The role of urinary N-acetyl-ß-D-glucosaminidase in early detection of acute kidney injury among pediatric patients with neoplastic disorders in a retrospective study.

BACKGROUND: The 1-year cumulative incidence of AKI reportedly is high (52%) in pediatric neoplastic disorders. About half of these events occur within 2 weeks. However, subclinical AKI episodes may remain unrecognized by the conventional creatinine-based approaches. We investigated the diagnostic value of urinary N-acetyl-ß-D-glucosaminidase (uNAG) as an early marker of acute kidney injury (AKI). METHODS: In our retrospective study, 33 children with neoplastic disorders were inculded who had serial uNAG tests (at least 5 samples/patient) with a total of 367 uNAG measurements. Renal function was determined by cystatin-C and creatinine based GFR, and relative increase of uNAG index (uNAGRI). We focused on detecting both clinical and subclinical AKI episodes (according to Biomarker-Guided Risk Assessment using pRIFLE criteria and /or elevated uNAG levels) and the incidence of chronic kidney damage. RESULTS: Sixty episodes in 26 patients, with positivity at least in one parameter of kidney panel, were identified during the observation period. We detected 18/60 clinical and 12/60 subclinical renal episodes. In 27/60 episodes only uNAG values was elevated with no therapeutic consequence at presentation. Two patients were detected with decreased initial creatinine levels with 3 "silent" AKI. In 13 patients, modest elevation of uNAG persisted suggesting mild, reversible tubular damage, while chronic tubuloglomerular injury occurred in 5 patients. Based on ROC analysis for the occurence of AKI, uNAGRI significantly indicated the presence of AKI, the sensitivity and specificity are higher than the changes of GFRCreat. Serial uNAG measurements are recommended for  the reduction of the great amount of false positive uNAG results, often due to overhydratation. CONCLUSION: Use of Biomarker-guided Risk Assessment for AKI identified 1.5 × more clinical and subclinical AKI episodes than with creatinine alone in our pediatric cancer patients. Based on the ROC curve for the occurence of AKI, uNAGRI has relatively high sensitivity and specificity comparable to changes of GFRCysC. The advantage of serial uNAG measurements is to decrease the number of false positive results. TRIAL REGISTRATION: The consent to participate is not applicable because it was not reqired for ethical approval and it is a retrospectiv study.

Urinary retinol binding protein predicts renal outcome in systemic immunoglobulin light-chain (AL) amyloidosis.

Renal risk stratification in systemic immunoglobulin light-chain (AL) amyloidosis is according to estimated glomerular filtration rate (eGFR) and urinary protein creatinine ratio (uPCR), the latter attributed to glomerular dysfunction, with proximal tubular dysfunction (PTD) little studied. Urinary retinol binding protein 4 (uRBP), a low molecular weight tubular protein and highly sensitive marker of PTD, was prospectively measured in 285 newly diagnosed, untreated patients with systemic AL amyloidosis between August 2017 to August 2018. At diagnosis, the uRBP/creatinine ratio (uRBPCR) correlated with serum creatinine (r = 0·618, P < 0·0001), uPCR (r = 0·422, P < 0·0001) as well as both fractional excretion of phosphate and urate (r = 0·563, P < 0·0001). Log uRBPCR at diagnosis was a strong independent predictor of end-stage renal disease {hazard ratio [HR] 2·65, [95% confidence interval (CI) 1·06-6·64]; P = 0·038}, particularly in patients with an eGFR >30 ml/min/1.73 m2 [HR 4·11, (95% CI 1·45-11·65); P = 0·008] and those who failed to achieve a deep haematological response to chemotherapy within 3 months of diagnosis [HR 6·72, (95% CI 1·83-24·74); P = 0·004], and also predicted renal progression [HR 1·91, (95% CI 1·18-3·07); P = 0·008]. Elevated uRBPCR indicates PTD and predicts renal outcomes independently of eGFR, uPCR and clonal response in systemic AL amyloidosis. The role of uRBPCR as a novel prognostic biomarker merits further study, particularly in monoclonal gammopathies of renal significance.

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.

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.

TGFß1-mediated PI3K/Akt and p38 MAP kinase dependent alternative splicing of fibronectin extra domain A in human podocyte culture.

