Diabetes is the major cause of end stage renal disease, and tubular alterations are now considered to participate in the development and progression of diabetic nephropathy (DN). Here, we report for the first time that expression of the insulin receptor (IR) in human kidney is altered during diabetes. We detected a strong expression in proximal and distal tubules from human renal cortex, and a significant reduction in type 2 diabetic patients. Moreover, isolated proximal tubules from type 1 diabetic rat kidney showed a similar response, supporting its use as an excellent model for in vitro study of human DN. IR protein down-regulation was paralleled in proximal and distal tubules from diabetic rats, but prominent in proximal tubules from diabetic patients. A target of renal insulin signaling, the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), showed increased expression and activity, and localization in compartments near the apical membrane of proximal tubules, which was correlated with activation of the GSK3β kinase in this specific renal structure in the diabetic condition. Thus, expression of IR protein in proximal tubules from type 1 and type 2 diabetic kidney indicates that this is a common regulatory mechanism which is altered in DN, triggering enhanced gluconeogenesis regardless the etiology of the disease.

The importance of renal and hepatic gluconeogenesis in glucose homeostasis is well established, but the cellular localization of the key gluconeogenic enzymes liver fructose-1,6-bisphosphatase (FBPase) and cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in these organs and the potential contribution of other tissues in this process has not been investigated in detail. Therefore, we analyzed the human tissue localization and cellular distribution of FBPase and PEPCK immunohistochemically. The localization analysis demonstrated that FBPase was expressed in many tissues that had not been previously reported to contain FBPase activity (e.g., prostate, ovary, suprarenal cortex, stomach, and heart). In some multicellular tissues, this enzyme was detected in specialized areas such as epithelial cells of the small intestine and prostate or lung pneumocytes II. Interestingly, FBPase was also present in pancreas and cortex cells of the adrenal gland, organs that are involved in the control of carbohydrate and lipid metabolism. Although similar results were obtained for PEPCK localization, different expression of this enzyme was observed in pancreas, adrenal gland, and pneumocytes type I. These results show that co-expression of FBPase and PEPCK occurs not only in kidney and liver, but also in a variety of organs such as the small intestine, stomach, adrenal gland, testis, and prostate which might also contribute to gluconeogenesis. Our results are consistent with published data on the expression of glucose-6-phosphatase in the human small intestine, providing evidence that this organ may play an important role in the human glucose homeostasis.

The expression of aldolase A and B isoenzyme transcripts was confirmed by RT-PCR in rat kidney and their cell distribution was compared with characteristic enzymes of the gluconeogenic and glycolytic metabolic pathway: fructose-1,6-bisphosphatase (FBPase), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate kinase (PK). We detected aldolase A isoenzyme in the thin limb and collecting ducts of the medulla and in the distal tubules and glomerula of the cortex. The same pattern of distribution was found for PK, but not for aldolase B, PEPCK, and FBPase. In addition, co-localization studies confirmed that aldolase B, FBPase, and PEPCK are expressed in the same proximal cells. This segregated cell distribution of aldolase A and B with key glycolytic and gluconeogenic enzymes, respectively, suggests that these aldolase isoenzymes participate in different metabolic pathways. In order to test if FBPase interacts with aldolase B, FBPase was immobilized on agarose and subjected to binding experiments. The results show that only aldolase B is specifically bound to FBPase and that this interaction was specifically disrupted by 60 microM Fru-1,6-P2. These data indicate the presence of a modulated enzyme-enzyme interaction between FBPase and isoenzyme B. They affirm that in kidney, aldolase B specifically participates, along the gluconeogenic pathway and aldolase A in glycolysis.

Nuclear localization has been observed for glycolytic enzymes but not for key gluconeogenic enzymes. We report our ¢ndings on the intracellular localization of liver FBPase in rat liver and kidney, the main organs in the endogenous glucose production. Immuno£uorescence and confocal analysis revealed that FBPase was present in the cytosol and, unexpectedly, inside the nucleus of hepatocytes and proximal cells of the nephron. Additionally, FBPase was found in the plasma membrane area of adjacent hepatocytes where glycogen is synthesized and in the apical region of proximal kidney cells. This subcellular distribution in multiple compartments suggests the presence of di¡er-ent localization signals on FBPase for diverse metabolic functions. ß

Expression of the sodium and ascorbic acid (AA) cotransporter SVCT2 is induced during the period of cellular arborization and synaptic maturation of early postnatal (P1-P5) rat cerebral neurons. The physiological importance of the transporter for neurons is evidenced by the lethality and delayed neuronal differentiation detected in mice with ablation of SVCT2. The mechanism(s) involved in these defects and the role of SVCT2 in neuronal branching have not been determined yet. To address this, we used lentiviral expression vectors to increase the levels of SVCT2 in N2a cells and analyzed the effects on neurite formation. Expression of a fusion protein containing the human SVCT2wt and EYFP induced an increase in the number of MAP2+ neurites and filopodia in N2a cells. Overexpression of SVCT2 and treatment with AA promoted ERK1/2 phosphorylation. Our data suggest that enhanced expression of the high affinity AA transporter SVCT2, which tightly regulates intracellular AA concentrations, induces neuronal branching that then activates key signaling pathways that are involved in the differentiation and maturation of cortical neurons during postnatal development.

