J Cell Physiol. 2015 Oct;230(10):2437-46.

In vivo sodium tungstate treatment prevents E-cadherin loss induced by diabetic serum in HK-2 cell line

 

Romina Bertinat12*, Pamela Silva1, Elizabeth Mann3, Xuhang Li3, Francisco Nualart2, Alejandro J. Yáñez12*

1 Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia, Chile.

2 Centro de Microscopía Avanzada (CMA)-Bío Bío, Universidad de Concepción, Concepción, Chile.

3 Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, MD, United States.

* Corresponding author: Dr. Romina Bertinat, Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Los robles s/n, Valdivia, Chile. romibert@gmail.com; +56954043743; ayanez@uach.cl; +5663221065

 

ABSTRACT.

Diabetic nephropathy (DN) is characterized by interstitial inflammation and fibrosis, which is the result of chronic accumulation of extracellular matrix produced by activated fibroblasts in the renal tubulointerstitium. Renal proximal tubular epithelial cells (PTECs), through the process of epithelial-to-mesenchymal transition (EMT), are the source of fibroblasts within the interstitial space, and loss of E-cadherin has shown to be one of the earliest steps in this event. Here, we studied the effect of the anti-diabetic agent sodium tungstate (NaW) in the loss of E-cadherin induced by transforming growth factor (TGF) b-1, the best-characterized in vitro EMT promoter, and serum from untreated or NaW-treated diabetic rats in HK-2 cell line, a model of human kidney PTEC. Our results showed that both TGFb-1 and serum from diabetic rat induced a similar reduction in E-cadherin expression. However, E-cadherin loss induced by TGFb-1 was not reversed by NaW, whereas sera from NaW-treated rats were able to protect HK-2 cells. Searching for soluble mediators of NaW effect, we compared secretion of TGFb isoforms and vascular endothelial growth factor (VEGF)-A, which have opposite actions on EMT. 1 mM NaW alone reduced secretion of both TGFb-1 and -2, and stimulated secretion of VEGF-A after 48 h. However, these patterns of secretion were not observed after diabetic rat serum treatment, suggesting that protection from E-cadherin loss by serum from NaW-treated diabetic rats originates from an indirect rather than a direct effect of this salt on HK-2 cells, via a mechanism independent of TGFb and VEGF-A functions.

PMID: 25728412

 

SUPPLEMENT.

Diabetes is a progressive metabolic disease, characterized by chronic hyperglycemia and disturbances of carbohydrate, fat, and protein metabolism that is the major cause of end stage renal disease. Diabetic nephropathy (DN) is characterized by fibrosis, a progressive and generally irreversible process leading to renal failure as a consequence of the chronic accumulation of extracellular matrix proteins in the renal tubulointerstitium. Enhanced production of extracellular matrix proteins is achieved by resident renal fibroblasts but also by new fibroblasts that are abnormally generated during epithelial-to-mesenchymal transition (EMT), a process by which fully differentiated epithelial cells lose their epithelial characteristics and acquire migratory mesenchymal properties. One of the earliest events in EMT is the loss of epithelial adhesion, which occurs after down-regulation of components of cell-to-cell contact, such as E-cadherin. Diabetes is an inflammatory disease and renal fibrosis is induced by inflammatory mediators such as transforming growth factor-β1 (TGF-β1), a potent EMT inducer. On the contrary, vascular endothelial growth factor isoform A (VEGF-A) supplementation suppresses EMT and ameliorates tubulointerstitial damage in experimental animal models.

Although there are several commercially available anti-diabetic drugs, the search of new agents has never stopped because no ideal anti-diabetic drug has been found yet. Also, understanding of the pathogenesis of renal fibrosis is of key importance in order to develop effective new therapeutic strategies. For this purpose, experimental animal models are important tools to pre-clinical studies. Sodium tungstate (NaW) is an inorganic salt that exerts potent anti-diabetic and anti-obesity activity in animal models of diabetes (1). In the diabetic rat kidney, NaW treatment completely reverses the most characteristic histopathological feature, the so-called Armanni-Ebstein lesion, which is an extensive glycogen deposition in tubular cells (2). NaW treatment has also been shown to restore the number and function of immune cells as well as the immunoglobulin level in diabetic rats (3). Hence, we explored the effect of NaW on in vitro EMT by analyzing E-cadherin expression in the human renal cell line HK2, using TGFb1 as the EMT inducer.

