Metformin attenuates the effect of Staphylococcus aureus on airway tight junctions by increasing PKCζ-mediated phosphorylation of occludin.

Airway epithelial tight junction (TJ) proteins form a resistive barrier to the external environment, however, during respiratory bacterial infection TJs become disrupted compromising barrier function. This promotes glucose flux/accumulation into the lumen which acts as a nutrient source for bacterial growth. Metformin used for the treatment of diabetes increases transepithelial resistance (TEER) and partially prevents the effect of bacteria but the mechanisms of action are unclear. We investigated the effect of metformin and Staphylococcus aureus on TJ proteins, zonula occludins (ZO)-1 and occludin in human airway epithelial cells (H441). We also explored the role of AMP-activated protein kinase (AMPK) and PKCζ in metformin-induced effects. Pretreatment with metformin prevented the S. aureus-induced changes in ZO-1 and occludin. Metformin also promoted increased abundance of full length over smaller cleaved occludin proteins. The nonspecific PKC inhibitor staurosporine reduced TEER but did not prevent the effect of metformin indicating that the pathway may involve atypical PKC isoforms. Investigation of TJ reassembly after calcium depletion showed that metformin increased TEER more rapidly and promoted the abundance and localization of occludin at the TJ. These effects were inhibited by the AMPK inhibitor, compound C and the PKCζ pseudosubstrate inhibitor (PSI). Metformin increased phosphorylation of occludin and acetyl-coA-carboxylase but only the former was prevented by PSI. This study demonstrates that metformin improves TJ barrier function by promoting the abundance and assembly of full length occludin at the TJ and that this process involves phosphorylation of the protein via an AMPK-PKCζ pathway.

New perspectives in application of kidney biomarkers in mycotoxin induced nephrotoxicity, with a particular focus on domestic pigs.

The gradual spread of Aspergilli worldwide is adding to the global shortage of food and is affecting its safe consumption. Aspergillus-derived mycotoxins, including aflatoxins and ochratoxin A, and fumonisins (members of the fusariotoxin group) can cause pathological damage to vital organs, including the kidney or liver. Although the kidney functions as the major excretory system in mammals, monitoring and screening for mycotoxin induced nephrotoxicity is only now a developmental area in the field of livestock feed toxicology. Currently the assessment of individual exposure to mycotoxins in man and animals is usually based on the analysis of toxin and/or metabolite contamination in the blood or urine. However, this requires selective and sensitive analytical methods (e.g., HPLC-MS/MS), which are time consuming and expensive. The toxicokinetic of mycotoxin metabolites is becoming better understood. Several kidney biomarkers are used successfully in drug development, however cost-efficient, and reliable kidney biomarkers are urgently needed for monitoring farm animals for early signs of kidney disease. ß2-microglobulin (ß2-MG) and N-acetyl-ß-D-glucosaminidase (NAG) are the dominant biomarkers employed routinely in environmental toxicology research, while kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) are also emerging as effective markers to identify mycotoxin induced nephropathy. Pigs are exposed to mycotoxins due to their cereal-based diet and are particularly susceptible to Aspergillus mycotoxins. In addition to commonly used diagnostic markers for nephrotoxicity including plasma creatinine, NAG, KIM-1 and NGAL can be used in pigs. In this review, the currently available techniques are summarized, which are used for screening mycotoxin induced nephrotoxicity in farm animals. Possible approaches are considered, which could be used to detect mycotoxin induced nephropathy.

Assessing the validity and reliability of the MUST and MST nutrition screening tools in renal inpatients.

