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

Investigation of the barium enema x-ray examination as a significant contributor to the genetically-significant dose from diagnostic radiology

The results of a study conducted by Maree (1995) indicated that the genetically-significant dose (GSD) for the white, female population in South Africa was considerably higher than the GSD for females in Great Britain, France and the United States of America. Further to this finding, Maree's study demonstrated that the barium enema x-ray examination was the major contributor to the GSD for this population group. A study of barium enema examinations was embarked on in order to explain the findings of Maree. The study was designed to include dose-area product measurements on patients having the barium enema procedure. In addition patient data and technique factors were recorded. The x-ray equipment used for the investigation was one digital and two non-digital fluoroscopic systems in the Western Cape. The digital unit utilised an overhead tube as did one of the conventional units. The other unit had an undercouch fluoroscopic tube and an overhead tube used for the standard radiography views. Comparison of the dose-area product measurements demonstrated that the unit having an undercouch tube had a mean dose-area product of 99.69 Gy cm² which culminates in a higher dose to the patient than the equipment utilising an overhead tube. The mean dose-area product of the two units with an overhead tube was 56.57 Gy cm² and 51.94 Gy cm² respectively. Free Air Exposure tables based on "RADCOMP Entrance Skin Exposure Software Program" (Nuclear Associates and Zamenhof, 1990) were used together with average technique factors to calculate skin entrance doses. These skin entrance doses were used to calculate gonad doses with the aid of a computer program from the Food and Drug Administration in the USA (Peterson and Rosenstein, 1989). The results were compared with the results of the barium enema component of the research conducted by Maree. The comparison indicated an average gonad dose for males of 242 μGy x 10⁻¹ (present study) compared to 485 μGy x 10⁻¹ (Maree) and an average gonad dose for females of 11185 μGy x 10⁻¹ (present study) compared to 16111 μGy x 10⁻¹ (Maree). Air-kerma at skin entrance was calculated using dose-area product measurements, recorded during the present study, for individual exposures and screening. These values were used to calculate the gonad dose. A discrepancy was demonstrated between the calculation of gonad dose from calculated as opposed measured skin entrance dose. The average gonad dose calculated by Maree is 16111 μGy x 10⁻¹ and the average gonad dose calculated for the present study using the measured skin entrance dose is 4236 μGy x 10⁻¹. This seems to explain the larger GSD estimated by Maree for the white female patients. A national protocol for measuring patient doses from x-ray examinations is proposed for South Africa

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

'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

The amalgamation of secondary schools : a case study of amalgamation culture shock in a rural New Zealand Catholic community : a thesis presented in partial fulfillment of the requirements for the degree of Master of Educational Administration at Massey University, Palmerston North, New Zealand

This case study examines the process which led to the formation of Chanel College by the amalgamation of St Bride's and St Joseph's colleges in 1978. From 1970 - 1999 a radical restructuring of Catholic secondary education in New Zealand resulted in the closure of twelve single sex secondary schools for girls and the amalgamation of twenty-six secondary schools. Chanel College was the first New Zealand Catholic secondary school to bypass the co-institutional transitional phase of amalgamation and to be a co-educational college from its beginning. As time passes there is the possibility that important understandings that were part of the history of the merger might be lost and stakeholders in each of the merging organisations might feel that their own roots and mission have not been given enough recognition and respect in the new organisation. The community which provides the focus of this case study had experienced a long period of stability followed by years of rapid cultural, educational, and leadership change. The tortuous progress of amalgamation for the Catholic community in the Wairarapa provides an ideal opportunity to examine the importance of leadership and process in the management of significant educational change. The importance of managing the culture shock of amalgamations is often underestimated or overlooked. This form of culture shock involves the confusion, disorientation and severe emotional stress associated with moving from a familiar culture to one most unlike the old environment. If this management issue is not addressed effectively there can be a significant area of 'unfinished business' which leaves a bitter legacy for a new school struggling to create an accepted culture of its own. The stakeholders also find themselves involved in a situation which is often not of their choosing where they face the often unwelcome task and ongoing process of creating a new culture where the unconscious taken for granted beliefs, thoughts and values which had provided the foundation for the merging schools must be revisited until a new culture develops which is accepted by the new community as appropriate to its needs. In the Conclusions and Recommendations section the stakeholder and community management issues often encountered in the amalgamation process are summarised and management recommendations are made and solutions proposed

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

Glucose-evoked changes in Transforming Growth Factor Beta1 modulate cell-substrate binding in human proximal tubule derived epithelial cells.

