TGF-beta 1-induced epithelial-to-mesenchymal transition and therapeutic intervention in diabetic nephropathy

Background/Aims: Epithelial-to-mesenchymal cell transformation (EMT) is the trans-differentiation of tubular epithelial cells into myofibroblasts, an event underlying progressive chronic kidney disease in diabetes, resulting in fibrosis. Mainly reported in proximal regions of the kidney, EMT is now recognized as a key contributor to the loss of renal function throughout the nephron in diabetic nephropathy (DN). Concomitant upregulation of TGF-beta in diabetes makes this pro-fibrotic cytokine an obvious candidate in the development of these fibrotic complications. This article reviews recent findings clarifying our understanding of the role of TGF-beta and associated sub-cellular proteins in EMT. Methods: To understand the pathology of EMT and the role of TGF-beta, we reviewed the literature using PubMed for English language articles that contained key words related to EMT, TGF-beta and DN. Results: EMT and phenotypic plasticity of epithelial cells throughout the nephron involves cytoskeletal reorganization and de novo acquisition of classic mesenchymal markers. Concurrent downregulation of epithelial adhesion molecules results in a loss of function and decreased cell coupling, contributing to a loss of epithelial integrity. TGF-beta 1 is pivotal in mediating these phenotypic changes. Conclusion: TGF-beta-induced EMT is a key contributor to fibrotic scar formation as seen in DN, and novel routes for future therapeutic intervention are discussed

SGK1 in the kidney: disrupted sodium transport in diabetes and beyond

Renal complications of diabetes can be severe; however, the mechanisms that underlie the development and progression of diabetic nephropathy are poorly understood. Recent evidence suggests that the serum and glucocorticoid induced kinase-1 (SGK1) may be key to this process. SGK1 expression and function are increased in models of diabetes and polymorphisms of the SGK1 gene are associated with type 2 diabetes mellitus. A key regulator of sodium transport within the renal epithelium of the distal nephron, SGK1 was originally isolated as a glucocorticoid-sensitive gene that regulated the epithelial sodium channel (ENaC; known also as the sodium channel, nonvoltage-gated 1, SCNN1). It is now apparent that SGK1 modulates sodium re-absorption by a number of sodium transporters/channels throughout the length of the nephron including; the Na+/H+ exchange isoform 3 (NHE3), the Na+Cl- co-transporter (NCC) and the Na+/K+-ATPase. In addition, SGK1 is regulated by a diverse range of factors including; insulin, glucose, intracellular calcium, transforming growth factor-beta1, flow rate and osmolality. This brief review examines the evidence supporting an involvement of SGK1 in diabetic nephropathy and discusses how dysregulated sodium transport may account for the development of secondary hypertension associated with the condition. Furthermore, the article examines how aberrant SGK1 expression and activity may be responsible for the cellular changes seen in the damaged nephron

Functional expression of TRPV4 channels in human collecting duct cells: implications for secondary hypertension in diabetic nephropathy

Background. The Vanilloid subfamily of transient receptor potential (TRPV) ion channels has been widely implicated in detecting osmotic and mechanical stress. In the current study, we examine the functional expression of TRPV4 channels in cell volume regulation in cells of the human collecting duct. Methods. Western blot analysis, siRNA knockdown, and microfluorimetry were used to assess the expression and function of TRPV4 in mediating Ca2+-dependent mechanical stimulation within a novel system of the human collecting duct (HCD). Results. Native and siRNA knockdown of TRPV4 protein expression was confirmed by western blot analysis. Touch was used as a cell-directed surrogate for osmotic stress. Mechanical stimulation of HCD cells evoked a transient increase in [Ca2+]i that was dependent upon thapsigargin-sensitive store release and Ca2+ influx. At 48 hrs, high glucose and mannitol (25 mM) reduced TRPV4 expression by 54% and 24%, respectively. Similar treatment doubled SGK1 expression. Touch-evoked changes were negated following TRPV4 knockdown. Conclusion. Our data confirm expression of Ca2+-dependent TRPV4 channels in HCD cells and suggest that a loss of expression in response to high glucose attenuates the ability of the collecting duct to exhibit regulatory volume decreases, an effect that may contribute to the pathology of fluid and electrolyte imbalance as observed in diabetic nephropathy

Visfatin reduces gap junction mediated cell-to-cell communication in proximal tubule-derived epithelial cells