OBJECTIVE: The aim of this study was to determine the validity and reliability of the Malnutrition Universal Screening Tool (MUST) and the Malnutrition Screening Tool (MST) in hospital inpatients with renal disease. DESIGN: A cross-sectional and longitudinal study. SETTING: The study took place on 3 renal inpatient wards in a tertiary hospital in south London. PATIENTS: A total of 276 participants were recruited. INTERVENTION: Not applicable. MAIN OUTCOME MEASURE: Concurrent validity was assessed by comparing the MUST and MST tools completed by nursing staff with the subjective global assessment tool completed by dietetic staff. Predictive validity was evaluated by assessing the association between malnutrition and length of hospital stay. Mid-upper arm circumference and bioelectrical impedance spectroscopy were used to assess construct validity. In the reliability study, the MUST and MST tools were repeated on the same day by nursing staff. OBJECTIVE: MUST had a sensitivity of 53.8% (95% confidence interval [CI], 46.6% to 60.0%) and a specificity of 78.3% (95% CI, 70.1% to 85.2%), and MST had a sensitivity of 48.7% (95% CI, 41.7% to 54.0%) and a specificity of 85.5% (95% CI, 77.9 to 91.3) when compared with subjective global assessment. Risk of malnutrition as identified by MUST but not the MST tools had a significantly longer length of hospital stay (P = .038 and .061). Both MUST and MST tools identified patients at risk of malnutrition had a significantly lower mid-upper arm circumference (P = .005 and P = .029, respectively) and percent fat mass (P = .023 and P = .052, respectively). Reliability assessed by kappa was 0.58 for MUST (95% CI, 0.20 to 0.80) and 0.33 for MST (95% CI, -0.03 to 0.54). CONCLUSIONS: The MUST and MST nutrition tools are not sensitive enough to identify all of the malnourished renal inpatients, despite being fairly reliable and related to other nutrition status markers.

Nephrotoxic Biomarkers with Specific Indications for Metallic Pollutants: Implications for Environmental Health.

Environmental and occupational exposure to heavy metals and metalloids is a major global health risk. The kidney is often a site of early damage. Nephrotoxicity is both a major consequence of heavy metal exposure and potentially an early warning of greater damage. A paradigm shift occurred at the beginning of the 21st century in the field of renal medicine. The medical model of kidney failure and treatment began to give way to a social model of risk factors and prevention with important implications for environmental health. This development threw into focus the need for better biomarkers: markers of exposure to known nephrotoxins; markers of early damage for diagnosis and prevention; markers of disease development for intervention and choice of therapy. Constituents of electronic waste, e-waste or e-pollution, such as cadmium (Cd), lead (Pb), mercury (HG), arsenic (As) and silica (SiO2) are all potential nephrotoxins; they target the renal proximal tubules through distinct pathways. Different nephrotoxic biomarkers offer the possibility of identifying exposure to individual pollutants. In this review, a selection of prominent urinary markers of tubule damage is considered as potential tools for identifying environmental exposure to some key metallic pollutants.

High glucose-induced Smad3 linker phosphorylation and CCN2 expression are inhibited by dapagliflozin in a diabetic tubule epithelial cell model.

BACKGROUND: In the kidney glucose is freely filtered by the glomerulus and, mainly, reabsorbed by sodium glucose cotransporter 2 (SGLT2) expressed in the early proximal tubule. Human proximal tubule epithelial cells (PTECs) undergo pathological and fibrotic changes seen in diabetic kidney disease (DKD) in response to elevated glucose. We developed a specific in vitro model of DKD using primary human PTECs with exposure to high D-glucose and TGF-ß1 and propose a role for SGLT2 inhibition in regulating fibrosis. METHODS: Western blotting was performed to detect cellular and secreted proteins as well as phosphorylated intracellular signalling proteins. qPCR was used to detect CCN2 RNA. Gamma glutamyl transferase (GT) activity staining was performed to confirm PTEC phenotype. SGLT2 and ERK inhibition on high D-glucose, 25 mM, and TGF-ß1, 0.75 ng/ml, treated cells was explored using dapagliflozin and U0126, respectively. RESULTS: Only the combination of high D-glucose and TGF-ß1 treatment significantly up-regulated CCN2 RNA and protein expression. This increase was significantly ameliorated by dapagliflozin. High D-glucose treatment raised phospho ERK which was also inhibited by dapagliflozin. TGF-ß1 increased cellular phospho SSXS Smad3 serine 423 and 425, with and without high D-glucose. Glucose alone had no effect. Smad3 serine 204 phosphorylation was significantly raised by a combination of high D-glucose+TGF-ß1; this rise was significantly reduced by both SGLT2 and MEK inhibition. CONCLUSIONS: We show that high D-glucose and TGF-ß1 are both required for CCN2 expression. This treatment also caused Smad3 linker region phosphorylation. Both outcomes were inhibited by dapagliflozin. We have identified a novel SGLT2 -ERK mediated promotion of TGF-ß1/Smad3 signalling inducing a pro-fibrotic growth factor secretion. Our data evince support for substantial renoprotective benefits of SGLT2 inhibition in the diabetic kidney.

