Effects of Curcumin Analogues DMC and EF24 in Combination with the Cytokine TRAIL against Kidney Cancer.

The natural compound curcumin has been shown to have therapeutic potential against a wide range of diseases such as cancer. Curcumin reduces cell viability of renal cell carcinoma (RCC) cells when combined with TNF-related apoptosis-inducing ligand (TRAIL), a cytokine that specifically targets cancer cells, by helping overcome TRAIL resistance. However, the therapeutic effects of curcumin are limited by its low bioavailability. Similar compounds to curcumin with higher bioavailability, such as demethoxycurcumin (DMC) and 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), can potentially have similar anticancer effects and show a similar synergy with TRAIL, thus reducing RCC viability. This study aims to show the effects of DMC and EF24 in combination with TRAIL at reducing ACHN cell viability and ACHN cell migration. It also shows the changes in death receptor 4 (DR4) expression after treatment with these compounds individually and in combination with TRAIL, which can play a role in their mechanism of action.

Neutrophil gelatinase-associated lipocalin (NGAL) is localised to the primary cilium in renal tubular epithelial cells - A novel source of urinary biomarkers of renal injury.

BACKGROUND: Primary cilia have been shown to play a central role in regulating epithelial cell differentiation during injury and repair. Growing evidence implicates structural and functional abnormalities of primary cilia in kidney epithelial cells in the onset and development of various kidney diseases including polycystic kidney disease (PKD). Neutrophil-gelatinase associated lipocalin (NGAL) has been identified as a reliable urinary biomarker of kidney injury. However, the mechanism by which this protein accumulates in patient urine samples has not been fully elucidated. METHODS: Human renal tubular epithelial cells (RPTECs) were exposed to previously characterized deciliating agents to assess mechanisms of primary cilium loss. Confocal immunofluorescent imaging was employed to visualise the effects on cilia. Western blot analysis was utilised to quantify the ciliary protein Arl13b in both RPTEC whole cell lysates and supernatants. Co-immunoprecipitation was used to demonstrate co-localisation of Arl13b and NGAL in urinary samples from a clinical Chronic Allograft Nephropathy (CAN) cohort. RESULTS: Immunofluorescent analysis revealed that NGAL was localised to the primary cilium in RPTECs, co-localizing with a ciliary specific protein, Arl13b. Deciliation experiments showed that loss of the cilia coincided with loss of NGAL from the cells. CONCLUSION: The accumulation of NGAL in supernatants in vitro and in the urine of CAN patients was concurrent with loss of Arl13b, a specific ciliary protein. The findings of this study propose that increased NGAL urinary concentrations are directly linked to deciliation of the renal epithelial cells as a result of injury.

Reactive carbonyl compounds impair wound healing by vimentin collapse and loss of the primary cilium.

In renal pathologies tubulo-interstitial fibrosis results from an aberrant wound-healing ability where the normal epithelial tissue is substituted for scar tissue caused by accumulation of extracellular matrix proteins (ECM). During the wound-healing process, epithelial cells may undergo epithelial-mesenchymal transition (EMT) acquiring a mesenchymal-like phenotype that allows cells to migrate and re-epithelialize the wound site. It has been reported that chronic inflammation and uremic milieu are involved in wound-healing and enhanced kidney damage in chronic kidney disease (CKD) patients. In this study we evaluated reactive carbonyl compounds (RCC) effects on renal wound healing. The compounds resulting from carbonyl stress evaluated in this study were glyoxal (GO), methylglyoxal (MGO), malondialdehyde (MDA) and 4-hydroxy-hexenal (HHE). Wound repair ability was evaluated by the wound healing assay using HK-2 cells. EMT was evaluated by morphological, protein and transcriptional changes using microscopy, western blot, zymography and RT-qPCR. Changes in the vimentin network and primary cilia were assessed by immunofluorescence. Our data demonstrated that MDA and GO delay wound closure mediated by vimentin disruption, which caused collagen I mRNA decrease, and deciliation. In contrast, HHE treatment (and MGO to a minor degree) induced morphological changes and increased mesenchymal marker expression and gelatinase activity in HK-2 cells. In this study, we have demonstrated for the first time that exposure to RCC differentially affects wound healing in proximal tubular epithelia. A better comprehension of effects of uremic toxins on wound healing and fibrosis and migration is necessary to seek mechanisms to slow down renal fibrosis.

