Activation of glycogen synthase kinase 3β ameliorates diabetes-induced kidney injury

Abstract

Increase in protein synthesis contributes to kidney hypertrophy and matrix protein accumulation in diabetes. We have previously shown that high glucose-induced matrix protein synthesis is associated with inactivation of glycogen synthase kinase 3β (GSK3β) in renal cells and in the kidneys of diabetic mice. We tested whether activation of GSK3β by sodium nitroprusside (SNP) mitigates kidney injury in diabetes. Studies in kidney-proximal tubular epithelial cells showed that SNP abrogated high glucose-induced laminin increment by stimulating GSK3β and inhibiting Akt, mTORC1, and events in mRNA translation regulated by mTORC1 and ERK. NONOate, an NO donor, also activated GSK3β, indicating that NO may mediate SNP stimulation of GSK3β. SNP administered for 3 weeks to mice with streptozotocin-induced type 1 diabetes ameliorated kidney hypertrophy, accumulation of matrix proteins, and albuminuria without changing blood glucose levels. Signaling studies showed that diabetes caused inactivation of GSK3β by activation of Src, Pyk2, Akt, and ERK; GSK3β inhibition activated mTORC1 and downstream events in mRNA translation in the kidney cortex. These reactions were abrogated by SNP. We conclude that activation of GSK3β by SNP ameliorates kidney injury induced by diabetes.

Keywords: Cell Signaling; Diabetic Nephropathy; Eukaryotic Translation Initiation; Extracellular Matrix Protein; Glycogen Synthase Kinase 3 (GSK-3); Kidney; Kidney Hypertrophy; Mammalian Target of Rapamycin (mTOR); Sodium Nitroprusside; Type 1 Diabetes.

FIGURE 1.

SNP activates GSK3β in MCT cells. A, equal amounts of cell lysate protein were immunoblotted with specific antibodies to detect HG (30 mm)-induced changes in phosphorylation (P) of GSK3α/β at Ser-21/9 and at Tyr-279/216 and eIF2Bϵ at Ser-535. B, dose-dependent effect of SNP on phosphorylation of GSK3β at Ser-9 and eIF2Bϵ at Ser-535 in MCT cells. C, effect of SNP on HG-induced changes in phosphorylation of GSK3β on Ser-9 and eIF2Bϵ on Ser-535. Loading of equal protein was assessed by immunoblotting using antibodies against the respective proteins. Representative blots and histograms of composite data from 3–4 experiments are shown in A–C. Error bars, S.E.

FIGURE 2.

SNP inhibits high glucose stimulation of matrix protein expression in MCT cells. MCT cells were treated with or without high glucose for 15 min following preincubation with or without SNP for 30 min. Equal amounts of cell lysate protein were immunoblotted with specific antibodies to detect changes in laminin β1 (A) and laminin γ1 (B). Immunoblotting showed that SNP blocked high glucose-induced phosphorylation (P) and activation of Akt (C) and ERK (D). Loading of equal protein was assessed by immunoblotting using antibodies against respective proteins. Representative blots and histogram of composite data from 3–4 experiments are shown. Error bars, S.E.

FIGURE 3.

High glucose-induced activation of Src and Pyk2 is required for matrix protein synthesis via GSK 3β inactivation. Equal amounts of cell lysate protein were immunoblotted with their respective antibodies. Preincubation of cells with SNP inhibited HG-induced activation of Src (A) and Pyk2 (B). Preincubation with PP2 (a Src inhibitor) and BAPTA/AM (a Pyk2 inhibitor) blocked HG-induced GSK3β phosphorylation (P) at Ser-9 (C and D) and synthesis of laminin γ1 (E and F). HG-induced activation of Akt and ERK was blocked by PP2 (G and I) and BAPTA/AM (H and J). Immunoblotting using antibody against the respective total proteins or actin was done to assess loading. Representative blots and histograms of composite data from 3–4 experiments are shown. Error bars, S.E.

FIGURE 4.