Urinary Biomarkers of Mycotoxin Induced Nephrotoxicity-Current Status and Expected Future Trends.

The intensifying world-wide spread of mycotoxigenic fungal species has increased the possibility of mycotoxin contamination in animal feed and the human food chain. Growing evidence shows the deleterious toxicological effects of mycotoxins from infants to adults, while large population-based screening programs are often missing to identify affected individuals. The kidney functions as the major excretory system, which makes it particularly vulnerable to nephrotoxic injury. However, few studies have attempted to screen for kidney injury biomarkers in large, mycotoxin-exposed populations. As a result, there is an urgent need to screen them with sensitive biomarkers for potential nephrotoxicity. Although a plethora of biomarkers have been tested to estimate the harmful effects of a wide spectrum of toxicants, ß2-microglobulin (ß2-MG) and N-acetyl-ß-D-glucosaminidase (NAG) are currently the dominant biomarkers employed routinely in environmental toxicology research. Nevertheless, kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) are also emerging as useful and informative markers to reveal mycotoxin induced nephrotoxicity. In this opinion article we consider the nephrotoxic effects of mycotoxins, the biomarkers available to detect and quantify the kidney injuries caused by them, and to recommend biomarkers to screen mycotoxin-exposed populations for renal damage.

Evaluation of Urinary Biomarkers of Proximal Tubular Injury, Inflammation, and Fibrosis in Patients With Albuminuric and Nonalbuminuric Diabetic Kidney Disease.

INTRODUCTION: Albuminuric and nonalbuminuric pathways contribute to diabetic kidney disease. Proximal tubule and inflammation play important roles in these processes. Urinary biomarker(s) to detect early kidney damage and predict progression are needed. METHODS: Nine urinary biomarkers were measured at baseline in 400 patients with diabetes. Correlation and multivariate logistic and linear regression analyses were performed to assess the association of biomarkers with chronic kidney disease and progression. RESULTS: In the albumin/creatinine ratio (ACR) <3 cohort, the only biomarker significantly associated with estimated glomerular filtration rate < 60 ml/min was N-acetyl-ß-d-glucosaminidase. A combination of ACR and monocyte chemoattractant protein 1 (MCP1) were significantly associated with stage 2 chronic kidney disease in this cohort. Logistic models showed that in patients with all levels of albuminuria, ACR, retinol binding protein (RBP), and MCP1 were associated with progression. A model including MCP1, interleukin 6, and neutrophil gelatinase-associated lipocalin showed significant association with progression to chronic kidney disease 3/4 in the ACR <3 cohort. Linear mixed-model regression analyses demonstrated MCP1, RBP, and ACR as significant proteins associated with progression to stage 3 or worse, whereas MCP1 was the only significant biomarker in the ACR <3 cohort. Time-to-event and Cox proportional hazard models confirmed significant hazard ratios for progression for ACR, RBP, and MCP1, with significant differences noted between quantiles of biomarkers for ACR, RBP, and MCP1. CONCLUSION: In this study of diabetic patients with single baseline measurements of urinary biomarkers, albumin, RBP, and MCP1 were significantly associated with chronic kidney disease progression at all levels of albuminuria. Inflammatory cytokines, neutrophil gelatinase-associated lipocalin, and MCP1 were associated with progression in patients without albuminuria. N-acetyl-ß-d-glucosaminidase demonstrated a significant association with an estimated glomerular filtration rate < 60 ml/min in the ACR <3 cohort.