Alternative splicing is an important gene regulation process to distribute proteins in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin (Fn) protein, present in the extra cellular matrix (ECM) and a recognised marker of various pathologies. TGFß1 has been shown to induce alternative splicing of EDA+Fn in many cell types. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. In our previous study we have demonstrated expression and alternative splicing of EDA+Fn in basal condition in human podocytes culture. TGFß1 further induced the basal expression and alternative splicing of EDA+Fn through Alk5 receptor and SR proteins. In this study, we have investigated TGFß1 mediated signalling involved in alternative splicing of EDA+Fn in human podocytes. We have performed western blotting to characterise the expression of the EDA+Fn protein and other signalling proteins and RT-PCR to look for signalling pathways involved in regulation of alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We have used TGFß1 as a stimulator and SB431542, SB202190 and LY294002 for inhibitory studies. In this work, we have demonstrated in human podocytes culture TGFß1 2.5ng/ml induced phosphorylation of Smad1/5/8, Smad2 and Smad3 via the ALK5 receptor. TGFß1 significantly induced the PI3K/Akt pathway and the PI3K/Akt pathway inhibitor LY294002 significantly downregulated basal as well as TGFß1 induced alternative splicing of EDA+Fn in human podocytes. In addition to this, TGFß1 significantly induced the p38 MAP kinase signalling pathway and p38 MAP kinase signalling pathway inhibitor SB202190 downregulated the TGFß1-mediated alternative splicing of EDA+Fn in human podocytes. The results with PI3K and p38 MAP kinase signalling pathway suggest that inhibiting PI3K signalling pathway downregulated the basal alternative splicing of EDA+Fn in human podocytes and its the inhibition of p38 Map Kinase signalling pathway which had specifically downregulated the TGFß1 mediated alternative splicing of EDA+Fn in human podocytes culture. Activation of TGFß1-mediated Smad1/5/8 via Alk5 receptor suggests that TGFß1 signalling pathway involved Alk5/Alk1 receptor axis signalling in human podocytes.

CCN3, a key matricellular protein, distinctly inhibits TGFß1-mediated Smad1/5/8 signalling in human podocyte culture.

Growth factors like TGFß and CTGF (CCN2) plays a vital role in various cellular functions. TGFß and CTGF are overexpressed in renal fibrosis. CTGF act as profibrotic stimuli to TGFß. CCN3 is a member of CCN family which also comprises CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2) and CCN6 (WISP-3). CCN3 has been shown to antagonise CTGF. In this study, we investigated the role of CCN3 in TGFß1-mediated signalling in human podocytes culture. This study describes the novel function of CCN3 in regulation of TGFß1 mediated non-canonical Smad signalling in human podocytes culture. Experiments were conducted on conditionally immortalised human podocytes incubated with TGFß1 (1.25ng/ml and 2.5ng/ml) and CCN3 (360ng/ml). Western blot study was performed to study signalling proteins. RT-PCR was performed to study alternative splicing of Fibronectin (Fn). Real time PCR was performed to look for gene expression of Fn and collagen IV and collagen I. TGFß1 induced the Smad1/5/8, Smad3 and p38 phosphorylation and CCN3 downregulated the TGFß1 induced Smad1/5/8 phosphorylation and did not affect Smad3 and p38 phosphorylation. In addition to this CCN3 induced alternative splicing of Extra domain A Fibronectin (EDA+Fn). CCN3 also induced collagen IV, Collagen I and Fn gene expression. This is the first evidence of downregulation of TGFß-mediated activation of a Smad1/5/8 signalling pathway by CCN3 in human podocytes and in any cell type. Targeting CCN3-mediated events could provide exciting outcomes in the understanding of molecular mechanism of fibrosis.

The Regulation of TGFß1 Induced Fibronectin EDA Exon Alternative Splicing in Human Renal Proximal Tubule Epithelial Cells.

