Renal Epithelial Cells In Culture Research Articles

Abstract 86: Tumor Necrosis Factor-alpha Suppresses miR-29 and Up-regulates Fibrotic Genes in the Kidney

Tumor necrosis factor-alpha (TNF-a) is a pleiotropic inflammatory cytokine. Interstitial fibrosis is prominent in the Dahl salt-sensitive (SS) rat, including in the outer medullary region where thick ascending limbs (TALs) are abundant. We found previously that members of the miR-29 microRNA family, which have potent anti-fibrotic effects, were expressed at a lower level in the kidneys of SS rats. In the present study, we examined the role of TNF-a in the regulation of miR-29 and fibrotic genes in the kidney. RNA-seq analysis showed that SS rats fed a high salt diet had higher expression levels of TNF-a and TNFRSF1a which is one of its main receptors compared with a 0.4% salt diet group. Rat medullary thick ascending limb (mTAL) cells were treated with TNF-a at 25ng/ml for 24h or 48h. At 24h, TNF-a significantly decreased the abundance of miR-29b (to 57% ± 9% of control, n=14, p<0.05) and miR-29c (to 74% ± 6% of control, p<0.01) and significantly increased alpha-smooth muscle actin (a-SMA) by more than two fold and up-regulated extracellular matrix genes such as Col4a1 (to 161% ± 25% of control, n=10, p<0.05) and Col4a2 (to 172%± 18% of control, p<0.01). At 48h, TNF-a significantly suppressed miR-29a and again up-regulated a-SMA and Col4a2. Similarly, TNF-a suppressed miR-29 in primary cultures of human renal epithelial cells, an effect that was attenuated by inhibition of NF-kB. This study shows that TNF-a can suppress miR-29 and up-regulate fibrotic genes in renal epithelial cells including mTAL cells, which may play an important role in the development of renal tubulointerstitial injury in SS rats.

Heparan Sulfate‐Bound Dimeric IL‐2: A Potential Mediator of Renal Ischemic Injury

Interleukin‐2 (IL‐2) is a cytokine critical to normal immune function. Our laboratory has shown that IL‐2 is retained in tissues by heparan sulfate (HS). While known as a monomer, a dimeric form of IL‐2 was previously identified in fish optic neurons, and this form was toxic to oligodendrocytes. Given this observation, we asked whether dimeric IL‐2 is found in mammalian tissues. Murine and human aorta and kidney were assessed by Western blot for the presence of IL‐2. Dimeric (30 kD) IL‐2 was identified in each tissue. Heparinase digestion of tissues liberated dimeric IL‐2, suggesting that the dimer is bound, at least in part, by HS. To ascertain whether dimeric IL‐2 is cytotoxic, we treated cultures of renal epithelial cells with increasing concentrations of dimeric IL‐2, isolated by electroelution. Signs of cytotoxicity were evident within 15 minutes of dimer addition. Commercial IL‐2, isolated identically to dimeric IL‐2, was not cytotoxic. Systemic administration to mice of 10 μg of dimeric IL‐2 induced vacuolization of renal epithelium, a morphology typically seen with ischemic injury. Finally, murine kidneys subjected to 60 minutes of ischemia, compared to sham controls, expressed greatly increased amounts of dimeric IL‐2 in tissue homogenates. These results suggest that dimeric IL‐2 may contribute to acute tubular necrosis and, in turn, renal dysfunction. This work was supported by institutional funds.

Study on therapeutic action of bone marrow derived mesenchymal stem cell combined with vitamin E against acute kidney injury in rats