Aims: The tubular-basement membrane is a highly regulated microenvironment that facilitates numerous cell-matrix interactions critical in maintaining epithelial phenotype. Currently, we know little of how cell substrate and cell-cell interactions are modulated in diabetic nephropathy. This study identifies a role for glucose-evoked changes in TGF-β1, in modulation of interactions between proximal tubule derived epithelial cells and key components of the extracellular matrix. Methods: HK2 cells were cultured in either 5mM-glucose +/- TGF-β1 (2-10ng/mL) or 25mM-glucose. Glucose evoked increases in TGF-β1 secretion were determined by ELISA. HK2-ECM interactions were assessed via ECM arrays. Results: Cell culture supernatant of HK2-cells cultured in 25mM-glucose exhibited increased TGF-β1 secretion from 334pg/mL±4.1% to 994pg/mL ±4.3% as compared to 5mM-control (n=3 PCollagenIV>Collagen I>Laminin as determined by an ECM assay. TGF-β1 treated HK2 cells (48hrs) evoked increased binding to Collagen I, Collagen IV and Laminin to 340±26%, 228±38% and 289±42% respectively, whilst binding to fibronectin was unaltered as compared to control (n=3 P<0.01). HK2 cells cultured in 25mM glucose exhibited increased binding to Collagen I, Collagen IV and Laminin to 183±4%, 157±3% and 175±20% respectively, whilst binding to fibronectin was reduced to 80±5% as compared to control (n=3 P<0.001). Conclusions: The current study suggests that glucose-evoked changes in TGF-β1 are instrumental in reorganizing the extracellular-matrix and cell-substrate interactions in proximal tubule epithelial cells, changes that alter cell architecture, integrity and function, which can ultimately result in kidney damage ahead of overt renal failure in Diabetic-Nephropathy. This work was supported wholly or in part by the generous support of Diabetes UK (BDA: 11/0004215), and The Warwickshire Private Hospital (WPH) Charitable Trust

Connexins and gap-junction mediated intercellular communication in the diabetic kidney

Background: Altered connexin expression and/or function is linked to the development and progression of secondary microvascular complications associated with diabetes. Despite this, we know little for the role of these small membrane proteins in the diabetic kidney. This study examines if glucose-evoked changes in TGF-β1 modulate connexin expression and gap junction-mediated intercellular communication in diabetic nephropathy. Methods: Biopsy material was isolated from patients with diabetic nephropathy and stained for connexin-26 and connexin-43. Changes in expression, were corroborated by immunoblot analysis in model epithelial cells from human renal proximal tubules (HK2) cultured in either low glucose (5 mmol/l) +/− TGF-β1 (2–10 ng/ml) or high glucose (25 mmol/l) for 7 days. ELISA was used measured TGF-β1 secretion and paired-patch electrophysiology recorded junctional conductance in control versus TGF-β1 treated (10 ng/ml) HK2 cells. Results: Connexin-26 expression was significantly up regulated in biopsy material from patients with diabetic nephropathy, compared to normal control (102 700±6226 vs 21 030±4727; n=5, P<0.01). Similarly, connexin-43 expression increased to 116 300±5908 as compared to control 21 460±10 920 (n=5, P<0.01). In response to high glucose (25 mmol/l) treatment for 7 days, HK2 cells increased TGFβ1 secretion to 994.4±43.6 pg/ml compared to 5 mmol/l glucose (334±14.9 pg/ml; n=3; P<0.01). Immunoblot analysis confirmed that TGFβ1 (10 ng/ml) up-regulates expression of connexin-26 and connexin-43 to 203.9±7.5% and 151.1±7.1% respectively compared to control (n=4; P<0.001). Whole cell paired-patch electrophysiology was used to determine the junctional conductance between coupled HK2 cells ± TGF-β1 (10 ng/ml). TGF-β1 produced decreased junctional conductance to 0.42±0.2 nS compared to control 4.5±1.3 nS (n=5; P≤0.05). Conclusion: Expression of connexin-26 and connexin-43 increased in biopsy material isolated form patients with diabetic nephropathy, changes corroborated in HK2 cells treated chronically with TGF-β1. Despite this gain in expression, gap junction mediated intercellular conductance was reduced, a feature linked to increased hemi-channel activity