Differential regulation of E-cadherin and alpha-smooth muscle actin by BMP 7 in human renal proximal tubule epithelial cells and its implication in renal fibrosis.

Chronic kidney diseases are characterized by progressive tubulointerstitial fibrosis, and TGFbeta1 plays a crucial role in its development. Bone morphogenic protein 7 (BMP 7), another member of the TGF superfamily, antagonized the profibrotic effects of TGFbeta1, including epithelial mesenchymal transition and E-cadherin loss, in the previous studies from animal models. We investigated the effect of BMP 7 on TGFbeta1-mediated E-cadherin loss in two different transformed human adult proximal tubule epithelia. We found that BMP 7 not only failed to prevent TGFbeta1-mediated E-cadherin loss but itself downregulated E-cadherin levels and that it had an additive effect with TGFbeta1 in inducing E-cadherin loss. The downregulation of E-cadherin by BMP 7 was mediated through the Smad1/5 pathway. BMP 7-mediated E-cadherin loss was not followed by de novo alpha-smooth muscle actin (alpha-SMA) expression (a marker of myofibroblastic phenotype), which was due to the concurrent induction of Inhibitor of DNA binding 1 (Id1, a basic helix loop helix class transcriptional regulator) through a non-Smad pathway. Concurrent treatment of BMP 7 and TGFbeta1 prevented TGFbeta1-mediated alpha-SMA induction. In summary, our results suggest that E-cadherin loss, the key feature of epithelial mesenchymal transition, will not necessarily be followed by total phenotype change; rather, cells may undergo some loss of phenotypic marker in a ligand-dependent manner and participate in reparative processes. The inhibition of de novo expression of alpha-SMA could explain the antifibrotic effect of BMP 7. Id1 might play a crucial role in maintaining proximal tubule epithelial cell phenotype and its signaling regulation could be a potential therapeutic target.

Tgf-beta auto-induction and connective tissue growth factor expression in human renal tubule epithelial cells requires N-ras.

BACKGROUND/AIMS: Transforming growth factor (TGF) beta is strongly implicated in the progression of renal fibrosis. TGFbeta1 is reported to cause epithelial-mesenchymal transition, inhibition of epithelial cell proliferation, increased apoptosis, auto-induction of TGFbeta production and induction of secondary mediators of tissue fibrosis such as connective tissue growth factor (CTGF, CCN2). The aims of this study were to investigate the role of the Ras/MAP kinase pathway in TGFbeta1 inhibition of proliferation, TGFbeta auto-induction and TGFbeta1-induced CTGF expression in HKC human renal tubule epithelial cells. METHODS AND RESULTS: TGFbeta1 (0-25 ng/ml) inhibited proliferation of HKC cells and at 25 ng/ml also induced apoptosis. After 5-10 min of incubation, TGFbeta1 increased cellular levels of phospho-ERK1/2 and phospho-AKT with a bell-shaped dose-response curve with a maximally effective concentration of 2.5 ng/ml. TGFbeta3 caused an increase in extracellular TGFbeta1, which was significantly reduced in the presence of PD 98059. TGFbeta1 increased cellular and secreted CTGF protein in HKC cells in a MEK-dependent manner. To identify the Ras isoform involved, specific antisense oligonucleotides targeted to Ha-Ras, Ki-Ras and N-Ras were employed. Only inhibition of N-Ras resulted in a significant reduction of auto-induced TGFbeta1 secretion and TGFbeta1-induced cellular and secreted CTGF. CONCLUSION: These results establish that the Ras/MAP kinase pathway, specifically through N-Ras, mediates TGFbeta1 auto-induction and TGFbeta1-induced CTGF expression in human renal tubule epithelial cells.

Role played by disabled-2 in albumin induced MAP Kinase signalling.