Prolyl hydroxylase-1 regulates hepatocyte apoptosis in an NF-κB-dependent manner.

Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1(-/-) hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease.

Tacrolimus Modulates TGF-ß Signaling to Induce Epithelial-Mesenchymal Transition in Human Renal Proximal Tubule Epithelial Cells.

Epithelial-mesenchymal transition (EMT), a process which describes the trans-differentiation of epithelial cells into motile mesenchymal cells, is pivotal in stem cell behavior, development and wound healing, as well as contributing to disease processes including fibrosis and cancer progression. Maintenance immunosuppression with calcineurin inhibitors (CNIs) has become routine management for renal transplant patient, but unfortunately the nephrotoxicity of these drugs has been well documented. HK-2 cells were exposed to Tacrolimus (FK506) and EMT markers were assessed by RT PCR and western blot. FK506 effects on TGF-ß mRNA were assessed by RT PCR and TGF-ß secretion was measured by ELISA. The impact of increased TGF-ß secretion on Smad signaling pathways was investigated. The impact of inhibition of TGF-ß signaling on EMT processes was assessed by scratch-wound assay. The results presented in this study suggest that FK506 initiates EMT processes in the HK-2 cell line, with altered expression of epithelial and myofibroblast markers evident. Additionally, the study demonstrates that FK506 activation of the TGF-ß/ SMAD pathways is an essential step in the EMT process. Overall the results demonstrate that EMT is heavily involved in renal fibrosis associated with CNI nephrotoxicity.

Cystine accumulation attenuates insulin release from the pancreatic ß-cell due to elevated oxidative stress and decreased ATP levels.

The pancreatic ß-cell has reduced antioxidant defences making it more susceptible to oxidative stress. In cystinosis, a lysosomal storage disorder, an altered redox state may contribute to cellular dysfunction. This rare disease is caused by an abnormal lysosomal cystine transporter, cystinosin, which causes excessive accumulation of cystine in the lysosome. Cystinosis associated kidney damage and dysfunction leads to the Fanconi syndrome and ultimately end-stage renal disease. Following kidney transplant, cystine accumulation in other organs including the pancreas leads to multi-organ dysfunction. In this study, a Ctns gene knockdown model of cystinosis was developed in the BRIN-BD11 rat clonal pancreatic ß-cell line using Ctns-targeting siRNA. Additionally there was reduced cystinosin expression, while cell cystine levels were similarly elevated to the cystinotic state. Decreased levels of chronic (24 h) and acute (20 min) nutrient-stimulated insulin secretion were observed. This decrease may be due to depressed ATP generation particularly from glycolysis. Increased ATP production and the ATP/ADP ratio are essential for insulin secretion. Oxidised glutathione levels were augmented, resulting in a lower [glutathione/oxidised glutathione] redox potential. Additionally, the mitochondrial membrane potential was reduced, apoptosis levels were elevated, as were markers of oxidative stress, including reactive oxygen species, superoxide and hydrogen peroxide. Furthermore, the basal and activated phosphorylated forms of the redox-sensitive transcription factor NF-κB were increased in cells with silenced Ctns. From this study, the cystinotic-like pancreatic ß-cell model demonstrated that the altered oxidative status of the cell, resulted in depressed mitochondrial function and pathways of ATP production, causing reduced nutrient-stimulated insulin secretion.

New developments concerning the proximal tubule in diabetic nephropathy: in vitro models and mechanisms.