AimsThe study aims to investigate the effect to treat acute kidney injury (AKI) with bone marrow derived mesenchymal stem cells (BMSCs) combined with vitamin E and to develop a new treatment mode for AKI preclinical study. Main methodsBMSCs were separated from rat bone marrow. Gentamicin was used as a damage factor in the culture of renal tubular epithelial cells (RTECs) in vitro. After co-cultured with BMSCs and vitamin E, cell proliferation of each group was detected with CCK-8. In vivo, BMSCs (3.3×106cells/kg) combined with vitamin E (80mg/kg) were administered in AKI rats induced by gentamicin intravenously. The pathological changes, biochemical parameters and apoptosis genes after treatment were investigated furthermore. Key findingsIn co-cultured system, proliferating ability of RTECs was improved by BMSCs or vitamin E, especially for the combined group (P<0.05). The treated rats in combined group presented the lowest serum creatinine and the highest urea nitrogen compared to non-treated rats. The improvement in renal pathological changes was followed by less necrosis, degeneration and expansion of renal tubule. Under transmission electron microscope, unclear cell structure and reduction of endoplasmic reticulum in the cytoplasm of RTECs were ameliorated with the treatment. Most apoptosis genes were up-regulated in model group while down-regulated with the therapy. Further analysis showed that the two treatments may act independently with each other. SignificanceOur data demonstrated that both BMSC and vitamin E hold therapeutic action to AKI induced by gentamicin. Especially, the combined treatment is better than BMSC or vitamin E alone.

Short and long term gene expression variation and networking in human proximal tubule cells when exposed to cadmium

Cadmium (Cd2+) is a known nephrotoxin causing tubular necrosis during acute exposure and potentially contributing to renal failure in chronic long-term exposure. To investigate changes in global gene expression elicited by cadmium, an in-vitro exposure system was developed from cultures of human renal epithelial cells derived from cortical tissue obtained from nephrectomies. These cultures exhibit many of the qualities of proximal tubule cells. Using these cells, a study was performed to determine the cadmium-induced global gene expression changes after short-term (1 day, 9, 27, and 45 μM) and long-term cadmium exposure (13 days, 4.5, 9, and 27 μM). These studies revealed fundamental differences in the types of genes expressed during each of these time points. The obtained data was further analyzed using regression to identify cadmium toxicity responsive genes. Regression analysis showed 403 genes were induced and 522 genes were repressed by Cd2+ within 1 day, and 366 and 517 genes were induced and repressed, respectively, after 13 days. We developed a gene set enrichment analysis method to identify the cadmium induced pathways that are unique in comparison to traditional approaches. The perturbation of global gene expression by various Cd2+ concentrations and multiple time points enabled us to study the transcriptional dynamics and gene interaction using a mutual information-based network model. The most prominent network module consisted of INHBA, KIF20A, DNAJA4, AKAP12, ZFAND2A, AKR1B10, SCL7A11, and AKR1C1.

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.

Negative regulatory responses to metabolically triggered inflammation impair renal epithelial immunity in diabetes mellitus

Diabetes mellitus is characterized by chronic inflammation and increased risk of infections, particularly of tissues exposed to the external environment. However, the causal molecular mechanisms that affect immune cells and their functions in diabetes are unclear. Here we show, by transcript and protein analyses, signatures of glucose-induced tissue damage, chronic inflammation, oxidative stress, and dysregulated expression of multiple inflammation- and immunity-related molecules in diabetic kidneys compared with non-diabetic controls. Abnormal signaling involving cytokines, G-protein coupled receptors, protein kinase C isoforms, mitogen-activated protein kinases, nuclear factor-κB (NFκB), and Toll-like receptors (TLR) were evident. These were accompanied by overexpression of negative regulators of NFκB, TLR, and other proinflammatory pathways, e.g., A20, SOCS1, IRAK-M, IκBα, Triad3A, Tollip, SIGIRR, and ST2L. Anti-inflammatory and immunomodulatory molecules, e.g., IL-10, IL-4, and TSLP that favor TH2 responses were strongly induced. These molecular indicators of immune dysfunction led us to detect the cryptic presence of bacteria and human cytomegalovirus in more than one third of kidneys of diabetic subjects but none in non-diabetic kidneys. Similar signaling abnormalities could be induced in primary human renal tubular epithelial (but not mesangial) cell cultures exposed to high glucose, proinflammatory cytokines and methylglyoxal, and were reversed by combined pharmacological treatment with an antioxidant and a PKC inhibitor. Our results suggest that diabetes impairs epithelial immunity as a consequence of chronic and inappropriate activation of counter-regulatory immune responses, which are otherwise physiological protective mechanisms against inflammation. The immune abnormalities and cryptic renal infections described here may contribute to progression of diabetic nephropathy.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-012-0969-x) contains supplementary material, which is available to authorized users.

