Targeting alternative splicing of fibronectin in human renal proximal tubule epithelial cells with antisense oligonucleotides to reduce EDA+ fibronectin production and block an autocrine loop that drives renal fibrosis.

TGFß1 is a powerful regulator of fibrosis; secreted in a latent form, it becomes active after release from the latent complex. During tissue fibrosis, the EDA + isoform of cellular fibronectin is overexpressed. In pulmonary fibrosis it has been proposed that the fibronectin splice variant including an EDA domain (FN EDA+) activates latent TGFß. Our work investigates the potential of blocking the 'splicing in' of EDA with antisense oligonucleotides to inhibit TGFß1-induced EDA + fibronectin and to prevent the cascade of events initiated by TGFß1 in human renal proximal tubule cells (PTEC). Human primary PTEC were treated with TGFß1 for 48 h, medium removed and the cells transfected with RNase H-independent antisense oligonucleotides (ASO) designed to block EDA exon inclusion (ASO5). The efficacy of ASO to block EDA exon inclusion was assessed by EDA + fibronectin RNA and protein expression; the expression of TGFß, αSMA (α smooth muscle actin), MMP2 (matrix metalloproteinse-2), MMP9 (matrix metalloproteinse-9), Collagen I, K Cadherin and connexin 43 was analysed. Targeting antisense oligonucleotides designed to block EDA exon inclusion in fibronectin pre mRNA were effective in reducing the amount of TGFß1 -induced cellular EDA + fibronectin RNA and secreted EDA + fibronectin protein (assessed by western immunoblotting and immunocytochemistry) in human proximal tubule cells in an in vitro cell culture model. The effect was selective for EDA + exon with no effect on EDB + fibronectin RNA and total fibronectin mRNA. Exogenous TGFß1 induced endogenous TGFß, αSMA, MMP2, MMP9 and Col I mRNA. TGFß1 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGFß1-induced pro-fibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGFß, further increases in αSMA, MMP2, MMP9 was observed; ASO5 significantly inhibited this subsequent increase. ASO5 treatment also significantly inhibited ability of the cell culture medium harvested at the end of the experiment (96h) to stimulate SMAD3 reporter cells. The role of endogenous TGFß1 was confirmed by the use of a TGFß receptor inhibitor. Our results demonstrate a critical role of FN EDA+ in a cycle of TGFß driven pro-fibrotic responses in human PTEC and blocking its production with ASO technology offers a potential therapy to interrupt this vicious circle and hence limit the progression of renal fibrosis.

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.

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.

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 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.

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.

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.

Time-resolved fluorescence immunoassay for urine retinol-binding protein is more sensitive than polyclonal and monoclonal assays.

BACKGROUND: Retinol-binding protein4 (RBP) assays using polyclonal antibodies (pRBP) have major problems of non-linearity of dilution and a very small useable dynamic range. Our objective was to develop a specific assay with a wider dynamic range to detect tubular proteinuria. METHODS: mRBP (monoclonal capture and second antibody with colorimetric detection) and fluoroimmunoassays for RBP (fRBP) (polyclonal capture and monoclonal second antibody with fluorescence detection) were developed and compared with pRBP. Four hundred and eighty-eight patient samples were collected; 290 samples were analysed by mRBP and 198 samples with fRBP and compared with pRBP. RESULTS: mRBP assay has the advantages of better linearity on dilution and wider analytical range over pRBP. It is limited by poor signal in the patients with albuminuria and glomerular proteinuria and inferior discrimination between patient groups. fRBP had an intra-assay and inter-assay CV of <6% and <8%, respectively, and analytical range was 2.3-599 µg/L. fRBP was linear on dilution within the analytical range. Correlation (r) was 0.8722 (95% CI 0.7621 to 0.9333, P< 0.0001); Mann-Whitney test revealed no significant difference (U = 18,877, n = 198, P = 0.5244) asserting that the medians of the two samples were identical. Bland-Altman test between pRBP and fRBP showed a mean negative bias of 16.43 (CI -994 to 1027) µg/mmol. CONCLUSIONS: The combination assay with fluorescence detection (fRBP) proved more discriminatory than a purely monoclonal system especially in patients with significant proteinuria and has advantages of better linearity on dilution and wider analytical range than the existing pRBP assay and compared extremely well with pRBP.