Albumin has been shown to activate the mitogen activated protein kinase (MAPK) pathway in proximal tubular cells (PTECs) of the kidney. Megalin, the putative receptor for albumin has potential signalling properties. However, the mechanisms by which megalin signals are unclear. The adaptor phosphoprotein Disabled-2 (Dab2) is known to interact with the cytoplasmic tail of megalin and may be involved in albumin-mediated MAPK signalling. In this study, we investigated the role of Dab2 in albumin-mediated MAPK signalling and further studied the role of Dab2 in albumin-induced TGFbeta-1 secretion, a MAPK dependent event. We used RNA interference to knockdown Dab2 protein abundance in HKC-8 cells a model of human PTECs. Albumin activated ERK1,2 and Elk-1 in a MEK-1 dependent manner and resulted in secretion of TGFbeta-1. In the absence of albumin, knockdown of Dab2 resulted in a trend towards increase in pERK1,2 consistent with its putative role as an inhibitor of cell proliferation. However albumin-induced ERK1,2 activation was completely abolished by Dab2 knockdown. Dab2 knockdown did not however result in inhibition of albumin-induced TGFbeta-1 secretion. These results suggest that Dab2 is a ligand dependent bi-directional regulator of ERK1,2 activity by demonstrating that in addition to its more traditional role as an inhibitor of ERK1,2 it may also activate ERK1,2.

TGF-beta activates ERK5 in human renal epithelial cells.

The role of the MAP kinase, extracellular signal-regulated kinase 5 (ERK5) remains unknown, however it is involved in cell differentiation and survival as highlighted by the embryonic lethality of the ERK5 knockout. ERK5 can be activated by growth factors and other extracellular signals. TGF-beta, a powerful controller of epithelial cell phenotype, is known to activate the MAP kinase, ERK1/2 however its effect on ERK5 remains unknown. This study demonstrates, fort the first time, ERK5 activation by TGF-beta, observed in both transformed and primary adult human PTEC; activation required ALK-5 receptor activity. In addition this work demonstrates expression of myocyte enhancer factor-2 (MEF2C) by PTEC and that TGF-beta increased the association of MEK5 with phospho-ERK5 and MEF2C. ERK5 activation by either TGF-beta or epidermal growth factor (EGF) was also inhibited by the p38 MAP kinase inhibitor, SB-202190.

Albumin induces interleukin-6 release from primary human proximal tubule epithelial cells.

BACKGROUND: Proximal tubule epithelial cells (PTECs) release proinflammatory and profibrogenic mediators when exposed to serum albumin that may contribute to progression of kidney disease. Interleukin 6 (IL-6) may influence renal fibrosis by modulating transforming growth factor beta1 (TGFbeta1) signalling. PTECs have been demonstrated to produce IL-6 in response to albumin treatment, but the mechanism has not been investigated. We hypothesized that albumin would induce release of IL-6 from PTECs, which would be sensitive to inhibition of PI3K, ERK1,2, p38 MAPK and NFkB. METHODS: Primary human PTECs were exposed to albumin (0.75-150 micronM) for 8 and 24 hours. IL-6 release was determined using enzyme-linked immunosorbent assay (ELISA). The effects of LY294002 (10 micronM), NH4Cl (10 mM), pyrrolidine dithiocarbamate (PDTC) (20 micronM), CAPE (17.5 micronM), PD098059 (20 micronM), SB202190 (5 micronM) and MG132 (10 micronM) on albumin-mediated IL-6 release were studied. RESULTS: Albumin caused a significant time- and concentration-dependent increase in IL-6 release by PTECs. LY294002, NH4Cl, CAPE, PD098059 and SB202190 all reduced albumin-mediated IL-6 release, but neither PDTC nor MG132 had any effect. CONCLUSIONS: These data demonstrate that albumin induces IL-6 release by primary human PTECs, and support a role for endocytosis, p38 MAPK, ERK1,2 and in this process.

The TGFbeta1-induced fibronectin in human renal proximal tubular epithelial cells is p38 MAP kinase dependent and Smad independent.