The EDA+ splice variant of fibronectin (Fn) is an early and important component of the extracellular matrix in renal fibrosis. In this work, we investigate cellular mechanisms of EDA+Fn production in human primary proximal tubule epithelial cells (PTECs). TGFß1-induced EDA+Fn production was assessed by immunocytochemistry, PCR, and Western blotting. SRp40 knockdown was achieved by siRNA. The role of the PI3 kinase-AKT signalling and splicing regulatory protein SRp40 in the production of EDA+Fn was studied by using the chemical inhibitor LY294002 and siRNA targeted to SRp40 respectively. Interaction between PI3 kinase-AKT signalling and SRp40 were assessed by immunofluorescence and immunoprecipitation. To assess the specificity of SRp40 in regulating the splicing of EDA+ exon, we studied the effect of SRp40 knockdown on TGFß1 induced splicing of FGF receptor 2. Primary human PTECs expressed EDA+ and EDA- Fn. TGFß1 treatment resulted in increases in the production and deposition of EDA+ Fn as well as an increase in the ratio of EDA+/EDA- Fn mRNA. The TGFß1 induced EDA+ production was dependent on PI3 kinase-AKT signalling and SRp40 expression. Immunoprecipitation experiments demonstrated direct binding between AKT and SRp40 with an increase in the amount of SRp40 bound to AKT upon TGFß1 treatment. TGFß1 treatment resulted in reduction in the FGF receptor2 IIIb splice variant which was unaffected by SRp40 knockdown. In this work, we have presented the first evidence for the regulation of Fn pre-mRNA splicing by PI3 kinase-AKT signalling and SRp40 in human PTECs. Targeting the splicing of Fn pre-mRNA to skip the EDA exon is an attractive option to combat fibrosis.

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.

Genetic loci influencing kidney function and chronic kidney disease.

Using genome-wide association, we identify common variants at 2p12-p13, 6q26, 17q23 and 19q13 associated with serum creatinine, a marker of kidney function (P = 10(-10) to 10(-15)). Of these, rs10206899 (near NAT8, 2p12-p13) and rs4805834 (near SLC7A9, 19q13) were also associated with chronic kidney disease (P = 5.0 x 10(-5) and P = 3.6 x 10(-4), respectively). Our findings provide insight into metabolic, solute and drug-transport pathways underlying susceptibility to chronic kidney disease.

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.

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.

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.

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.

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 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.

The role played by endocytosis in albumin-induced secretion of TGF-beta1 by proximal tubular epithelial cells.

Proteinuria predicts the decline of renal function in chronic kidney disease. Reducing albuminuria has been shown to be associated with a reduction in this rate of decline. Proximal tubular epithelial cells (PTECs), when exposed to albumin produce matrix proteins, proinflammatory and profibrotic cytokines like TGF-beta(1). Some of these effects are dependent on endocytosis of albumin by PTECs. However, conditions like diabetic nephropathy, believed to be associated with reduced albumin endocytosis, are associated with interstitial fibrosis. Moreover, megalin, the putative albumin binding receptor in PTECs, has potential signaling motifs in its cytoplasmic domain, suggesting its ability to signal in response to ligand binding from the apical surface of PTECs. Hence, we looked to see whether albumin-induced secretion of TGF-beta(1) by PTECs is dependent on albumin endocytosis or whether it could occur in the absence of albumin endocytosis. We studied the production of TGF-beta(1) in two accepted models of PTECs, opossum kidney cells and human kidney cell clone-8 cells, with widely varying degrees of endocytosis. We then studied the effect of inhibiting albumin endocytosis with various inhibitors on albumin-induced TGF-beta(1) secretion. Our results indicate that albumin-induced TGF-beta(1) secretion by PTECs does not require albumin endocytosis and therefore the mechanism for the induction of some profibrotic responses by albumin may differ from those required for some of the inflammatory responses. Moreover, we found that albumin-induced TGF-beta(1) secretion by PTECs is not dependent on its interaction with megalin.

Prenylation is not necessary for endogenous Ras activation in non-malignant cells.

Ras monomeric GTPases are pivotal to many core cellular processes such as proliferation and differentiation. The post-translational prenylation of Ras with a farnesyl or a geranylgeranyl moiety is thought to be critical for its membrane binding and consequent signaling activity. Inhibitors of Ras prenylation have an anti-proliferative effect in some Ras-transformed cells. We present a study of the effects of prenylation inhibitors on endogenous, wild-type Ras in three renal cell types, namely primary adult human renal fibroblasts, primary adult human mesangial cells, and a primate renal fibroblast cell line (Vero cells). We have previously demonstrated that Ras is necessary for normal proliferation in these cells. Here we show that Ras is farnesylated and not geranylgeranylated in all three cell types. Furthermore, inhibiting Ras farnesylation has no effect on cell proliferation or Ras activation. Although inhibiting geranylgeranylation in these cells does inhibit proliferation, this is through an Ras-independent mechanism. Non-prenylated Ras is able to localize to the plasma membrane, bind Raf when cells are stimulated by epidermal growth factor or platelet-derived growth factor, and activate the Ras downstream effectors mitogen-activated protein kinase and phosphotidylinositol 3-kinase. We conclude that in wild-type cells, endogenous Ras does not need to be prenylated to be active.

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.