DNAM-1 - a New Player in Renal Allograft Rejection

Despite effective treatment protocols to prevent rejection many renal allografts are lost due to the toxicity of immunosuppressants. Thus, more specific and less toxic immunosuppression is needed. DNAM-1 (CD226) on T cells has been shown to play an important role for allogeneic graft-versus-host responses. DNAM-1 has two ligands: the adhesion molecules CD155 and CD112. Both are expressed on dendritic, epithelial, and endothelial cells. The importance of DNAM-1and its ligands for renal allograft rejection is unknown. Primary cultures of murine renal tubular epithelial cells (rTECs) were prestimulated with IFN-β and IFN-γ to induce high surface expression of MHC. Surface expression of CD155 and CD112 was tested by FACS. Responder T cells were stimulated in vitro with allogeneic fully MHC-mismatched splenocytes. From these stimulation cocultures we measured proliferation (thymidine incorporation), cytokine production (ELISA) and cytotoxicity against IFN-stimulated rTECs from WT or CD155−/− mice (51Cr-release-assay). To test for a role of this pathway in vivo, we performed fully MHC-mismatched skin and kidney grafts from WT and CD155−/− donors. CD155 and CD112 were both highly expressed on rTECs and could be further increased by IFN-stimulation. Also, in acutely rejected murine renal allografts an upregulation of both molecules was detected. However, when CD155 was missing on targets in an allospecific chromium-release-assay, no difference in cytotoxicity was measured compared to WT targets. Also, allospecific T cell proliferation and IFN-γ secretion were not altered, when stimulator cells lacked CD155. When testing for the role of this pathway for allograft rejection in vivo, no difference between skin graft survival of WT and CD155−/−was observed. Renal allografts from CD155−/− donors show similar amounts of infiltrates and interestingly higher incidence of infarcts compared to WT donors. The reason for this is under investigation. In contrast to these results, allospecific T cell proliferation was be reduced with the use of a blocking antibody against DNAM-1. Also allospecific cytotoxicity was reduced, when DNAM-1 was blocked during stimulation. Experiments blocking DNAM1 or CD112 in vivo are ongoing. Thus, DNAM-1 is an important costimulatory receptor for alloreactive T cells. However, the important ligand might be CD112 rather than CD155. Thus, blocking DNAM-1 or CD112 might be a therapeutically interesting option to prevent renal allograft rejection.

Human Platelet Lysates as a Serum Substitute in Renal Epithelial Cell Culture

Cultured renal epithelia form monolayers of differentiated, polarized cells. On permeable supports, an apical and a basolateral compartment are separated by the cultured epithelium to study epithelial transport in vitro. However, culture conditions and culture media have a significant impact. In this respect, the quality of fetal bovine serum (FBS) seemed to be of major importance. The high lot‐to‐lot variablity of FBS was found to substantially influence the differentiation of epithelial cultures with regard to the generation of a transepithelial electrical resistance (TEER). Thus, batch‐testing of FBS was required. We recently reported on the use of human platelet lysates (PL) as a replacement for FBS. The growth promoting capacity of PL was tested on renal epithelial cell lines, for which differentiation end points are well established. PL support growth, proliferation and differentiation of LLC‐PK1, HK‐ 2 and MDCK cells. In addition, TEER was monitored in filter‐grown LLC‐PK1 and in MDCK II and MDCK I epithelia. Low‐resistance epithelia generated a TEER of 150–250 Ω•cm2 in PL‐media, as seen with FBS, and high‐resistance MDCK I retained their TEER of 8,000 Ω•cm2. PL are a valuable, animal‐derived component‐free substitute for FBS in renal epithelial cell culture with a proven high batch‐to batch uniformity.