BACKGROUND/AIM: Transforming growth factor beta 1 (TGFbeta1) is a fibrokine implicated in the progression of renal fibrosis. Following TGFbeta1 receptor activation, a number of signalling pathways are stimulated. This study investigates the role of p38 mitogen-activated protein (MAP) kinase and Smad pathways in the TGFbeta1-induced fibronectin (FN) production. METHODS: Transformed human proximal tubular epithelial cells of the line HKC were used. Secreted FN was analyzed by enzyme-linked immunosorbent assay and Smad proteins by Western blotting. Chemical inhibitors were used to study the role of p38 MAP kinase and the TGFbeta receptor ALK5. The Smad pathway was studied using a cell line overexpressing Smad7 and small interfering RNAs (siRNA). The FN mRNA expression was assessed by reverse transcription-polymerase chain reaction. RESULTS: TGFbeta1 produced a significant increase in FN secretion in both HKC and Smad7-HKC cells, and the p38 MAP kinase inhibitor SB202190 markedly reduced this (n = 3, p < 0.05 and p < 0.01). ALK5 inhibition also reduced the TGFbeta1-induced FN (n = 3, p < 0.05). Smad knockdown using the siRNA did not reduce the TGFbeta1-induced FN secretion. TGFbeta1 induced FN mRNA expression in HKC cells, and SB202190 decreased this induction (n = 5, p < 0.05). CONCLUSIONS: These results suggest that TGFbeta1-induced FN production in HKC cells is p38 MAP kinase dependent and Smad independent. Targeting p38 MAP kinase may be of therapeutic value in renal fibrosis.

A practical method of measuring glomerular filtration rate by iohexol clearance using dried capillary blood spots.

BACKGROUND: Exogenous tracer-based methods of measuring glomerular filtration rate (GFR) are difficult to perform, whilst creatinine-based estimation formulae are inaccurate. METHODS: We assessed a new technique of measuring iohexol clearance using timed dried capillary blood spots. A reference GFR was measured in 81 subjects (GFR 15-124 ml/min/1.73 m(2)) by iohexol clearance using three venous samples (2, 3 and 4 h after an intravenous bolus). GFR was estimated by six test methods; iohexol clearance using (i) 3 blood spots (2, 3, 4 h); (ii) 2 blood spots (2, 4 h) and (iii) 1 blood spot (4 h); (iv) the Modification of Diet in Renal Disease (MDRD) formula; (v) the Cockcroft-Gault formula, and (vi) a formula estimating GFR from serum cystatin C concentration. For each test method the bias and precision were calculated as the mean and standard deviation (SD) of the 'GFR differences' (test method GFR - reference GFR). RESULTS: The limits of agreement (bias +/-1.96 x SD; in ml/min/1.73 m(2)) were: (i) 1.1 +/- 15.1 for 3-spot iohexol clearance; (ii) 0.6 +/- 14.9 for 2-spot iohexol clearance; (iii) 4.5 +/- 21.2 for 1-spot iohexol clearance; (iv) -15.7 +/- 33.3 for the MDRD formula; (v) -9.6 +/- 32.9 for the Cockcroft-Gault formula, and (vi) -12.1 +/- 31.7 for the Cystatin C formula. The accuracy of all six test methods was similar among individuals with GFR <60 ml/min/ 1.73 m(2); however, in individuals with GFR > or =60 ml/min/ 1.73 m(2), the MDRD, Cockcroft-Gault and Cystatin C formulae were all imprecise and systematically underestimated GFR. CONCLUSIONS: Blood spot iohexol clearance provides a potentially practical method of estimating GFR accurately in large-scale epidemiological studies especially among individuals without established chronic kidney disease.

The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFbeta1 responses in human proximal-tubule epithelial cells.

In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGFbeta1-Smad (transforming growth factor-beta1-Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial-mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGFbeta are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, alpha-SMA (alpha-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGFbeta1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, alpha-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGFbeta1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and alpha-SMA, and decreased expression of E-cadherin. TGFbeta1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in alpha-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGFbeta1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulo-interstitial fibrosis.

