1. Technical Field
The present invention relates to a model system for simulating diabetes. More specifically, the present invention relates to an isolated and stable clone of cells which exhibit characteristics of diabetic cells for use in testing anti-diabetic drugs, and the pathogenesis of diabetic nephropathy. Further, the present invention relates to the use of a stable, permanent clone of mesangial cells which underexpresses GLUT1 mRNA and protein and which can be used therapeutically.
2. Background Art
Diabetic nephropathy, neuropathy, and retinopathy are the three most common tissue complications of diabetes. Diabetic kidney disease leads to end stage renal disease (ESRD) in approximately one-third of all patients suffering from diabetes. It has recently been established that hyperglycemia per se plays a significant role in the development of diabetic glomerulopathy and renal failure, however, the mechanisms by which it exerts its damaging effects are unknown (Diabetes Control and Complications Trial Research Group, 1993). Therefore, significant research efforts have been directed toward in vitro and in vivo investigation of the aforementioned mechanisms in order to discover the pathogenesis of diabetic nephropathy to develop therapies which may prevent or delay the complications of this disorder.
However, while researchers have attempted to develop therapies which might delay or prevent diabetic nephropathy, such as aldose reductase inhibitors and ACEIs (angiotensin converting enzyme inhibitors), no treatment to date has been optimal or highly effective in preventing the disease. Tight control of blood glucose levels puts patients at significant risk for hypoglycemia-induced symptoms or death. Therefore, new and more effective therapies are needed, based upon a better understanding of the disease mechanisms mediating diabetic nephropathy.
The central tenet of current theories addressing the mechanisms of development of diabetic nephropathy involves mesangial cell expansion and glomerular scarring due to increased production of extracellular matrix (ECM) components by mesangial cells (MCS). This has been proposed to be caused by several different mechanisms, however most of them either require or are facilitated by enhanced glucose uptake and metabolism. For instance, high extracellular glucose concentrations stimulate excessive ECM (scar tissue) production (Ayo et al., 1990; Ayo et al., 1991). The mechanisms by which this process is mediated are not clear, however enhanced mesangial cell aldose reductase (AR) activity with increased production of sorbitol, oxygen radicals, and vasodilator prostaglandins have been implicated, as have glycosylation of proteins, PKC (protein kinase C) activation, and TGF.beta..sub.1 stimulation.
The renal glomerular lesion of human and experimental diabetes mellitus is characterized by glomerular hypertrophy (Bilous et al., 1989a; Seyer-Hansen et al., 1980) and the deposition of extracellular matrix in the form of diffuse thickening of the peripheral basement membrane and mesangial expansion (Mauer et al., 1984). The progressive accumulation of matrix in the mesangial areas, and the associated encroachment on neighboring capillaries with loss of filtration surface area, is considered as the main structural lesion responsible for the relentless decline in glomerular function (Mauer et al., 1984; Steffes et al., 1992). There is persuasive evidence that this critical change may be the result of an altered mesangial cell (MC) metabolism involving the extracellular matrix. MCs in tissue culture synthesize proteoglycans, fibronectin, laminin, thrombospondin and various forms of collagens, primarily type IV and type I (Yaoita et al., 1990; Scheinman et al., 1978; Ayo et al., 1990). Therefore, a metabolic derangement of these cells in diabetes resulting in the excessive formation and deposition of these matrix components is a likely determinant of mesangial expansion and glomerulosclerosis.
The knowledge on how extracellular matrix synthesis is controlled in MCs is only fragmentary. It is known that the synthetic activity may be stimulated by very diverse factors, notably the mechanical strain induced by distending forces during glomerular hypertension (Riser et al., 1992) and the action of TGF-.beta.1 (Okuda et al., 1990). In diabetes, an obvious injurious alteration could be the continued presence of an abnormally high concentration of extracellular glucose. Recent evidence in humans (Bilous et al., 1989b; Barbosa et al., 1994) has confirmed early findings in animal studies (Rash, 1979; Steffes et al., 1980; Petersen et al., 1988) indicating that strict control of glycemia with insulin administration or successful pancreas transplantation may delay the onset and slow the progression of the characteristic mesangial matrix expansion. In addition, it has been amply demonstrated that MCs in tissue culture increase the production of fibronectin, laminin, collagen type IV as well as mRNA levels for these matrix proteins when incubated in the presence of elevated concentrations of glucose (Ayo et al., 1990; Ayo et al., 1991; Danne et al., 1993; Haneda et al., 1991; Nahman et al., 1992). Present indications are that even the effects of TGF-.beta. and mechanical strain are modulated by the level of extracellular glucose. (Riser, et al., 1995) Although it has been suggested that the increase in matrix synthesis may be partially due to the osmolar effect caused by high glucose concentrations (Nahman et al., 1992), most of the evidence accumulated thus far indicates that the change is related to the metabolism of glucose (Danne et al., 1993; Studer et al., 1993; Fumo et al., 1994). Glucose enters MCs by a facilitated diffusion process which is independent of insulin action (Kreisberg and Ayo, 1993). It has, therefore, been proposed that intracellular concentrations of glucose may approach those in the extracellular environment in diabetes (Kreisberg and Ayo, 1993). High intracellular concentrations of glucose may then increase extracellular matrix formation by activating the polyol pathway, inducing myo-inositol depletion, increasing non-enzymatic glycosylation of proteins, or by generation of the second messengers inositol triphosphate and diacylglycerol followed by transcriptional activation of extracellular matrix genes (Fumo et al., 1994; Kreisberg, 1992).
Interrelating the above, diabetes is a complex disorder associated with the aforementioned hyperglycemia, altered expression of growth factors, signal transducers, cytokines, and hypertension with mesangial cell stretch.
Recent work by several investigators has provided strong evidence of a role, still undefined, for hyperglycemia per se in the development of diabetic tissue complications (Ayo et al., 1990; Ayo et al., 1991). This is further supported by the results from the DCCT trial (Diabetes Control and Complications Trial Research Group, 1993) which demonstrated that strict control of blood glucose levels in diabetic patients led to slowing of the development of renal, nerve, and other diabetic tissue complications which commonly develop. It must be concluded that there is considerable evidence demonstrating several mechanisms by which high extracellular glucose concentrations play a key role in the development of diabetic tissue complications.
It is presently not known whether glucose acts at the cell membrane to elicit the effects described above or whether it must be taken up by the cell. Because glucose is a substrate for sorbitol synthesis and because high extracellular glucose stimulates sorbitol and extracellular matrix production by mesangial cells, it is desirable to develop a model which would allow the investigation of the role of glucose uptake and transport separate from the extracellular effects of high glucose, such as osmolality, glycosylation of proteins, stimulation of phospholipase-C, as well as other effects (Larkin and Dunlop, 1992). Additionally, it is desirable to develop new drugs and test existing drugs that are potentially useful in the treatment of diabetes, and specifically, the treatment of diabetic nephropathy, by the use of such a cell line. Finally, it would be useful to develop such a cell line to facilitate the study of the pathogenesis, progression, prevention and therapy of diabetic nephropathy. Current therapies for the treatment of diabetic complications are suboptimal. Even patients who have relatively well regulated blood glucose levels may still be at risk for diabetic nephropathy, and other tissue complications.