Background/Aims: In the current study we examined if the adipocytokine, visfatin, alters connexin-mediated intercellular communication in proximal tubule-derived epithelial cells. Methods: The effects of visfatin (10-200ng/mL) on cell viability and cytotoxicity in HK2-cells were assessed by MTT, crystal violet and lactate dehydrogenase assays. Western blot analysis was used to confirm expression of Cx26, Cx40 and Cx43. The effect of visfatin (10-200ng/mL) on TGF-β1 secretion was confirmed by ELISA, and the effects of both TGF-β1 (2-10ng/mL) and visfatin (10-200ng/mL) on connexin expression were assessed by western blot. Functional intercellular communication was determined using transfer of Lucifer Yellow and paired-whole cell patch clamp electrophysiology. Results: In low glucose (5mM), visfatin (10-200ng/mL) did not affect membrane integrity, cytotoxicity or cell viability at 48hrs, but did evoke a concentration-dependent reduction in Cx26 and Cx43 expression. The expression of Cx40 was unaffected. At 48hrs, visfatin (10-200ng/mL) increased the secretion of TGF-β1 and the visfatin-evoked changes in connexin expression were mimicked by exogenous application of the pro-fibrotic cytokine (2-10ng/ml). Visfatin reduced dye transfer between coupled cells and decreased functional conductance, with levels falling by 63% as compared to control. Although input resistance was increased following visfatin treatment by 166%, the change was not significant as compared to control. The effects of visfatin on Cx-expression and cell-coupling were blocked in the presence of a TGF-β1 specific neutralizing antibody. Conclusions: The adipocytokine visfatin selectively evoked a non-toxic reduction in connexin expression in HK2-cells. The loss in gap-junction associated proteins was mirrored by a loss in functional conductance between coupled cells. Visfatin increased TGF-β secretion and the pattern of change for connexins expression was mimicked by exogenous application of TGF-β1. The effect of visfatin on Cx-expression and dye transfer were negated in the presence of a TGF-β1 neutralising antibody. These data suggest that visfatin reduces connexin-mediated intercellular communication in proximal tubule-derived epithelial cells via a TGF-β dependent pathway. © 2013 S. Karger AG, Base

SGK and disrupted renal sodium handling in diabetes

Diabetic nephropathy is associated with secondary hypertension arising from aberrant sodium reabsorption in the kidney. This thesis characterises a novel human cell line derived from the human cortical collecting duct (HCD) to assess glucoseevoked changes in key elements, such as the serum and glucocorticoid inducible kinase (SGKI) and the epithelial sodium channel (ENaC), involved in the regulation of sodium transport. In addition I have also examined the effects of TGF-f3I and [Ci+]i on SGKI and ENaC expression. RT-PCR, western blot analysis, immunocytochemistry and single cell imaging were employed to determine presence, localisation and function of these elements under various glycaemic conditions. Our data suggest that high glucose, TGF-f3I and [Cl+]i up-regulate both SGKI and [alpha]ENaC protein expression, which in turn stimulates Na+ transport. In pathological conditions associated with aberrant Na + reabsorption, excessive levels of Na + may further exacerbate the state of hypertrophy, a common manifestation associated with diabetic nephropathy. Mechanical stress evoked TRPV4 m~diated changes in [Ca2+]i. Propagation of this Ca2+ signal via the gap junction protein connexin 43 (Cx-43) was enhanced following glucose treatment, as was Cx-43 expression. Under pathophysiological conditions these changes and the increased expression levels of our key signaling elements, may lead to deranged Na+ handling and inhibition of cell volume recovery mechanisms which together may further enhance the condition of diabetic nephropathy in Type 11 diabetes

'Special K' and a loss of cell-to-cell adhesion in proximal tubule-derived epithelial cells: modulation of the adherens junction complex by ketamine

Ketamine, a mild hallucinogenic class C drug, is the fastest growing ‘party drug’ used by 16–24 year olds in the UK. As the recreational use of Ketamine increases we are beginning to see the signs of major renal and bladder complications. To date however, we know nothing of a role for Ketamine in modulating both structure and function of the human renal proximal tubule. In the current study we have used an established model cell line for human epithelial cells of the proximal tubule (HK2) to demonstrate that Ketamine evokes early changes in expression of proteins central to the adherens junction complex. Furthermore we use AFM single-cell force spectroscopy to assess if these changes functionally uncouple cells of the proximal tubule ahead of any overt loss in epithelial cell function. Our data suggests that Ketamine (24–48 hrs) produces gross changes in cell morphology and cytoskeletal architecture towards a fibrotic phenotype. These physical changes matched the concentration-dependent (0.1–1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin. Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin. However, the loss in E-cadherin can be partially rescued pharmacologically by blocking p38 MAPK using SB203580. These data provide compelling evidence that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling in the proximal kidney via a non-classical pro-fibrotic mechanism and the data provides the first indication that this illicit substance can have major implications on renal function. Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention

Quantifying cellular mechanics and adhesion in renal tubular injury using single cell force spectroscopy