During brain development, radial glial (RG) cells and the different progenitor subtypes are characterized by their bipolar morphology that includes an ovoid cell body and one or two radial processes that span across the developing cerebral wall. Different cells transport the reduced form of vitamin C, ascorbic acid (AA), using sodium-dependent ascorbic acid cotransporters (SVCT1 or SVCT2). SVCT2 is mainly expressed in the nervous system (CNS); however, its localization in the central nervous system during embryonic development along with the mechanism by which RG take up vitamin C and its intracellular effects is unknown. Thus, we sought to determine the expression and localization of SVCT2 during CNS development. SVCT2 is preferentially localized in the RG body at the ventricular edge of the cortex during the neurogenic stage (E12 to E17). The localization of SVCT2 overexpressed by in utero electroporation of E14 embryos is consistent with ventricular polarization. A similar distribution pattern was observed in human brain tissue sections at 9 weeks of gestation; however, SVCT2 immunoreaction was also detected in the inner and outer subventricular zone (SVZ). Finally, we used C17.2 neural stem cell line, J1ES cells and primary cell cultures derived from the brain cortex to analyze functional SVCT2 activity, AA effects in progenitor cells bipolar morphology, and SVCT2 expression levels in different culture conditions. Our results indicate that basal RG cells and apical intermediate and subapical progenitors are the main cell types expressing SVCT2 in the lissencephalic brain. SVCT2 was mainly detected in the apical region of the ventricular zone cells, contacting the cerebrospinal fluid. In gyrencephalic brains, SVCT2 was also detected in progenitor cells located in the inner and outer SVZ. Finally, we defined that AA has a strong radializing (bipolar morphology) effect in progenitor cells in culture and the differentiation condition modulates SVCT2 expression.

Isoform 1 of the sodium-vitamin C co-transporter (SVCT1) is expressed in the apical membrane of proximal tubule epithelial cells in adult human and mouse kidneys. This study is aimed at analyzing the expression and function of SVCTs during kidney development. RT-PCR and immunohistochemical analyses revealed that SVCT1 expression is increased progressively during postnatal kidney development. However, SVCT1 transcripts were barely detected, if not absent, in the embryonic kidney. Instead, the high-affinity transporter, isoform 2 (SVCT2), was strongly expressed in the developing kidney from E15; its expression decreased at postnatal stages. Immunohistochemical analyses showed a dynamic distribution of SVCT2 in epithelial cells during kidney development. In renal cortex tubular epithelial cells, intracellular distribution of SVCT2 was observed at E19 with distribution in the basolateral membrane at P1. In contrast, SVCT2 was localized to the apical and basolateral membranes between E17 and E19 in medullary kidney tubular cells but was distributed intracellularly at P1. In agreement with these findings, functional expression of SVCT2, but not SVCT1 was detected in human embryonic kidney-derived (HEK293) cells. In addition, kinetic analysis suggested that an ascorbate-dependent mechanism accounts for targeted SVCT2 expression in the developing kidney during medullary epithelial cell differentiation. However, during cortical tubular differentiation, SVCT1 was induced and localized to the apical membrane of tubular epithelial cells. SVCT2 showed a basolateral polarization only for the first days of postnatal life. These studies suggest that the uptake of vitamin C mediated by different SVCTs plays differential roles during the ontogeny of kidney tubular epithelial cells.

Diabetic nephropathy (DN) is a major complication of diabetic patients and the leading cause of end-stage renal disease. Glomerular dysfunction plays a critical role in DN, but deterioration of renal function also correlates with tubular alterations. Human DN is characterized by glycogen accumulation in tubules. Although this pathological feature has long been recognized, little information exists about the triggering mechanism. In this study, we detected over-expression of muscle glycogen synthase (MGS) in diabetic human kidney. This enhanced expression suggests the participation of MGS in renal metabolic changes associated with diabetes. HK2 human renal cell line exhibited an intrinsic ability to synthesize glycogen, which was enhanced after over-expression of protein targeting to glycogen. A correlation between increased glycogen amount and cell death was observed. Based on a previous transcriptome study on human diabetic kidney disease, significant differences in the expression of genes involved in glycogen metabolism were analyzed. We propose that glucose, but not insulin, is the main modulator of MGS activity in HK2 cells, suggesting that blood glucose control is the best approach to modulate renal glycogen-induced damage during long-term diabetes.