Drug transporter expression and function in primary cultures of human renal epithelial cells

Primary epithelial cell cultures are an important model for studying drug transporters in vitro. They can be used as a replacement for animal models and possibly are more suitable due to variation in transporter expression between animal and human tissue. The aim of this study was to validate the use of primary epithelial cell cultures as a model for studying drug transporter expression and function in human kidney.Primary epithelial cells were isolated from human kidney tissue samples and cultured for 7 to 10 days. qPCR was used to determine mRNA expression of key transporters in the primary cells. Cells were grown on permeable filter supports to create polarised monolayers and transport of a range of radio‐labelled compounds was tested across the cell monolayers. Dye retention assays were used to determine the presence of functional efflux transporters.Expression of mRNA for a range of prototypic transporters including OCT‐2, MDR1 and MRP was confirmed. Basolateral to apical transport of various compounds including metformin and indomethacin was shown suggesting possible elimination via the kidney. Hoechst 33342 retention assay showed expression of functional MDR1. CMFDA dye retention assay showed presence of MK571 sensitive transporters, likely to be MRPs. These findings show that primary cell cultures are a good model for studying drug transporter expression and function in human kidney.

Epithelial Cells Activate Plasmacytoid Dendritic Cells Improving Their Anti-HIV Activity

Plasmacytoid dendritic cells (pDCs) play a major role in anti-viral immunity by virtue of their ability to produce high amounts of type I interferons (IFNs) and a variety of inflammatory cytokines and chemokines in response to viral infections. Since recent studies have established that pDCs accumulate at the site of virus entry in the mucosa, here we analyzed whether epithelial cells were able to modulate the function of pDCs. We found that the epithelial cell lines HT-29 and Caco-2, as well as a primary culture of human renal tubular epithelial cells (HRTEC), induced the phenotypic maturation of pDCs stimulating the production of inflammatory cytokines. By contrast, epithelial cells did not induce any change in the phenotype of conventional or myeloid DCs (cDCs) while significantly stimulated the production of the anti-inflammatory cytokine IL-10. Activation of pDCs by epithelial cells was prevented by Bafilomycin A1, an inhibitor of endosomal acidification as well as by the addition of RNase to the culture medium, suggesting the participation of endosomal TLRs. Interestingly, the cross-talk between both cell populations was shown to be associated to an increased expression of TLR7 and TLR9 by pDCs and the production of LL37 by epithelial cells, an antimicrobial peptide able to bind and transport extracellular nucleic acids into the endosomal compartments. Interestingly, epithelium-activated pDCs impaired the establishment of a productive HIV infection in two susceptible target cells through the stimulation of the production of type I IFNs, highlighting the anti-viral efficiency of this novel activation pathway.

C-Jun-N-Terminal Kinase Signaling Is Involved in Cyclosporine-Induced Epithelial Phenotypic Changes

Tubular epithelial cells play a central role in the pathogenesis of chronic nephropathies. Previous toxicogenomic studies have demonstrated that cyclosporine- (CsA-) induced epithelial phenotypic changes (EPCs) are reminiscent of an incomplete epithelial to mesenchymal transition (EMT) in a TGF-β-independent manner. Furthermore, we identified endoplasmic reticulum (ER) stress as a potential mechanism that may participate in the modulation of tubular cell plasticity during CsA exposure. Because c-jun-N-terminal kinase (JNK), which is activated during ER stress, is implicated in kidney fibrogenesis, we undertook the current study to identify the role of JNK signaling in EPCs induced by CsA. In primary cultures of human renal epithelial cells, CsA activates JNK signaling, and the treatment with a JNK inhibitor reduces the occurrence of cell shape changes, E-cadherin downregulation, cell migration, and Snail-1 expression. Our results suggest that CsA activates JNK signaling, which, in turn, may participate in the morphological alterations through the regulation of Snail-1 expression.

Gamma Interferon Controls Mouse Polyomavirus InfectionIn Vivo

Human polyomaviruses are associated with substantial morbidity in immunocompromised patients, including those with HIV/AIDS, recipients of bone marrow and kidney transplants, and individuals receiving immunomodulatory agents for autoimmune and inflammatory diseases. No effective antipolyomavirus agents are currently available, and no host determinants have been identified to predict susceptibility to polyomavirus-associated diseases. Using the mouse polyomavirus (MPyV) infection model, we recently demonstrated that perforin-granzyme exocytosis, tumor necrosis factor alpha (TNF-α), and Fas did not contribute to control of infection or virus-induced tumors. Gamma interferon (IFN-γ) was recently shown to inhibit replication by human BK polyomavirus in primary cultures of renal tubular epithelial cells. In this study, we provide evidence that IFN-γ is an important component of the host defense against MPyV infection and tumorigenesis. In immortalized and primary cells, IFN-γ reduces expression of MPyV proteins and impairs viral replication. Mice deficient for the IFN-γ receptor (IFN-γR(-/-)) maintain higher viral loads during MPyV infection and are susceptible to MPyV-induced tumors; this increased viral load is not associated with a defective MPyV-specific CD8(+) T cell response. Using an acute MPyV infection kidney transplant model, we further show that IFN-γR(-/-) donor kidneys harbor higher MPyV levels than donor kidneys from wild-type mice. Finally, administration of IFN-γ to persistently infected mice significantly reduces MPyV levels in multiple organs, including the kidney, a major reservoir for persistent mouse and human polyomavirus infections. These findings demonstrate that IFN-γ is an antiviral effector molecule for MPyV infection.

