Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy

Abstract

Transforming growth factor beta (TGF-beta) has been shown to participate in the pathophysiology of diabetic complications. As shown most recently, TGF-beta stimulates the expression of a distinct serine/threonine kinase (hSGK) which had previously been cloned as an early gene transcriptionally regulated by cell volume alterations. The present study was performed to elucidate transcription and function of hSGK in diabetic nephropathy. As shown by Northern blotting, an increase of extracellular glucose concentration increased hSGK mRNA levels in cultured cells, an effect qualitatively mimicked by osmotic cell shrinkage or treatment with TGF-beta (2 microgram/liter), phorbol 12,13-didecanoate (1 microM), or the Ca(2+) ionophore ionomycin (1 microM) and blunted by high concentrations of nifedipine (10 and 100 microM). In situ hybridization revealed that hSGK transcription was markedly enhanced in diabetic nephropathy, with particularly high expression in mesangial cells, interstitial cells, and cells in thick ascending limbs of Henle's loop and distal tubules. According to voltage clamp and tracer flux studies in Xenopus oocytes expressing the renal epithelial Na(+) channel ENaC or the mouse thick ascending limb Na(+),K(+),2Cl(-) cotransporter BSC-1, coexpression with hSGK stimulated ENaC and BSC-1 11-fold and 6-fold, respectively, effects reversed by kinase inhibitors staurosporine (1 microM) and chelerythrine (1 microM) and not elicited by inactive hSGK. In conclusion, excessive extracellular glucose concentrations enhance hSGK transcription, which in turn stimulates renal tubular Na(+) transport. These observations disclose an additional element in the pathophysiology of diabetic nephropathy.

Figure 1

Transcriptional regulation of the serine/threonine kinase hSGK in 3T3 fibroblasts. (A) Influence on hSGK mRNA of an 8-h increase of extracellular glucose concentration from 5.5 to 30 mM and of an 8-h exposure to 2 ng/ml TGF-β-1. (B) Decay of mRNA levels after addition of 5 μg/ml actinomycin D at 30 mM glucose and at 5.5 mM glucose in hypotonic extracellular fluid. Before the experimental period, the cells were exposed to 30 mM glucose. (C) Effect of an 8-h increase of extracellular glucose concentration from 5.5 to 30 mM in presence and absence of 30 μg/ml anti-TGF-β-1 antibody.

Figure 2

Transcriptional regulation of hSGK in endothelial cells. (A) Effect of 20 mM, 50 mM, or 100 mM glucose within 2 h. (B) Effect of the Ca²⁺ ionophore ionomycin (1 μM), of phorbol 12,13-didecanoate (PDD; 1 μM) and of both ionomycin and PDD (incubation time 2 h). (C) Effect of glucose (100 mM), nifedipine (10 μM or 100 μM), and glucose (100 mM) in the presence of nifedipine (10 μM or 100 μM) (incubation time 2 h).

Figure 3

In situ detection of hSGK mRNA in intact kidneys and diabetic nephropathy. In normal kidney significant transcription of hSGK is found in a few mesangial cells and single epithelial cells of the distal tubule (A) and thick ascending limbs of Henle's loop (B, arrowheads). In diabetic kidneys (C–F), however, high levels of hSGK transcripts are found in numerous glomerular cells (C) and epithelial cells from the thick ascending limb of Henle's loop (D) as well as in clusters of interstitial cells of fibrotic areas (E). No labeling of cells was observed after hybridization with the α-³⁵S-labeled sense hSGK RNA probe (F). (×300.)

Figure 4

Stimulation of ENaC by hSGK. Amiloride-sensitive currents (30 μM amiloride) in Xenopus oocytes injected with the rat epithelial Na⁺ channel α,β,γ ENaC or the α^S622A,β,γ ENaC mutant with or without hSGK, or with ENaC + hSGK in the presence of staurosporine (1 μM) or chelerythrine (1 μM). (A) Original traces. (B) Effect of hSGK on wild-type ENaC with and without protein kinase inhibitors (arithmetic means ± SEM). (C) Effect of hSGK and of inactive hSGK^K127R on ENaC α^S622A-mediated currents (arithmetic means ± SEM).

Figure 5

Stimulation of the thick ascending limb Na⁺,K⁺,2Cl⁻ cotransporter BSC-1 by hSGK ²²Na⁺ uptake in Xenopus oocytes injected with and without the thick ascending limb Na⁺,K⁺,2Cl⁻ cotransporter BSC-1 and/or the active kinase hSGK or inactive kinase hSGK^K127R. In some of the experiments furosemide (100 μM) was added to inhibit BSC-1. Arithmetic means ± SEM.