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.

TGFß1-mediated expression and alternative splicing of Fibronectin Extra Domain A in human podocyte culture.

Alternative splicing is a fundamental phenomenon to build protein diversity in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin protein present in the extra cellular matrix (ECM) in renal fibrosis. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. TGFß1 is a strong stimulator of ECM proteins in renal injury. In this study, we have investigated alternative splicing of EDA+ Fn in human podocytes in response to TGFß1. We have performed western blotting and immunofluorescence to characterise the expression of the EDA+Fn protein, real-time PCR for RNA expression and RT-PCR to look for alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We used TGFß1 as a stimulator and SB431542 and SRPIN340 for inhibitory studies. In this work, for the first time we have demonstrated in human podocytes culture EDA+Fn is expressed in the basal condition and TGFß1 2.5ng/ml induced the Fn mRNA and EDA+Fn protein expression demonstrated by real-time PCR, western blotting and immunofluorescence. TGFß1 2.5ng/ml induced the alternative splicing of EDA+Fn shown by conventional RT-PCR. Studies with ALK5 inhibitor SB431542 and SRPIN340 show that TGFß1 induced alternative splicing of EDA+Fn was by the ALK5 receptor and the SR proteins.  In human podocytes culture, alternative splicing of EDA+Fn occurs at basal conditions and TGFß1 further induced the alternative splicing of EDA+Fn via ALK5 receptor activation and SR proteins. This is the first evidence of basal and TGFß1 mediated alternative splicing of EDA+Fn in human podocytes culture.

Human podocytes responses to alternatively spliced Extra domain A Fibronectin in culture.

The interactions of the extracellular matrix (ECM) proteins with cells strongly regulate cell behaviour. The glomerular basement membrane (GBM) is a dynamic structure made up of protein secreted by endothelial cells and podocyte. These proteins could regulate the behaviour of these cells in health and diseases. Extra Domain A + Fn (EDA+Fn) is an alternatively spliced form of Fibronectin (Fn) recently identified in GBM and a recognised marker of various pathologies. In this study for the first time, we have investigated the responses of human podocytes cultured on different composition of GBM proteins which are cellular Fn (EDA+), plasma Fn (EDA-) and collagen IV. Conditionally immortalised human podocyte were grown on the dishes coated with different matrices; collagen IV (Col IV), cellular fibronectin (CFn) containing the EDA Exon, plasma fibronectin (PFn), which lacks the EDA Exon (EDA-Fn). We have performed western blotting to characterise the expression of the different proteins, real time PCR and RT-PCR to look for gene expression and alternative splicing of EDA+Fn. We have used TGFß1 as a stimulator. We have used HEK-Blue-hTLR4 cells to determine the biological activity of cellular Fn. Conditionally immortalised human podocyte show marked differences in their morphology grown on the dishes coated with different matrices; Col IV, CFn, and PFn. CFn was biologically active as it activated the TLR4 signalling in HEK-Blue-hTLR4 cells. Different matrices effects basal as well as TGFß1 mediated alternative splicing of EDA+Fn. TGFß1 was active on different matrices as it induced phosphorylation of pSmad3 however it did not affect phosphorylation of pAkt and p38. Interestingly, different cellular matrices affected basal phosphorylation of pAkt. CFn downregulated gene expression of synaptopodin and increased gene expression of collagen I and Fn. CFn increased cell death in detached human podocytes. Alteration of the constituents of the GBM is likely to significantly alter podocyte cellular responses to growth factors involved in podocytopathies, such as TGFß.

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.

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.

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.

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.