Abstract Tubulointerstitial fibrosis represents the major underlying pathology of diabetic nephropathy where loss of cell-to-cell adhesion is a critical step. To date, research has predominantly focussed on the loss of cell surface molecular binding events that include altered protein ligation. In the current study, atomic force microscopy single cell force spectroscopy (AFM- SCFS) was used to quantify changes in cellular stiffness and cell adhesion in TGF-β1 treated kidney cells of the human proximal tubule (HK2). AFM indentation of TGF-β1 treated HK2 cells showed a significant increase (42%) in the Elastic modulus (stiffness) compared to control. Fluorescence microscopy confirmed that increased cell stiffness is accompanied by reorganization of the cytoskeleton. The corresponding changes in stiffness, due to F-actin rearrangement, affected the work of detachment by changing the separation distance between two adherent cells. Overall, our novel data quantitatively demonstrate a correlation between cellular elasticity, adhesion and early morphologic/phenotypic changes associated with tubular injury

Bilateral Differences in Vascular Stiffness and Blood Pressure of Female College Tennis Players and Controls

Vascular stiffness is highly correlated to cardiovascular disease (CVD) and has been attenuated by regular aerobic exercise in older adults. Additionally, body composition strongly predicts vascular stiffness and CVD risk. Previous research has documented that bilateral differences in shear stress lead to differences in vascular endothelial function in tennis players. However, whether this translates into bilateral differences in vascular stiffness and blood pressure remains to be elucidated. The SphygmoCor is a gold standard for assessing arterial stiffness but is not readily accessible. In comparison, the TicWatch GTH Pro, which uses the SphygmoCor algorithm to provide a cardiovascular health (CVH) score (ARTY score), could be a feasible way to monitor CVH, but its accuracy is unknown. PURPOSE: This study aimed to evaluate bilateral differences in vascular stiffness and blood pressure in Division III female college tennis players and recreationally active female students. A secondary aim was to determine if the TicWatch GTH Pro ARTY score relates to SphygmoCor outcomes. METHODS: Subjects from the Skidmore women’s tennis team (n=10) and recreationally active female students (n=10) underwent a single testing session where anthropometrics, body composition, bilateral cardiovascular measurements, and a TicWatch analysis were performed. RESULTS: There were no significant differences in weight, body fat percentage, or BMI between the two groups (p=0.96; p=0.83; p=0.69). Augmentation pressure and augmentation index were different between the dominant and non-dominant arms (p=0.02; p=0.02), but no interactions of the group by arm were observed (all, p\u3e0.05). In addition, the TicWatch ARTY score related significantly with brachial diastolic pressure, mean arterial pressure, and central diastolic pressure (r²= -0.52; r²= -0.50; r²= -0.56). CONCLUSION: In groups well-matched for age and body composition, two indices of vascular stiffness significantly differed between the arms, but this did not appear specific to this cohort of tennis players. The results also indicate that the TicWatch is an accurate device for assessing CVH indicators and may predict brachial diastolic pressure, central diastolic pressure, and mean arterial pressure

Digital afx: digital dressing and affective shifts in Sin City and 300

In Sin City (Robert Rodriguez, 2005) and 300 (Zack Snyder, 2006) extensive post-production work has created stylised colour palettes, manipulated areas of the image, and added or subtracted elements. Framing a discussion around the terms ‘affect’ and ‘emotion’, this paper argues that the digital technologies used in Sin City and 300 modify conventional interactions between representational and aesthetic dimensions. Brian Massumi suggests affective imagery can operate through two modes of engagement. One mode is embedded in a meaning system, linked to a speci?c emotion. The second is understood as an intensi?cation whereby a viewer reacts but that reaction is not yet gathered into an alignment with meaning. The term ‘digital afx’ is used to describe manipulations that produce imagery allowing these two modes of engagement to coexist. Digital afx are present when two competing aesthetic strategies remain equally visible within sequences of images. As a consequence the afx mingle with and shift the content of representation

Quantitative investigation of calcimimetic R568 on beta-cell adhesion and mechanics using AFM single-cell force spectroscopy

In this study we use a novel approach to quantitatively investigate mechanical and interfacial properties of clonal b-cells using AFM-Single Cell Force Spectroscopy (SCFS). MIN6 cells were incubated for 48 h with 0.5 mMCa2+ ± the calcimimetic R568 (1 lM). AFM-SCFS adhesion and indentation experiments were performed by using modified tipless cantilevers. Hertz contact model was applied to analyse force–displacement (F–d) curves for determining elastic or Young’s modulus (E). Our results show CaSR-evoked increases in cell-to-cell adhesion parameters and E modulus of single cells, demonstrating that cytomechanics have profound effects on cell adhesion characterization