The incidence of Type 2 diabetes is increasing rapidly worldwide, and understanding the mechanisms of its complications including diabetic nephropathy (DN) is important in the discovery of early biomarkers, understanding the causative mechanisms of its complications and identifying therapeutic targets. DN is characterized by glomerulosclerosis, tubulointerstitial fibrosis and tubular atrophy. The tubular component of the disease is important in progression of disease. In vitro models are a valuable alternative to animal studies and an effective way to explore mechanisms of human disease. Several proximal tubular cell lines have been used in studying mechanisms of DN. Key extracellular conditions that contribute to damage to the proximal tubule in DN include hyperglycaemia, proteinuria, and hypoxia and inflammation. According to current knowledge, these exert their effects through changes in transforming growth factor beta signalling, the renin-angiotensin system, dysregulation of pathways such as the polyol pathway, hexosamine pathway and protein kinase C pathway and through formation of advanced glycation end products. Studies in cell culture models have been instrumental in the delineation of these processes. However, all of the existing cell culture models have limitations including dedifferentiation. To bring research forward along with technological advances, such as major advances in 'omics' methodologies, a more suitable model is necessary. The RPTEC/TERT1 cell line is a promising alternative to previous proximal tubular epithelial cell lines due to features that resemble the cell type in vivo, such as its epithelial characteristics, maintenance of functional capabilities, glucose handling, expression of the primary cilium and transport activity including albumin. This cell line will facilitate identification of mechanisms of DN with potential to identify new therapeutic targets.

Urinary biomarkers of chronic allograft nephropathy.

PURPOSE: Chronic allograft nephropathy (CAN) is widely accepted as the leading cause of renal allograft loss after the first year post transplantation. This study aimed to identify urinary biomarkers that could predict CAN in transplant patients. EXPERIMENTAL DESIGN: The study included 34 renal transplant patients with histologically proven CAN and 36 renal transplant patients with normal renal function. OrbiTrap MS was utilized to analysis a urinary fraction in order to identify other members of a previously identified biomarker tree . This novel biomarker pattern offers the potential to distinguish between transplant recipients with CAN and those with normal renal function. RESULTS: The primary node of the biomarker pattern was reconfirmed as ß2 microglobulin. Three other members of this biomarker pattern were identified: neutrophil gelatinase-associated lipocalin, clusterin, and kidney injury biomarker 1. Significantly higher urinary concentrations of these proteins were found in patients with CAN compared to those with normal kidney function. CONCLUSIONS AND CLINICAL RELEVANCE: While further validation in a larger more-diverse patient population is required to determine if this biomarker pattern provides a potential means of diagnosing CAN by noninvasive methods in a clinical setting, this study clearly demonstrates the biomarkers' ability to stratify patients based on transplant function.

Chloride channel ClC-5 binds to aspartyl aminopeptidase to regulate renal albumin endocytosis.

ClC-5 is a chloride/proton exchanger that plays an obligate role in albumin uptake by the renal proximal tubule. ClC-5 forms an endocytic complex with the albumin receptor megalin/cubilin. We have identified a novel ClC-5 binding partner, cytosolic aspartyl aminopeptidase (DNPEP; EC 3.4.11.21), that catalyzes the release of N-terminal aspartate/glutamate residues. The physiological role of DNPEP remains largely unresolved. Mass spectrometric analysis of proteins binding to the glutathione-S-transferase (GST)-ClC-5 C terminus identified DNPEP as an interacting partner. Coimmunoprecipitation confirmed that DNPEP and ClC-5 also associated in cells. Further experiments using purified GST-ClC-5 and His-DNPEP proteins demonstrated that the two proteins bound directly to each other. In opossum kidney (OK) cells, confocal immunofluorescence studies revealed that DNPEP colocalized with albumin-containing endocytic vesicles. Overexpression of wild-type DNPEP increased cell-surface levels of ClC-5 and albumin uptake. Analysis of DNPEP-immunoprecipitated products from rat kidney lysate identified ß-actin and tubulin, suggesting a role for DNPEP in cytoskeletal maintenance. A DNase I inhibition assay showed a significant decrease in the amount of G actin when DNPEP was overexpressed in OK cells, suggesting a role for DNPEP in stabilizing the cytoskeleton. DNPEP was not present in the urine of healthy rats; however, it was readily detected in the urine in rat models of mild and heavy proteinuria (diabetic nephropathy and anti-glomerular basement membrane disease, respectively). Urinary levels of DNPEP were found to correlate with the severity of proteinuria. Therefore, we have identified another key molecular component of the albumin endocytic machinery in the renal proximal tubule and describe a new role for DNPEP in stabilizing the actin cytoskeleton.