 

FIGURE 1

FIGURE 1. NaW does not reverse TGFb-1-induced E-cadherin loss in HK-2 cells. HK-2 cells were allowed to reach confluence and then serum-starved for 24 h. Cells were incubated with 5 ng/ml TGFb-1 for 48 h, in the presence or absence of 1 mM NaW. E-cadherin expression was studied by means of qRT-PCR (A), Western blot (B) and immunofluorescence (E-cadherin: green channel; TOPRO3 nuclear staining: blue channel) (C). Each experiment was performed in triplicate and 3 separate times. * p<0.05; ** p<0.01

 

In this study (4), we demonstrate that the serum from type 1 diabetic rats down-regulates the expression of E-cadherin in the human renal epithelial cell line HK-2, in a similar fashion to that of TGFb1, one of the best characterized inducers of EMT in vitro. Interestingly, co-incubation with NaW was unable to inhibit the loss of E-cadherin expression induced by TGFb1, however, it produced an effect when first administered in vivo to diabetic rats and then the serum added to HK-2 cells. This result clearly indicates that the effect of NaW over the expression of E-cadherin in HK-2 cells occurs via indirect mechanisms. A similar report has demonstrated that NaW alone induced a little increase in proliferation of the INS-1E rat b cell line, but when these cells were incubated with serum from NaW-treated diabetic rats, the rate of proliferation was greatly enhanced, explaining the total increase in b cell mass observed in vivo after NaW treatment (5). Together, these data strongly suggest beneficial in vivo effects of NaW that cannot be replicated in vitro, indicating that the study of NaW in vitro is underestimating its effects due to the lack of other interacting factors that may potentiate NaW therapeutic value.

Because treatment of human patients with NaW has been tried inconclusively only once (6) and further clinical studies have been delayed, experiments with serum from NaW-treated diabetic patients are not possible at the moment. Therefore, the importance of this study is that our approach offers a more physiological model to study NaW actions on human cells. At present, there is no effective therapy for treatment of fibrotic diseases. Since removal of deposited matrix is almost impossible as the fibrotic disease progresses, the sooner the disease is detected, the better the chances to stop or even reverse the damage. Given the urgent need for new therapies addressing this complex process, drugs targeting EMT have been the focus of intense research. This study describes novel functions of NaW in the context of diabetic EMT, renal inflammation and fibrosis, and suggests that treatments with NaW can prevent down-regulation of E-cadherin, a major player in EMT, that together with the well-known normoglycemic and normotriglyceridemic effects of this salt, may offers a promising therapy for DN in humans.

 

FIGURE 2

FIGURE 2. Serum from NaW-treated diabetic rats prevents loss of E-cadherin expression in HK-2 cells. HK-2 cells were allowed to reach confluence and then serum-starved for 24 h. Cells were incubated with 2.5% serum from untreated control (UC), NaW-treated control (TC), untreated diabetic (UD) and NaW-treated diabetic (TD) rats for 48 h, and E-cadherin expression was studied by means of qRT-PCR (A), Western blot (B) and immunofluorescence (E-cadherin: green channel; TOPRO3 nuclear staining: blue channel) (C). n=5. Each experiment was performed 3 separate times. * p<0.05; *** p<0.001: **** p<0.0001

 

REFERENCES

(1) Bertinat R, Nualart F, Li X, Yáñez AJ, Gomis R. 2015. Preclinical and clinical studies for sodium tungstate: Application in humans. J Clin Cell Immunol. 2015; 6(1). pii: 285.

(2) Barberà A, Gomis RR, Prats N, Rodríguez-Gil JE, Domingo M, Gomis R, Guinovart JJ. Tungstate is an effective antidiabetic agent in streptozotocin-induced diabetic rats: a long-term study. Diabetologia. 2001;44(4):507-13.

(3) Palanivel R, Sakthisekaran D. Immunomodulatory effect of tungstate on streptozotocin-induced experimental diabetes in rats. Ann N Y Acad Sci. 2002;958:382-6.

(4) Bertinat R, Silva P, Mann E, Li X, Nualart F, Yáñez AJ. 2015 In vivo sodium tungstate treatment prevents E-cadherin loss induced by diabetic serum in HK-2 cell line. J Cell Physiol. 230(10):2437-46.

(5) Altirriba J, Barbera A, Del Zotto H, Nadal B, Piquer S, Sánchez-Pla A, Gagliardino JJ, Gomis R. Molecular mechanisms of tungstate-induced pancreatic plasticity: a transcriptomics approach. BMC Genomics. 2009;28(10):406.

(6) Hanzu F, Gomis R, Coves MJ, Viaplana J, Palomo M, Andreu A, Szpunar J, Vidal J. Proof-of-concept trial on the efficacy of sodium tungstate in human obesity. Diabetes Obes Metab. 2010;12(11):1013-8.

 

 

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