shown that compared to serum samples, patients’ urine samples often have a longer duration of ZIKV persistency and higher viral load. This finding suggests that an independent viral reservoir may exist in the human urinary system. Despite the clinical observations, the host cells of ZIKV in the human urinary system are poorly characterized. In this study, we demonstrate that ZIKV can infect renal proximal tubular epithelial cells (RPTEpiCs) in immunodeficient mice in vivo and in both immortalized and primary human renal proximal tubular epithelial cells (hRPTEpiCs) in vitro. Importantly, ZIKV infection in mouse kidneys caused caspase-3-mediated apoptosis of renal cells. Similarly, in vitro infection of immortalized and primary hRPTEpiCs resulted in notable cytopathic effects. Consistent with the clinical observations, we found that ZIKV infection can persist with prolonged duration in hRPTEpiCs. RNA-Seq analyses of infected hRPTEpiCs revealed a large number of transcriptional changes in response to ZIKV infection, including type I interferon signaling genes and anti-viral response genes. Our results suggest that hRPTEpiCs are a potential reservoir of ZIKV in the human urinary system, providing a possible explanation for the prolonged persistency of ZIKV in patients’ urine.Emerging Microbes & Infections (2017) 6, e77; doi:10.1038/emi.2017.67; published online 23 August 2017

Carbon monoxide inhibits the nuclear-cytoplasmic translocation of HMGB1 in an in vitro oxidative stress injury model of mouse renal tubular epithelial cells.

Carbon monoxide (CO), as a vital small molecule in signaling pathways, is found to be involved in ischemia-reperfusion injury (IRI) in renal transplantation. CO-releasing molecule-2 (CORM-2), a CO-releasing molecule, is a type of metal carbonyl complexes which can quickly release CO in vivo. In this study, an in vitro oxidative stress injury model was established to examine the effect of CORM-2 pretreatment on the nuclear-cytoplasmic translocation of high mobility group box 1 protein (HMGB1) in mouse primary renal proximal tubular epithelial cells (RPTECs). Immunofluorescence staining showed that HMGB1 in the medium- and CORM-2-treated groups was predominantly localized in the nucleus of the cells, whereas higher amounts of HMGB1 translocated to the cytoplasm in the H2O2- and inactive CORM-2 (iCORM-2)-treated groups. Western blotting of HMGB1 showed that the total amounts of cytoplasmic HMGB1 in the H2O2-treated (0.59±0.27) and iCORM-2-treated (0.57±0.22) groups were markedly higher than those in the medium-treated (0.19±0.05) and CORM-2-treated (0.21±0.10) groups (P<0.05). Co-immunoprecipitation showed that the levels of acetylated HMGB1 in the H2O2-treated (642.98±57.25) and iCORM-2-treated (342.11±131.25) groups were markedly increased as compared with the medium-treated (78.72±74.17) and CORM-2-treated (71.42±53.35) groups (P<0.05), and no significant difference was observed between the medium-treated and CORM-2-treated groups (P>0.05). In conclusion, our study demonstrated that in the in vitro oxidative stress injury model of primary RPTECs, CORM-2 can significantly inhibit the nuclear-cytoplasmic translocation of HMGB1, which is probably associated with the prevention of HMGB1 acetylation.

The protective role of G protein-coupled estrogen receptor 1 (GPER-1) on methotrexate-induced nephrotoxicity in human renal epithelium cells