Abstract SY23-01: Early endosomes: Platforms for the induction of oncogenic phenotypes

Abstract Oncogenic signaling markedly impacts the adhesive and migratory potential of tumor cells. Many of the resulting cellular changes are elicited by coordinated alterations in endosomal membrane trafficking. Although long regarded as a means for cells to internalize nutrients and cell surface molecules, emerging evidence suggests that the endosomal circuitry is also an essential platform for signal transduction and a conduit for the transport of cellular machinery, including adhesion proteins and signaling molecules, appropriate compartmentalization of which appears to be essential for eliciting changes that accompany disease progression. The disruption of epithelial glandular architecture and the acquisition of aggressive invasive phenotypes, two hallmarks of tumor progression, exemplify the above paradigm. Our efforts are directed at elucidating and defining the role of ARF6-coupled endocytic pathways in the acquisition of migratory/invasive phenotypes. Recent work using three-dimensional basement membrane cultures of renal and mammary epithelial cells reveals that the formation of signaling endosomes during glandular cyst morphogenesis, serve as platforms for hyperactive receptor signaling leading to the disruption of epithelial cysts and generation of certain tumorigenic phenotypes. We are also investigating the structural and molecular changes at the invasive front of tumor cells. Our recent studies, and others, have shown that selective sorting of specific cargo such as the membrane-type proteases along early endocytic recycling pathways, is tightly coupled to the biogenesis and function of invasive structures such as invadopodia and microvesicles. Invadopodia are actin-rich protrusions that form at the adherent cell surface and mediate pericellular proteolysis. Microvesicles, generated by the outward budding and fission of the plasma membrane, facilitate the horizontal transfer of bioactive molecules and are now thought to have vital roles in tumor invasion and metastasis, inflammation and stem-cell expansion. Since endosomes confer spatial and temporal dimensions to oncogenic signaling, understanding the interplay between endocytic trafficking and the activation of invasive cell programs could provide new and important insights into the progression toward pathophysiological states. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY23-01. doi:10.1158/1538-7445.AM2011-SY23-01

Microarray Analysis of Gene Expression Patterns in Human Proximal Tubule Cells Over a Short and Long Time Course of Cadmium Exposure

Numerous studies showed that renal proximal tubules cells are the cell type critically affected by chronic exposure to cadmium (Cd2+). The aim of the present study was to apply global gene expression technology and a human renal epithelial cell culture model (HPT) to determine whether time of exposure to Cd2+ exerts a major influence on the resulting pattern of global gene expression. HPT cells were exposed to Cd2+ for a short, 1-d, period of exposure (9, 27, and 45 μM) versus a longer, 13-d, period (4.5, 9, and 27 μM), with the hypothesis being that the stress response of the cells would be more active during the short time of exposure. The results showed that the differential expression of genes was very extensive for HPT cells exposed to Cd2+ for 1 d, with more than 1848 genes displaying alterations compared to control and with the major categories of genes being involved in stress responses; cell death; checkpoint arrest, DNA repair, and the cell cycle; inflammatory responses; and cell adhesion, motion and differentiation. In contrast, HPT cells exposed to Cd2+ for 13 d showed 923 genes to be differentially expressed, with a marked reduction in the number of differentially expressed stress response genes and a significant increase in the number of genes involved in development and differentiation. There were 387 differentially expressed genes common to both times of exposure. Data suggest that unless one is actively seeking to study the acute stress response, global gene expression technology should not be applied within an early time course of toxicant exposure.