Mechanisms of chemical carcinogenesis in the kidneys.

Chemical carcinogens are substances which induce malignant tumours, increase their incidence or decrease the time taken for tumour formation. Often, exposure to chemical carcinogens results in tissue specific patterns of tumorigenicity. The very same anatomical, biochemical and physiological specialisations which permit the kidney to perform its vital roles in maintaining tissue homeostasis may in fact increase the risk of carcinogen exposure and contribute to the organ specific carcinogenicity observed with numerous kidney carcinogens. This review will address the numerous mechanisms which play a role in the concentration, bioactivation, and uptake of substances from both the urine and blood which significantly increase the risk of cancer in the kidney.

High-Throughput Proteomic Approaches to the Elucidation of Potential Biomarkers of Chronic Allograft Injury (CAI).

This review focuses on the role of OMICs technologies, concentrating in particular on proteomics, in biomarker discovery in chronic allograft injury (CAI). CAI is the second most prevalent cause of allograft dysfunction and loss in the first decade post-transplantation, after death with functioning graft (DWFG). The term CAI, sometimes referred to as chronic allograft nephropathy (CAN), describes the deterioration of renal allograft function and structure as a result of immunological processes (chronic antibody-mediated rejection), and other non-immunological factors such as calcineurin inhibitor (CNI) induced nephrotoxicity, hypertension and infection. Current methods for assessing allograft function are costly, insensitive and invasive; traditional kidney function measurements such as serum creatinine and glomerular filtration rate (GFR) display poor predictive abilities, while the current "gold-standard" involving histological diagnosis with a renal biopsy presents its own inherent risks to the overall health of the allograft. As early as two years post-transplantation, protocol biopsies have shown more than 50% of allograft recipients have mild CAN; ten years post-transplantation more than 50% of the allograft recipients have progressed to severe CAN which is associated with diminishing graft function. Thus, there is a growing medical requirement for minimally invasive biomarkers capable of identifying the early stages of the disease which would allow for timely intervention. Proteomics involves the study of the expression, localization, function and interaction of the proteome. Proteomic technologies may be powerful tools used to identify novel biomarkers which would predict CAI in susceptible individuals. In this paper we will review the use of proteomics in the elucidation of novel predictive biomarkers of CAI in clinical, animal and in vitro studies.

Sgk-1 is a positive regulator of constitutive albumin uptake in renal proximal tubule cells.

BACKGROUND/AIMS: Receptor-mediated endocytosis of albumin by the renal proximal tubule requires a number of proteins including megalin/cubilin, sodium/hydrogen exchanger isoform 3 (NHE3) and ClC-5, as well as the PSD-95/Dlg/Zo-1 (PDZ) scaffold sodium/hydrogen exchanger regulatory factor 2 (NHERF2). Despite members from the AGC kinase family, v-Akt Murine Thymoma Viral Oncogene (Akt or Protein Kinase B) and Serum/Glucocorticoid regulated Kinase 1 (Sgk-1) regulating a number of essential proteins in the albumin handling pathway, their role in uptake is largely unknown. METHODS: Opossum kidney (OK) cells were exposed to Texas-Red albumin, in the presence of silencing constructs against Sgk-1, Akt and NHERF2, in addition to the NHE3 inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and NHE3 activator dexamethasone. Target protein was also measured by Western blot analysis in OK cells following exposure to dexamethasone and albumin. RESULTS: Silencing Sgk-1 or overexpression of a dominant negative mutant (DN-Sgk-1) led to a significant reduction of albumin endocytosis compared to control. Conversely, over-expression of wildtype (WT) or constitutively active (CA) Sgk-1 significantly increased uptake. Previous reports have shown Sgk-1 can activate NHE3 through an interaction mediated by NHERF2. We found that silencing both Sgk-1 and NHERF2 demonstrated no additive effect on uptake, suggesting signaling via similar endpoints. Treatment with dexamethasone increased Sgk-1 protein levels and increased albumin endocytosis in OK cells. Interestingly, silencing Akt also lead to a reduction in albumin endocytosis, however in cells silenced for both Sgk-1 and Akt, the additive change in albumin uptake demonstrated that these proteins may act via separate pathways. CONCLUSIONS: We have characterized a Sgk-dependent pathway that regulates albumin uptake in the proximal tubule which also includes NHE3 and NHERF2. These data provide further insights into this essential tubular process.