Nephrotoxicity is an important problem during methotrexate (MTX) treatment, which has been widely used for the treatment of several cancer types. Females are less susceptible to kidney diseases; however, the reason for this condition has not to be fully clarified. But sex hormones such as estrogen may have a protective effect on the kidney. We aimed to evaluate the possible protective role of estrogen on the MTX-induced renal epithelial cell death. Primary renal proximal tubular epithelial cells (RPTEC) were incubated with MTX (1, 10 and 100 μM), either alone or in combination with the 17β-estradiol, G protein-coupled estrogen receptor 1 (GPER1) agonist G-1, estrogen receptor alpha agonist propyl pyrazole triol (PPT), estrogen receptor beta agonist diarylpropionitrile (DPN). Cell viability was determined by MTT assays. Interleukin (IL)-1β, IL-6, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were determined in RPTEC. Approximately half of the cell death was observed with 10 μM MTX incubation for 48 h. The cell death was prevented by co-incubating with17β-estradiol, PPT and G-1. MTX was significantly induced IL-1β and IL-6.17β-estradiol, PPT and G-1 significantly decreased effects of MTX. SOD activity was significantly decreased treatment with MTX compared to control group. SOD activity was increased with co-incubation with 17β-estradioland G-1 compared to treatment with MTX. MDA levels significantly increased in treatment with MTX compared with the control group. Increased MDA levels by MTX-induced was decreased significantly by the treatment with 17β-estradiol and G-1. These data indicate that especially 17β-estradiol and G-1 may be useful in preventing undesirable effects of MTX in renal failure.

Proteasome or immunoproteasome inhibitors cause apoptosis in human renal tubular epithelial cells under normoxic and hypoxic conditions.

Ischemic acute kidney injury is characterized by apoptosis of tubular epithelial cells. Proteasome plays a key role in cellular processes such as proliferation, apoptosis and inflammation. The results of animal studies about the effect of proteasome inhibitors on the course of ischemic acute kidney injury are controversial. Primary human renal tubular epithelial cells were cultured with or without the hypoxia mimetic CoCl2 and with or without the proteasome inhibitor CEP-18770 and/or the immunoproteasome inhibitor ONX-0914. The level of the proteasome subunit β5, the immunoproteasome subunits LMP7 and LMP2, the function of these proteolytic machines, HIF-1α and its transcriptional target lactate dehydrogenase-A, p53 and its transcriptional targets TP53-inducible glycolysis and apoptosis regulator and p21, and finally of activated cleaved caspase-3 were assessed by means of western blotting. CoCl2 decreased the expression of β5, LMP7 and LMP2, as well as the activity of proteasome and immunoproteasome. It increased HIF-1α and its function, along with p53 and its function and induced apoptosis. CEP-18770 and ONX-0914 induced the above alterations toward the same directions as CoCl2 does. In CoCl2-treated cells, pretreatment with CEP-18770 and/or ONX-0914 potentiates the changes induced by CoCl2 alone. CoCl2, CEP-18770 and ONX-0914 induce apoptosis in human renal tubular epithelial cells. Importantly, proteasome or immunoproteasome inhibitors are rather toxic than beneficial in human renal tubular epithelial cells treated with the hypoxia mimetic CoCl2.

Human proximal tubule cells form functional microtissues.

The epithelial cells lining the proximal tubules of the kidney mediate complex transport processes and are particularly vulnerable to drug toxicity. Drug toxicity studies are classically based on two-dimensional cultures of immortalized proximal tubular cells. Such immortalized cells are dedifferentiated, and lose transport properties (including saturable endocytic uptake) encountered in vivo. Generating differentiated, organotypic human microtissues would potentially alleviate these limitations and facilitate drug toxicity studies. Here, we describe the generation and characterization of kidney microtissues from immortalized (HK-2) and primary (HRPTEpiC) human renal proximal tubular epithelial cells under well-defined conditions. Microtissue cultures were done in hanging drop GravityPLUS™ culture plates and were characterized for morphology, proliferation and differentiation markers, and by monitoring the endocytic uptake of albumin. Kidney microtissues were successfully obtained by co-culturing HK-2 or HRPTEpiC cells with fibroblasts. The HK-2 microtissues formed highly proliferative, but dedifferentiated microtissues within 10 days of culture, while co-culture with fibroblasts yielded spherical structures already after 2 days. Low passage HRPTEpiC microtissues (mono- and co-culture) were less proliferative and expressed tissue-specific differentiation markers. Electron microscopy evidenced epithelial differentiation markers including microvilli, tight junctions, endosomes, and lysosomes in the co-cultured HRPTEpiC microtissues. The co-cultured HRPTEpiC microtissues showed specific uptake of albumin that could be inhibited by cadmium and gentamycin. In conclusion, we established a reliable hanging drop protocol to obtain functional kidney microtissues with proximal tubular epithelial cell lines. These microtissues could be used for high-throughput drug and toxicology screenings, with endocytosis as a functional readout.