TGF-beta1-induced connective tissue growth factor (CCN2) expression in human renal proximal tubule epithelial cells requires Ras/MEK/ERK and Smad signalling.

BACKGROUND: Connective tissue growth factor (CTGF, CCN2) plays a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and mediating many of the pro-fibrotic effects of transforming growth factor (TGF)-beta. CCN2 induction by TGF-beta in renal proximal tubule epithelial cells (PTECs) is likely to play an important role in the development of tubulointerstitial fibrosis. In this study, we investigated the induction of CCN2 by TGF-beta1 and the possible mechanisms of this induction in human PTECs. METHODS: Experiments were performed on primary and transformed (human kidney cell (HKC)-clone 8) human PTECs. Induction of CCN2 in response to TGF-beta1 was studied at the gene promoter level by reporter gene assay, mRNA by semi-quantitative RT-PCR and protein by immunoblotting. While chemical inhibitors were used to assess the role of Ras/MEK/ERK1,2 signalling, an HKC cell line over-expressing Smad7 was used to assess the role of Smad signalling in induction of CCN2 by TGF-beta1. RESULTS: TGF-beta1 induced CCN2 promoter activity, mRNA and protein in human PTECs. TGF-beta1-dependent CCN2 promoter activity was reduced by inhibiting Ras and MEK activation. MEK inhibition also resulted in inhibition of the TGF-beta1-induced secreted CCN2 protein. There was no significant increase in CCN2 gene promoter activity or protein by TGF-beta1 in Smad7 over-expressing HKCs. CONCLUSIONS: TGF-beta1 induces the expression of CCN2 in human PTECs. This induction is dependent on Ras/MEK/ERK and Smad signalling. Inhibiting TGF-beta induced CCN2 by targeting Smad and/or Ras/MEK/ERK1,2 signalling pathways could be of therapeutic value in renal fibrosis.

Erk5 is a mediator to TGFß1-induced loss of phenotype and function in human podocytes.

BACKGROUND: Podocytes are highly specialized cells integral to the normal functioning kidney, however, in diabetic nephropathy injury occurs leading to a compromised phenotype and podocyte dysfunction which critically produces podocyte loss with subsequent renal impairment. TGFß1 holds a major role in the development of diabetic nephropathy. Erk5 is an atypical mitogen-activated protein (MAP) kinase involved in pathways modulating cell survival, proliferation, differentiation, and motility. Accordingly, the role of Erk5 in mediating TGFß1-induced podocyte damage was investigated. METHODS: Conditionally immortalized human podocytes were stimulated with TGFß1 (2.5 ng/ml); inhibition of Erk5 activation was conducted with the chemical inhibitor BIX02188 (10 µM) directed to the upstream Mek5; inhibition of Alk5 was performed with SB431542 (10 µM); Ras signaling was inhibited with farnesylthiosalicylic acid (10 µM). Intracellular signaling proteins were investigated by western blotting; phenotype was explored by immunofluorescence; proliferation was assessed with a MTS assay; motility was examined with a scratch assay; barrier function was studied using electric cell-substrate impedance sensing; apoptosis was studied with annexin V-FITC flow cytometry. RESULTS: Podocytes expressed Erk5 which was phosphorylated by TGFß1 via Mek5, whilst not involving Ras. TGFß1 altered podocyte phenotype by decreasing P-cadherin staining and increasing α-SMA, as well as reducing podocyte barrier function; both were prevented by inhibiting Erk5 phosphorylation with BIX02188. TGFß1-induced podocyte proliferation was prevented by BIX02188, whereas the induced apoptosis was not. Podocyte motility was reduced by BIX02188 alone and further diminished with TGFß1 co-incubation. CONCLUSION: These results describe for the first time the expression of Erk5 in podocytes and identify it as a potential target for the treatment of diabetic renal disease.

Smad mediated regulation of inhibitor of DNA binding 2 and its role in phenotypic maintenance of human renal proximal tubule epithelial cells.