Oxidative stress induced by potassium bromate exposure results in altered tight junction protein expression in renal proximal tubule cells.

Potassium bromate (KBrO(3)) is an oxidising agent that has been widely used in the food and cosmetic industries. It has shown to be both a nephrotoxin and a renal carcinogen in in vivo and in vitro models. Here, we investigated the effects of KBrO(3) in the human and rat proximal tubular cell lines RPTEC/TERT1 and NRK-52E. A genome-wide transcriptomic screen was carried out from cells exposed to a sub-lethal concentration of KBrO(3) for 6, 24 and 72 h. Pathway analysis identified "glutathione metabolism", "Nrf2-mediated oxidative stress" and "tight junction (TJ) signalling" as the most enriched pathways. TJ signalling was less impacted in the rat model, and further studies revealed low transepithelial electrical resistance (TEER) and an absence of several TJ proteins in NRK-52E cells. In RPTEC/TERT1 cells, KBrO(3) exposure caused a decrease in TEER and resulted in altered expression of several TJ proteins. N-Acetylcysteine co-incubation prevented these effects. These results demonstrate that oxidative stress has, in conjunction with the activation of the cytoprotective Nrf2 pathway, a dramatic effect on the expression of tight junction proteins. The further understanding of the cross-talk between these two pathways could have major implications for epithelial repair, carcinogenesis and metastasis.

The role of MAPK in drug-induced kidney injury.

This paper focuses on the role that mitogen-activated protein kinases (MAPKs) play in drug-induced kidney injury. The MAPKs, of which there are four major classes (ERK, p38, JNK, and ERK5/BMK), are signalling cascades which have been found to be broadly conserved across a wide variety of organisms. MAPKs allow effective transmission of information from the cell surface to the cytosolic or nuclear compartments. Cross talk between the MAPKs themselves and with other signalling pathways allows the cell to modulate responses to a wide variety of external stimuli. The MAPKs have been shown to play key roles in both mediating and ameliorating cellular responses to stress including xenobiotic-induced toxicity. Therefore, this paper will discuss the specific role of the MAPKs in the kidney in response to injury by a variety of xenobiotics and the potential for therapeutic intervention at the level of MAPK signalling across different types of kidney disease.

Sirolimus enhances cyclosporine a-induced cytotoxicity in human renal glomerular mesangial cells.

End Stage Renal Disease (ESRD) is an ever increasing problem worldwide. However the mechanisms underlying disease progression are not fully elucidated. This work addressed nephrotoxicity induced by the immunosuppressive agents' cyclosporine A (CsA) and sirolimus (SRL). Nephrotoxicity is the major limiting factor in long term use of CsA. SRL causes less nephrotoxicity than CsA. Therefore investigations into the differential effects of these agents may identify potential mechanisms of nephrotoxicity and means to prevent ESRD induced by therapeutic drugs. Using ELISA, Western blotting, quantitative PCR and a reporter gene assay we detailed the differential effects of CsA and SRL in human renal mesangial cells. CsA treatment increased profibrotic TGF-ß1 secretion in human mesangial cells whereas SRL did not, indicating a role for TGF-ß in CsA toxicity. However we observed a synergistic nephrotoxic effect when CsA and SRL were co-administered. These synergistic alterations may have been due to an increase in CTGF which was not evident when the immunosuppressive drugs were used alone. The CsA/SRL combination therapy significantly enhanced Smad signalling and altered the extracellular matrix regulator matrix metalloproteinase 9 (MMP-9). Inhibition of the ERK 1/2 pathway, attenuated these CsA/SRL induced alterations indicating a potentially significant role for this pathway.