Bioengineering of living renal membranes consisting of hierarchical, bioactive supramolecular meshes and human tubular cells

Maintenance of polarisation of epithelial cells and preservation of their specialized phenotype are great challenges for bioengineering of epithelial tissues. Mimicking the basement membrane and underlying extracellular matrix (ECM) with respect to its hierarchical fiber-like morphology and display of bioactive signals is prerequisite for optimal epithelial cell function in vitro. We report here on a bottom-up approach based on hydrogen-bonded supramolecular polymers and ECM-peptides to make an electro-spun, bioactive supramolecular mesh which can be applied as synthetic basement membrane. The supramolecular polymers used, self-assembled into nano-meter scale fibers, while at micro-meter scale fibers were formed by electro-spinning. We introduced bioactivity into these nano-fibers by intercalation of different ECM-peptides designed for stable binding. Living kidney membranes were shown to be bioengineered through culture of primary human renal tubular epithelial cells on these bioactive meshes. Even after a long-term culturing period of 19 days, we found that the cells on bioactive membranes formed tight monolayers, while cells on non-active membranes lost their monolayer integrity. Furthermore, the bioactive membranes helped to support and maintain renal epithelial phenotype and function. Thus, incorporation of ECM-peptides into electro-spun meshes via a hierarchical, supramolecular method is a promising approach to engineer bioactive synthetic membranes with an unprecedented structure. This approach may in future be applied to produce living bioactive membranes for a bio-artificial kidney.

Dietary methionine restriction improves colon tight junction barrier function and alters claudin expression pattern

The beneficial effects of caloric restriction in increasing longevity and forestalling age-related diseases are well known. Dietary restriction of methionine also renders similar benefits. We recently showed in a renal epithelial cell culture system that reduction of culture medium methionine by 80% resulted in altered tight junctional (TJ) claudin composition and also improved epithelial barrier function (51). In the current study, we examined the effect of dietary restriction of methionine on TJ barrier function in rat gastrointestinal tissue to see whether this phenomenon also holds true in a tissue model and for a different epithelial cell type. After 28 days on methionine-restricted (MR) diet, rats showed small but significant reductions in the plasma and (intracellular) colonocyte levels of methionine. Colon mucosal sheets from rats on the MR diet showed increased transepithelial electrical resistance with concomitant decrease in paracellular diffusion of (14)C-D-mannitol, suggesting improved barrier function relative to rats on control diet. This improved barrier function could not be explained by changes in colon crypt length or frequency. Neither was the colonocyte mitotic index nor the apoptotic frequency altered significantly. However, TJ composition/structure was being altered by the MR diet. RT-PCR and Western blot analysis showed an increase in the abundance of claudin-3 and an apparent change in the posttranslational modification of occludin, data reinforcing a paracellular barrier alteration. Overall, our data suggest that reduction in dietary intake of methionine results in improved epithelial barrier function by inducing altered TJ protein composition.

Clostridium perfringens epsilon toxin is cytotoxic for human renal tubular epithelial cells

Clostridium perfringens epsilon toxin (ETX) is responsible for a fatal enterotoxemia in different animal species, producing extensive renal damage, neurological disturbance and edema of lungs, heart and kidneys. However, there is no information about the susceptibility of humans to ETX. Here, we report that primary cultures of human renal tubular epithelial cells (HRTEC) exposed to ETX showed a marked swelling with subsequent large blebs surrounding most cells. The incubation of HRTEC with ETX produced a reduction of cell viability in a dose- and time-dependent manner. The CD(50) after 1-hour and 24-hour incubation were 3 µg/mL and 0.5 µg/mL, respectively. The pulse with ETX for 3 min was enough to produce a significant cytotoxic effect on HRTEC after 1-hour incubation. ETX binds to HRTEC forming a large complex of about 160 kDa similar to what was found in the Madin-Darby canine kidney (MDCK) cell line. The HRTEC could be a useful cell model to improve the understanding of the mechanisms involved on the cell damage mediated by ETX.

