glycogen and Albuminuria

The redox sensitive glycogen synthase kinase (GSK) 3 has been recently implicated in the pathogenesis of proteinuric glomerulopathy. However, prior studies are less conclusive because they relied solely on chemical inhibitors of GSK3, which provide poor discrimination between the isoforms of GSK3 apart from potential off target activities. In murine kidneys, the β rather than the α isoform of GSK3 was predominantly expressed in glomeruli and distributed intensely in podocytes. By employing the doxycycline-activated Cre-loxP site specific gene targeting system, GSK3β was successfully knocked out (KO) selectively in podocytes in adult mice, resulting in a phenotype no different from control littermates. Electron microscopy of glomeruli in KO mice demonstrated more glycogen accumulation in podocytes but otherwise normal ultrastructures. Upon oxidative glomerular injury induced by protein overload, KO mice excreted significantly less albuminuria and had much attenuated podocytopathy and glomerular damage. The anti-proteinuric and glomerular protective effect was concomitant with diminished accumulation of reactive oxygen species in glomeruli in KO mice, which was likely secondary to a reinforced Nrf2 antioxidant response in podocytes. Collectively, our data suggest that GSK3β is dispensable for glomerular function and histology under normal circumstances but may serve as a therapeutic target for protecting from oxidative glomerular injuries.

Topics: Albuminuria; Animals; Antioxidants; Cells, Cultured; Glycogen; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Immunoblotting; Kidney Diseases; Kidney Glomerulus; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microscopy, Confocal; Microscopy, Electron, Transmission; Oxidation-Reduction; Podocytes; Reactive Oxygen Species

Glycogen synthase kinase (GSK)3 is a ubiquitously expressed serine/threonine kinase existing in two isoforms, namely GSK3α and GSK3β. Aside from the long-recognized role in insulin signal transduction and glycogen biosynthesis, GSK3β has been recently coined as a master control molecule in nuclear factor-κB activation and inflammatory kidney injury. Nevertheless, previous studies are less conclusive because they relied greatly on small molecule inhibitors, which lack selectivity and barely distinguish between the GSK3 isoforms. In addition, early embryonic lethality after global knockout of GSK3β precludes interrogation of the biological role of GSK3β in the adult kidney. To circumvent these issues, the Cre/loxP system was used to generate a conditional knockout mouse model in which the GSK3β gene was specifically deleted in kidney cortical tubules at postnatal mature stage. Kidney-specific ablation of GSK3β resulted in a phenotype no different from control littermates. Knockout mice (KO) were viable and exhibited normal development and normal kidney physiology in terms of kidney function, urine albumin excretion, and urine-concentrating ability. It is noteworthy that apart from normal glomerular and tubulointerstitial morphology, the kidneys from KO demonstrated more glycogen accumulation in the renal cortical tubules as assessed by both periodic acid-Schiff staining for light microscopy and direct biochemical assay, consistent with an elevated glycogen synthetic activity as evidenced by diminished inhibitory phosphorylation of glycogen synthase that occurred subsequent to GSK3β ablation. This finding was further validated by electron microscopic observations of increased deposition of glycogen particles in the renal tubules of KO, suggesting that GSK3α could not fully compensate for the loss of GSK3β in regulating glycogen metabolism in the kidney. Collectively, our study suggests that kidney-specific ablation of GSK3β barely affects kidney function and histology under normal circumstances. Extended examinations of these KO under diseased conditions are merited to understand the role of GSK3β in renal pathophysiology.