Carcinogens induce loss of the primary cilium in human renal proximal tubular epithelial cells independently of effects on the cell cycle.

The primary cilium is an immotile sensory and signaling organelle found on the majority of mammalian cell types. Of the multitude of roles that the primary cilium performs, perhaps some of the most important include maintenance of differentiation, quiescence, and cellular polarity. Given that the progression of cancer requires disruption of all of these processes, we have investigated the effects of several carcinogens on the primary cilium of the RPTEC/TERT1 human proximal tubular epithelial cell line. Using both scanning electron microscopy and immunofluorescent labeling of the ciliary markers acetylated tubulin and Arl13b, we confirmed that RPTEC/TERT1 cells express primary cilium upon reaching confluence. Treatment with the carcinogens ochratoxin A (OTA) and potassium bromate (KBrO(3)) caused a significant reduction in the number of ciliated cells, while exposure to nifedipine, a noncarcinogenic renal toxin, had no effect on primary cilium expression. Flow cytometric analysis of the effects of all three compounds on the cell cycle revealed that only KBrO(3) resulted in an increase in the proportion of cells entering the cell cycle. Microarray analysis revealed dysregulation of multiple pathways affecting ciliogenesis and ciliary maintenance following OTA and KBrO(3) exposure, which were unaffected by nifedipine exposure. The primary cilium represents a unique physical checkpoint with relevance to carcinogenesis. We have shown that the renal carcinogens OTA and KBrO(3) cause significant deciliation in a model of the proximal tubule. With KBrO(3), this was followed by reentry into the cell cycle; however, deciliation was not found to be associated with reentry into the cell cycle following OTA exposure. Transcriptomic analysis identified dysregulation of Wnt signaling and ciliary trafficking in response to OTA and KBrO(3) exposure.

Cyclosporine A--induced oxidative stress in human renal mesangial cells: a role for ERK 1/2 MAPK signaling.

Cyclosporine A (CsA) is a potent immunosuppressant used to prevent organ transplant rejection and in the treatment of autoimmune diseases. However, chronic CsA nephropathy is the major limiting factor to its widespread use. The exact mechanisms of CsA-induced renal damage remain to be fully elucidated. The objective of the current research was to examine whether CsA treatment induced any glomerular mesangial cell alterations. In this research goal, human mesangial cells (HMCs) were treated with CsA for various time points. CsA caused an increase in the production of reactive oxygen species (ROS). Microarray analysis of mesangial cells treated with CsA also indicated 282 dysregulated genes. Bioinformatic analysis of these 282 genes indicated enriched apoptotic oxidative stress, mitogen-activated protein kinase (MAPK), and transforming growth factor-ß signaling in response to CsA treatment. The focus of this study was directed on oxidative stress and MAPK signaling as potential novel mechanisms of CsA nephrotoxicity. One key contributor to oxidative stress, thioredoxin interacting protein, was significantly upregulated following CsA treatment. Inhibition of the MAPK pathway resulted in attenuation of the CsA-induced mesangial cell alterations. These findings suggest a major role for ROS, oxidative stress, and MAPK signaling in promoting CsA-induced glomerular dysfunction and subsequent nephrotoxicity.

Transcriptomic alterations induced by Ochratoxin A in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model.

