GLUT1 and TGF-ß: the link between hyperglycaemia and diabetic nephropathy

András Mogyorósi1 and Fuad N. Ziyadeh2

1 Division of Nephrology, Department of Medicine, Virginia Commonwealth University/Medical College of Virginia and McGuire VAMC, Richmond, 2 Renal-Electrolyte and Hypertension Division, Department of Medicine, and the Penn Center for the Molecular Studies of Kidney Diseases, University of Pennsylvania, Philadelphia, USA

Correspondence and offprint requests to: Fuad N. Ziyadeh, MD, Renal-Electrolyte & Hypertension Division, University of Pennsylvania, 415 Curie Boulevard, 700 Clinical Research Building, Philadelphia, PA 19104-6144, USA.

Abstract

Recent experimental work implicates transforming growth factor-ß (TGF-ß) as an aetiologic mediator of diabetic nephropathy and the ubiquitous glucose transporter GLUT1 as an important permissive factor for the tissue injury caused by hyperglycaemia. High ambient glucose increases GLUT1 expression and glucose transport activity when compared with physiologic glucose concentrations. Treatment of rat mesangial cells with TGF-ß up-regulates GLUT1 mRNA and protein levels and significantly increases glucose uptake. Addition of neutralizing anti-TGF-ß antibody prevents the stimulatory effects of high glucose on GLUT1 expression. Cultured rat mesangial cells transduced with the human GLUT1 gene and thus overexpressing the GLUT1 protein show marked increase in glucose uptake and the synthesis of extracellular matrix molecules, even when grown in normal ambient glucose concentrations. Thus, TGF-ß and GLUT1, two proteins that are up-regulated in glomerular mesangial cells in a hyperglycaemic milieu, can influence the expression of one another. It is therefore fair to conclude that, with successful interruption of the TGF-ß–GLUT1 axis, the beneficial effects of strict glucose control on the development of diabetic nephropathy could likely be augmented.

Keywords: diabetic nephropathy; fibrosis; GLUT1; hyperglycaemia, TGF-ß

Introduction

Diabetic nephropathy is the most frequent cause of end-stage renal disease. In the early 1990s it was convincingly proved that the single most important factor in the development of this nephropathy (diagnosed by the presence of microalbuminuria) is hyperglycaemia [1]. However, describing exactly how high ambient glucose exerts its deleterious effects remained an elusive aim. Recent experimental work implicates transforming growth factor-ß (TGF-ß) as an aetiologic mediator and the glucose transporter GLUT1 as an important permissive factor for the tissue injury caused by hyperglycaemia.

TGF-ß in diabetic nephropathy

There is ample evidence that TGF-ß plays an important role in in vitro and in vivo models of diabetic kidney disease as well as in human diabetic nephropathy [24]. TGF-ß is a central cytokine in cell growth and extracellular matrix production. The hypertrophic and prosclerotic effects of high ambient glucose in cultures of glomerular mesangial and tubular epithelial cells are mediated by increased production and activation of TGF-ß [58]. Elevated renal TGF-ß mRNA and protein levels have been found in various animal models and the human form of diabetic nephropathy [4,9]. Treatment of streptozotocin-diabetic mice with neutralizing anti-TGF-ß antibodies attenuates kidney hypertrophy and the increase in extracellular matrix molecules [10]. On the other hand, normoglycaemic mice, transgenic for active TGF-ß1, develop proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis with increased expression of collagens I and III, biglycan and decorin [11]—alterations that are also hallmarks of diabetic nephropathy [2,12]. Importantly, in a recent genetic study in type 1 (insulin-dependent) diabetic patients [13], the Thr263Ile mutation of the TGF-ß1 gene was found to be associated with the presence of diabetic nephropathy, further strengthening the case for a role for TGF-ß in human diabetic kidney disease.

GLUT1 in diabetic nephropathy

It is the increased rate of glucose metabolism intracellularly that promotes the pathological changes in diabetic nephropathy [2]. From this perspective it is clear that the activity of glucose transporters on the cell surface of target cells may be a rate-limiting factor in the development of diabetic changes in the kidney. Cloning studies have characterized a large gene family of facilitative glucose transporters (GLUT) that are expressed in a tissue-specific manner and two sodium–glucose cotransporters (SGLT) in brush-border membranes of intestinal and renal epithelia. These different transporters play specific roles in glucose transport and homeostasis owing to their different tissue distribution, kinetic properties, and regulation of function [14]. GLUT1 is widely expressed in many cell types in vivo and it is also expressed at very high levels in cultured or transformed cells. In the kidney, GLUT1 is constitutively expressed in both the glomerular and tubular compartments [15]. GLUT1 has a low Km for glucose (2–20 mM) and is a `high-affinity, low-capacity' transporter, operating at close to its maximal transport velocity and thus capable of effectively transporting glucose into most cells. The description of the pattern of expression of GLUT1 in the kidney in the diabetic state has proved to be difficult so far. Early in the course of streptozotocin-induced diabetes in the rat, kidney GLUT1 is up-regulated in the medulla but down-regulated in the cortical area [16]. At present, there is no information about the expression of GLUT1 in humans with diabetes or diabetic kidney disease. However, there is growing evidence from in vitro work that GLUT1 may play an important role in the genesis of the pathological changes in diabetic nephropathy. In cultured rat mesangial cells, high ambient glucose (20 mM) increases GLUT1 mRNA and protein levels as well as GLUT1 transport activity (as measured by the cellular uptake of [3H]2-deoxyglucose) when compared with physiologic glucose concentrations (8 mM) [17]. Cultured rat mesangial cells transduced with the human GLUT1 gene and thus overexpressing the GLUT1 protein show marked increase in glucose uptake and the synthesis of the extracellular matrix molecules collagen I, collagen IV, fibronectin, and laminin, even when grown in normal ambient glucose concentrations [18].