Topics: Albuminuria; Animals; Blotting, Western; Female; Glycogen; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Kidney; Kidney Concentrating Ability; Kidney Glomerulus; Kidney Tubules; Male; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Reverse Transcriptase Polymerase Chain Reaction; Time Factors

A subgroup of patients with type 2 diabetes shows a clustering of abnormalities such as peripheral insulin resistance, hypertension, and microalbuminuria. To evaluate whether these traits reflect intrinsic disorders of cell function rather than in vivo environmental effects, we studied a group of 7 nondiabetic hypertensive subjects with an altered albumin excretion rate (AER) (HyMA+) and 3 groups of patients with type 2 diabetes: 7 with normal blood pressure and normal AER (DH-MA-), 7 with high blood pressure and normal AER (DH+MA-), and 7 with both high blood pressure and altered AER (DH+MA+). Glucose disposal was measured during an hyperinsulinemic clamp (40 mU. m(2)(-1). min(-1)) with primed deuterated [6.6 (2)H(2)] glucose infusion. In the same subjects, a skin biopsy was performed and the following parameters were investigated: glucose transport (as determined by [(3)H]2-deoxyglucose uptake); glycogen synthase activity (as determined by [(14)C] glucose incorporation from UDP-[U-(14)C] glucose into glycogen); glycogen phosphorylase activity (as measured by the incorporation of [U-(14)C]glucose 1-phosphate into glycogen); and total glycogen content. In vivo glucose disposal was significantly reduced in DH+MA- and DH+MA+, with respect to DH-MA-, HyMA+, and controls. Insulin-stimulated glucose transport was similar in the 3 groups of patients with diabetes. A significant reduction of intracellular glycogen content was observed in DH+MA- and DH+MA+ compared with DH-MA- in both basal and insulin-stimulated conditions, probably because of a major impairment of glycogen synthase activity. Glycogen phosphorylase activity did not show differences between the groups. These results suggest that (1) the combination of type 2 diabetes with hypertension and altered AER is associated with impaired insulin sensitivity, and (2) intrinsic, possibly genetic, factors may account for increased peripheral insulin resistance in hypertensive microalbuminuric patients with type 2 diabetes, pointing to the reduction of glycogen synthase activity as a shared common defect.

Topics: Albuminuria; Cells, Cultured; Deoxyglucose; Diabetes Mellitus, Type 2; Female; Fibroblasts; Glucose; Glucose Clamp Technique; Glycogen; Glycogen Synthase; Humans; Hypertension; Insulin; Insulin Resistance; Liver; Male; Middle Aged

Tamm-Horsfall glycoprotein, present only in the kidney thick ascending limb of Henle's loop, was studied here in streptozotocin diabetic rats. Tamm-Horsfall glycoprotein mRNA in situ hybridization was performed on snap-frozen left kidneys; the right kidneys were perfusion-fixed with 4% paraformaldehyde and embedded either in paraffin, for Tamm-Horsfall glycoprotein immunohistochemistry, or in Epon for stereologic measurements. The length of the thick ascending limb of Henle's loop and the amount of glycogen were measured and the ultrastructure of the cells was evaluated. Urinary excretion of Tamm-Horsfall glycoprotein, calcium, magnesium and albumin was measured. After 10 and 50 days' duration of diabetes, kidney weight increased 20 and 41%, respectively and the length of the thick ascending limb of Henle's loop increased 28 and 56%, respectively, compared with controls. Substantial glycogen accumulations were present in the thick ascending limb of Henle's loop, and electron microscopy revealed a significant decrease in organelles and basolateral membranes. After 10 and 50 days' duration of diabetes, in situ hybridization of Tamm-Horsfall glycoprotein mRNA revealed a fourfold decrease, and the immunostaining for Tamm-Horsfall glycoprotein showed a threefold decrease as measured by densitometry. However, urinary Tamm-Horsfall glycoprotein excretion rate was increased fivefold and urinary concentration about twofold. Urinary calcium excretion increased three-fold and magnesium twofold, but urinary albumin excretion was not significantly increased. The increased amount of Tamm-Horsfall glycoprotein, calcium and magnesium in the urine in diabetes occurs here concomitant with severe cellular damage in the thick ascending limb of Henle's loop.

Topics: Albuminuria; Animals; Calcium; Diabetes Mellitus, Experimental; Diuresis; Female; Glycogen; Immunohistochemistry; In Situ Hybridization; Kidney; Kidney Cortex; Kidney Medulla; Loop of Henle; Magnesium; Microscopy, Electron; Organ Size; Orosomucoid; Rats; Rats, Wistar; Reference Values; RNA, Messenger