Ochratoxin A (OTA) is a widely studied compound due to its role in renal toxicity and carcinogenicity. However, there is still no consensus on the exact mechanisms of toxicity or carcinogenicity. In the current study, we analysed the effect of OTA on three human renal proximal tubular models (human primary, RPTEC/TERT1 and HK-2 cells) and two rat renal proximal tubular models (rat primary and NRK-52E cells). Global transcriptomics analysis at two exposure times was performed to generate a set of 756 OTA sensitive genes. This gene set was then compared in more detail across all models and additionally to a rat in vivo renal cortex model. The results demonstrate a well-conserved response across all models. OTA resulted in deregulation of a number of pathways including cytoskeleton, nucleosome regulation, translation, transcription, ubiquitination and cell cycle pathways. Interestingly, the oxidative stress activated Nrf2 pathway was not enriched. These results point to an epigenetic action of OTA, perhaps initiated by actin binding as the actin remodelling gene, advillin was the highest up-regulated in all models. The largest model differences were observed between the human and the rat in vitro models. However, since the human in vitro models were more similar to the rat in vivo model, it is more likely that these differences are model-specific rather than species-specific per se. This study demonstrates the usefulness of in vitro cell culture models combined with transcriptomic analysis for the investigation of mechanisms of toxicity and carcinogenicity. In addition, these results provide further evidence supporting a non-genotoxic mechanism of OTA-induced carcinogenicity.

TGF-ß1 mediates sirolimus and cyclosporine A-induced alteration of barrier function in renal epithelial cells via a noncanonical ERK1/2 signaling pathway.

The immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) used in combination demonstrated beneficial effects in organ transplantation, but this combination can also result in increased adverse effects. We previously showed that not only CsA treatment but also its combination with SRL decreased paracellular permeability in renal proximal tubular cells by modification of the tight junction proteins, claudins, through ERK1/2 signaling pathway. In this present study, evidence is presented that not only CsA but also the combination of CsA/SRL may have adverse effects on the barrier function of renal proximal cells, at least in part, through the expression of the cytokine transforming growth factor (TGF)-ß(1). CsA treatment upregulated TGF-ß(1) gene expression and this upregulation was enhanced when CsA and SRL were applied together. Addition of TGF-ß(1) (5 ng/ml) altered the barrier function with increased transepithelial electrical resistance (TER) and claudin-1 expression. Use of a TGF-ß(1)-blocking antibody or blockage of TGF-ß(1) receptor kinase activity with SD208 prevented the CsA- and CsA/SRL-induced increase in TER. No evidence was found in the present studies to indicate that CsA or CsA/SRL treatment activated the TGF-ß(1) Smad canonical signaling pathway, whereas addition of TGF-ß(1) (5 ng/ml) did activate the Smad pathway. Addition of the ERK1/2 signaling inhibitor U0126 was able to prevent the TGF-ß(1)-mediated increase in TER and claudin expression. It is most likely that the CsA- and CsA/SRL-induced increases in TGF-ß(1) expression may not be sufficient to trigger the Smad pathway but however may trigger other TGF-ß(1) receptor-mediated signaling including the ERK1/2 signaling pathway.

Identification of novel indicators of cyclosporine A nephrotoxicity in a CD-1 mouse model.

The calcineurin inhibitor cyclosporine A (CsA) is a widely used immunosuppressive agent. However, nephrotoxicity is a serious side effect observed in patients which limits clinical use of CsA. CsA nephrotoxicity is associated with tubulointerstitial injury progressing to nephropathy. This is typically diagnosed by invasive renal biopsy and is often only detected when the disease process is well advanced. Therefore identification of novel, early indicators of CsA nephrotoxicity could be clinically advantageous. This study aimed to establish a murine model of CsA nephrotoxicity and to identify urinary proteins that may indicate the onset of CsA-induced nephropathy using 2-D gel electrophoresis. CsA nephrotoxicity was induced in CD-1 mice by daily CsA administration for 4weeks. By week 4, elevated serum creatinine and proteinuria were observed after CsA treatment indicating significant renal dysfunction. Decreased cadherin-1, increased α-smooth muscle actin and fibroblast specific protein 1 in kidney tissue indicated disruption of normal tubular architecture. Alterations in podocin and uromodulin were also observed which may indicate damage to other segments of the nephron. Proteomic analysis of urine identified a number of differentially regulated proteins that may be involved in early CsA nephropathy including cadherin 1, superoxide dismutase and vinculin. These findings suggest novel mechanisms of CsA nephrotoxicity and identify novel potential markers of the disease.