The Wnt11 Signaling Pathway in Potential Cellular EMT and Osteochondral Differentiation Progression in Nephrolithiasis Formation.

The molecular events leading to nephrolithiasis are extremely complex. Previous studies demonstrated that calcium and transforming growth factor-β1 (TGF-β1) may participate in the pathogenesis of stone formation, but the explicit mechanism has not been defined. Using a self-created genetic hypercalciuric stone-forming (GHS) rat model, we observed that the increased level of serous/uric TGF-β1 and elevated intracellular calcium in primary renal tubular epithelial cells (PRECs) was associated with nephrolithiasis progression in vivo. In the setting of high calcium plus high TGF-β1 in vitro, PRECs showed great potential epithelial to mesenchymal transition (EMT) progression and osteochondral differentiation properties, representing the multifarious increased mesenchymal and osteochondral phenotypes (Zeb1, Snail1, Col2A1, OPN, Sox9, Runx2) and decreased epithelial phenotypes (E-cadherin, CK19) bythe detection of mRNAs and corresponding proteins. Moreover, TGF-β-dependent Wnt11 knockdown and L-type Ca2+ channel blocker could greatly reverse EMT progression and osteochondral differentiation in PRECs. TGF-β1 alone could effectively promote EMT, but it had no effect on osteochondral differentiation in NRK cells (Rat kidney epithelial cell line). Stimulation with Ca2+ alone did not accelerate differentiation of NRK. Co-incubation of extracellular Ca2+ and TGF-β1 synergistically promotes EMT and osteochondral differentiation in NRK control cells. Our data supplied a novel view that the pathogenesis of calcium stone development may be associated with synergic effects of TGF-β1 and Ca2+, which promote EMT and osteochondral differentiation via Wnt11 and the L-type calcium channel.

Discovery and Characterization of Nonpeptidyl Agonists of the Tissue-Protective Erythropoietin Receptor.

Erythropoietin (EPO) and its receptor are expressed in a wide variety of tissues, including the central nervous system. Local expression of both EPO and its receptor is upregulated upon injury or stress and plays a role in tissue homeostasis and cytoprotection. High-dose systemic administration or local injection of recombinant human EPO has demonstrated encouraging results in several models of tissue protection and organ injury, while poor tissue availability of the protein limits its efficacy. Here, we describe the discovery and characterization of the nonpeptidyl compound STS-E412 (2-[2-(4-chlorophenoxy)ethoxy]-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine), which selectively activates the tissue-protective EPO receptor, comprising an EPO receptor subunit (EPOR) and the common β-chain (CD131). STS-E412 triggered EPO receptor phosphorylation in human neuronal cells. STS-E412 also increased phosphorylation of EPOR, CD131, and the EPO-associated signaling molecules JAK2 and AKT in HEK293 transfectants expressing EPOR and CD131. At low nanomolar concentrations, STS-E412 provided EPO-like cytoprotective effects in primary neuronal cells and renal proximal tubular epithelial cells. The receptor selectivity of STS-E412 was confirmed by a lack of phosphorylation of the EPOR/EPOR homodimer, lack of activity in off-target selectivity screening, and lack of functional effects in erythroleukemia cell line TF-1 and CD34(+) progenitor cells. Permeability through artificial membranes and Caco-2 cell monolayers in vitro and penetrance across the blood-brain barrier in vivo suggest potential for central nervous system availability of the compound. To our knowledge, STS-E412 is the first nonpeptidyl, selective activator of the tissue-protective EPOR/CD131 receptor. Further evaluation of the potential of STS-E412 in central nervous system diseases and organ protection is warranted.