Sodium nitroprusside inhibits high glucose-induced events in mRNA translation in MCT cells. MCT cells were treated with or without high glucose following preincubation with or without SNP for 15 min. Equal amounts of cell lysate protein were immunoblotted with specific antibodies to detect changes in phosphorylation (P) status of p70S6 kinase (A), ribosomal S6 protein (B), eIF4E (C), eIF4G (D), eEF2 (E), and eEF2 kinase (F). Loading of equal protein was assessed by immunoblotting using antibodies against respective proteins. Representative blots and histograms of composite data from 3–4 experiments are shown. Error bars, S.E.

FIGURE 5.

Determination of optimal dose of SNP. A, mice were injected intraperitoneally with saline or SNP (200, 400, and 800 μg/kg) in a similar volume daily for 6 days. Blood pressure was measured on alternate days by the tail cuff method in conscious mice using the CODA non-invasive blood pressure system (Kent Scientific Corp., Torrington, CT). Mice were trained for 1 week on the restrainer placed on a warm platform. Blood pressure (systolic, diastolic, and mean arterial pressure) in each mouse was recorded over 30 cuff inflations after 10 training cuff inflations (51). B, mice treated as above were sacrificed on day 6. Renal cortical homogenates were immunoblotted with antibodies against phospho (P)-Ser-9 GSK3β, phospho-Ser-535 eIF2Bϵ, GSK3β, or eIF2Bϵ. The histogram shows quantitative data from three mice in each group. Error bars, S.E.

FIGURE 6.

Sodium nitroprusside ameliorates albuminuria and kidney hypertrophy in diabetic mice. SNP or vehicle was administered to control (Con) and streptozotocin-induced type 1 diabetic (Diab) mice for 3 weeks as described under “Experimental Procedures.” SNP ameliorated kidney hypertrophy (A) (n = 5–8 in each group) and albuminuria (B) (n = 5–8 in each group). Error bars, S.E.

FIGURE 7.

Sodium nitroprusside inhibits kidney matrix protein increment in diabetic mice. Equal amounts of renal cortical lysate protein were immunoblotted with specific antibodies for matrix proteins fibronectin (A), laminin β1 (B), and laminin γ1 (C). D and E, regulation of TGFβ expression and SMAD3 phosphorylation (P), respectively. Loading was assessed by immunoblotting for actin or SMAD3. Representative blots and histograms are shown for n = 5–8 mice/group. Error bars, S.E.

FIGURE 8.

Immunohistochemical analysis of renal laminin expression. Immunoperoxidase staining with an antibody against laminin trimer showed increased deposition in glomerular mesangium in kidneys from diabetic mice (C) compared with non-diabetic control mice (A). SNP (D) ameliorated diabetes-induced changes in renal laminin expression when compared with kidney from diabetic mice (C). SNP did not affect laminin content in non-diabetic control mice (B). The histogram shows composite averages of renal glomerular tuft area (E) and immunoperoxidase staining intensities of laminin (F) in control and diabetic mice treated with or without SNP. For each mouse, 25–40 glomeruli were analyzed (n = 4 in each group). Error bars, S.E.

FIGURE 9.

Sodium nitroprusside inhibits diabetes-induced signaling reactions in the kidney cortex. Equal amounts of protein from renal cortical lysate were immunoblotted with corresponding antibodies to assess changes in phosphorylation (P) of GSK 3α/β (A) and eIF2Bϵ (B). Immunoblotting was done to identify upstream regulators of GSK 3β phosphorylation: Akt (C), ERK (D), Src (E), and Pyk2 (F). Immunoblotting with antibodies against respective total protein was done to assess loading. Representative blots and histograms are shown for 5–8 mice in each group. Error bars, S.E.

FIGURE 10.

Sodium nitroprusside inhibits diabetes-induced events in mRNA translation in kidney cortex. Equal amounts of renal cortical lysate protein were immunoblotted with antibodies to assess changes in phosphorylation (P) of p70S6 kinase (A) ribosomal S6 protein (B), eIF4E (C), and eEF2 kinase (D). Loading was assessed by immunoblotting with antibodies for respective total protein. Representative blots and histograms are shown for 5–8 mice in each group. Error bars, S.E.