In genetic studies using sib-pairs, two restriction fragment length polymorphisms (MspI and XbaI) of the GLUT1 gene did not show linkage to the presence of type 2 diabetes [19]. In a recent genetic study in Chinese type 2 diabetic patients, however, the XbaI(-) allele conferred a significantly higher susceptibility (odds ratio 1.9) to diabetic kidney disease [20]. It is not clear whether the presence of the XbaI(-) allele manifests in a higher in vivo expression and/or activity of GLUT1. It is also not known whether patients with this allele and normoglycaemia would develop any renal pathology. From this point of view it is particularly interesting, as mentioned above, that cultured mesangial cells transduced with the GLUT1 gene show marked increase in glucose uptake and extracellular matrix synthesis even when grown in a normal ambient glucose concentration [18].

TGF-ß and GLUT1

An exciting development is the recent notion that TGF-ß1 and GLUT1 can influence each others' biologic effects. The increased synthesis of TGF-ß in proximal tubular cells cultured under high glucose conditions [6] is inhibited by phlorizin, suggesting a dependence of TGF-ß synthesis on the basolateral activity of GLUT1 [21]. Additionally, treatment of rat mesangial cells with TGF-ß1 up-regulates GLUT1 mRNA and protein levels and significantly increases glucose uptake [22]. Furthermore, the expression of GLUT1 is more pronounced in cells cultured under high glucose condition (22 mM) and is inhibited by addition of neutralizing anti-TGF-ß antibody [22]. Thus, TGF-ß1 and GLUT1, two proteins that are up-regulated in kidney cells in a hyperglycaemic milieu, can influence the expression of one another.

How TGF-ß up-regulates GLUT1 abundance and activity is unclear. In Swiss 3T3 fibroblasts where TGF-ß also increases the glucose transport by increasing GLUT1 activity, TGF-ß may act through altering the glycosylation of GLUT1 [23,24]. Interestingly, tolazamide, a sulphonylurea antidiabetic agent, might have untoward effects on the kidney because it stimulates both GLUT1 expression and glucose transport as well as active and total TGF-ß secretion in mesangial cells [25]. This may lead to increased accumulation of collagen and fibronectin [25]. In view of the stimulating effect of TGF-ß on GLUT1 and the known prosclerotic actions of TGF-ß, tolazamide may well work through TGF-ß in this respect, but this has not yet been proven.

Is hyperglycaemia necessary for the development of diabetic kidney disease?

Interesting cell culture studies have shown that genetic overexpression of GLUT1 in rat mesangial cells can lead to the diabetic phenotype even in the absence of high surrounding glucose levels [18]. Similarly, high systemic TGF-ß levels in transgenic mice, even in the setting of normoglycaemia, result in glomerulosclerosis and tubulointerstitial fibrosis, increased matrix production and decreased matrix remodelling [11]. Does this mean that hyperglycaemia per se is not a necessary factor for the development of diabetic kidney disease? To refute this hypothesis it is important to note that the TGF-ß and GLUT1 levels achieved by genetic manipulation were exceedingly high compared with the normal phenotype [11,18], and it is questionable whether genetic mutation(s) in humans would lead to comparably up-regulated activity of TGF-ß and/or GLUT1. Anecdotal reports in the older literature have described the occurrence of diabetic nephropathy in non-diabetic patients based solely on the light microscopic finding of nodular glomerulosclerosis [26]. However, these reports did not formally exclude non-diabetic causes of such lesions (e.g. light-chain nephropathy and amyloidosis) because appropriate staining techniques were not applied. Consequently, `diabetic kidney disease without diabetes' has to be considered a rather unlikely phenomenon.

Taken all together, hyperglycaemia remains a necessary factor for the development of diabetic nephropathy, but a whole host of additional factors may modulate its deleterious effect. This is in agreement with the important finding of The Diabetes Control and Complications Trial that while some patients with excellent glucose control develop kidney disease, others with less strict or even poor blood glucose control do not [1]. By extrapolation it is fair to conclude that, with successful interruption of the TGF-ß–GLUT1 axis, the beneficial effects of strict glucose control on the development of diabetic nephropathy could likely be augmented.

Acknowledgments

Supported in part by the National Kidney Foundation of Virginia (A.M.), the Juvenile Diabetes Foundation International (F.N.Z.), and the National Institutes of Health (grants DK-44513, DK-45191 and DK-54608 to F.N.Z.; and training grant DK-07006).

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