Bardet‐Biedl Sydrome (BBS) proteins play a role in Centrosome Amplification

Many cancers result from mitotic or apoptotic abnormalities. We and others have previously shown that BBS proteins are involved in centrosome organization and regulation. We observed centrosomal clusters in renal epithelial primary cells obtained from BBS mutant mice. Along with the extra centrosomes, we observed a marked increase in the amount of gamma‐tubulin in BBS renal epithelial cells compared to wild type controls. It was unclear if these phenomena were related to the cell culture or a hallmark of BBS mutations. Therefore, we compared BBS mutant and wild type primary renal epithelial cells in culture with kidney sections of the same mice. Although the culture of cells exacerbates the phenotype, we demonstrate a definitive increase in centrosome number within cells in fixed kidneys obtained from BBS mutant mice as compared to wild type controls. We hypothesize that BBS proteins are involved in the regulation or transport of proteins destined for proteosome degradation. Specifically targeting cancer cells with multiple centrosomes presents a viable and novel therapy. Confirming BBS proteins role in centrosome regulation may help to better understand tumorigenesis.This work was supported by the NIH and HHMI.

Pals‐associated tight junction protein functionally links dopamine and angiotensin II to the regulation of sodium transport in renal epithelial cells

Dopamine inhibits renal cell Na(+),K(+)-ATPase activity and cell sodium transport by promoting the internalization of active molecules from the plasma membrane, whereas angiotensin II (ATII) stimulates its activity by recruiting new molecules to the plasma membrane. They achieve such effects by activating multiple and distinct signalling molecules in a hierarchical manner. The purpose of this study was to investigate whether dopamine and ATII utilize scaffold organizer proteins as components of their signalling networks, in order to avoid deleterious cross talk. Attention was focused on a multiple PDZ domain protein, Pals-associated tight junction protein (PATJ). Ectopic expression of PATJ in renal epithelial cells in culture was used to study its interaction with components of the dopamine signalling cascade. Similarly, expression of PATJ deletion mutants was employed to analyse its functional relevance during dopamine-, ATII- and insulin-dependent regulation of Na(+),K(+)-ATPase. Dopamine receptors and components of its signalling cascade mediating inhibition of Na(+),K(+)-ATPase interact with PATJ. Inhibition of Na(+),K(+)-ATPase by dopamine was prevented by expression of mutants of PATJ lacking PDZ domains 2, 4 or 5; whereas the stimulatory effect of ATII and insulin on Na(+),K(+)-ATPase was blocked by expression of PATJ lacking PDZ domains 1, 4 or 5. A multiple PDZ domain protein may add functionality to G protein-coupled and tyrosine kinase receptors signalling during regulation of Na(+),K(+)-ATPase. Signalling molecules and effectors can be integrated into a functional network by the scaffold organizer protein PATJ via its multiple PDZ domains.

MicroRNA expression profiles predictive of human renal allograft status

Immune rejection of organ transplants is a life-threatening complication and is exemplified by alterations in the expression of protein-encoding genes. Because microRNAs (miRNAs) regulate the expression of genes implicated in adaptive immunity, we investigated whether acute rejection (AR) is associated with alterations in miRNA expression within allografts and whether expression profiles are diagnostic of AR and predict allograft function. Seven of 33 renal allograft biopsies (12 AR and 21 normal) were profiled using microfluidic cards containing 365 mature human miRNAs (training set), and a subset of differentially expressed miRNAs were quantified in the remaining 26 allograft biopsies (validation set). We found a strong association between intragraft expression of miRNAs and messenger RNAs (mRNAs), and that AR, and renal allograft function, could be predicted with a high level of precision using intragraft levels of miRNAs. Our investigation of miRNA expression in normal human peripheral blood mononuclear cells (PBMCs) showed that miRNAs (miR-142-5p, -155, and -223) overexpressed in AR biopsies are highly expressed in PBMCs, and that stimulation with the mitogen phytohaemagglutinin results in an increase in the abundance of miR-155 and a decrease in miR-223 and let-7c. Quantification of miRNAs in primary cultures of human renal epithelial cells (HRECs) showed that miR-30a-3p, -10b, and let-7c are highly expressed in HRECs, and that stimulation results in a decreased expression of miR-30a-3p. Our studies, in addition to suggesting a cellular basis for the altered intragraft expression of miRNAs, propose that miRNA expression patterns may serve as biomarkers of human renal allograft status.