The basic-Helix-Loop-Helix family (bHLH) of transcriptional factors plays a major role in regulating cellular proliferation, differentiation and phenotype maintenance. The downregulation of one of the members of bHLH family protein, inhibitor of DNA binding 2 (Id2) has been shown to induce de-differentiation of epithelial cells. Opposing regulators of epithelial/mesenchymal phenotype in renal proximal tubule epithelial cells (PTEC), TGFß1 and BMP7 also have counter-regulatory effects in models of renal fibrosis. We investigated the regulation of Id2 by these growth factors in human PTECs and its implication in the expression of markers of epithelial versus myofibroblastic phenotype. Cellular Id2 levels were reduced by TGFß1 treatment; this was prevented by co-incubation with BMP7. BMP7 alone increased cellular levels of Id2. TGFß1 and BMP7 regulated Id2 through Smad2/3 and Smad1/5 dependent mechanisms respectively. TGFß1 mediated Id2 suppression was essential for α-SMA induction in PTECs. Although Id2 over-expression prevented α-SMA induction, it did not prevent E-cadherin loss under the influence of TGFß1. This suggests that the loss of gate keeper function of E-cadherin alone may not necessarily result in complete EMT and further transcriptional re-programming is essential to attain mesenchymal phenotype. Although BMP7 abolished TGFß1 mediated α-SMA expression by restoring Id2 levels, the loss of Id2 was not sufficient to induce α-SMA expression even in the context of reduced E-cadherin expression. Hence, a reduction in Id2 is critical for TGFß1-induced α-SMA expression in this model of human PTECs but is not sufficient in it self to induce α-SMA even in the context of reduced E-cadherin.

Primary culture of human renal proximal tubule epithelial cells and interstitial fibroblasts.

Renal physiology and pathology are complex systems that are best studied in whole living organisms. This, however, is often restricted by our desire to limit the number of animal experiments undertaken and to replace them with relevant in vitro models that can be used as surrogates for the system under test. Primary culture cells are derived directly from the relevant tissue and therefore correlate more closely with the system under examination. Although the tissue of origin is not always readily available for culture and cells may quickly change their phenotype after only a few passages, they can be used in many circumstances to validate results obtained from closely related cell lines and to confirm vital protein expression patterns. This chapter outlines methods by which proximal tubular epithelial cells and renal interstitial fibroblasts can be isolated and characterized from human renal nephrectomy tissue.

BMP-7 and proximal tubule epithelial cells: activation of multiple signaling pathways reveals a novel anti-fibrotic mechanism.

PURPOSE: Bone morphogenic protein-7 (BMP-7) is a member of the transforming growth factor beta (TGFbeta) superfamily involved in organogenesis. Recent work suggests that BMP-7 can reverse the fibrotic effects of TGFbeta but the underlying mechanism is unknown. We sought to determine BMP-7 signaling and its modulation of TGFbeta induced fibrotic outcomes in adult human proximal tubule epithelial cells (PTECs). METHODS: The effect of BMP-7 on phospho-p38 was assessed by Western blotting, p38 ELISA and Bio-plex phospho-protein assay. Secreted fibronectin (Fn) was measured by ELISA. RESULTS: BMP-7 had a concentration-dependent effect on intracellular signaling activating Smad 1/5/8 at higher concentrations and p38 mitogen activated protein (MAP) kinase at lower concentrations in both primary and transformed PTECs; BMP-7 caused phosphorylation of p38 at 2.5 ng/ml and Smads at 200 ng/ml. Similarly, nuclear accumulation of phospho-p38 and Smad were observed at these respective concentrations. These results suggested an inverse relationship between activation of Smads and p38 MAP kinase in this context. Consistent, with this BMP7 at 200 ng/ml reduced TGFbeta-induced p38 MAP activation and the p38-dependent TGFbeta-induced Fn secretion by PTECs. CONCLUSION: We have shown novel p38/Smad signaling along a BMP-7 gradient and demonstrated BMP-7 regulation of TGFbeta MAP kinase signaling and fibrotic outcomes.