Regulation of elongation phase of mRNA translation in diabetic nephropathy: amelioration by rapamycin

Abstract

High glucose and high insulin, pathogenic factors in type 2 diabetes, induce rapid synthesis of the matrix protein laminin-beta1 in renal proximal tubular epithelial cells by stimulation of initiation phase of mRNA translation. We investigated if elongation phase of translation also contributes to high glucose and high insulin induction of laminin-beta1 synthesis in proximal tubular epithelial cells. High glucose or high insulin rapidly increased activating Thr56 dephosphorylation of eEF2 and inactivating Ser366 phosphorylation of eEF2 kinase, events that facilitate elongation. Studies with inhibitors showed that PI3 kinase-Akt-mTOR-p70S6 kinase pathway controlled changes in phosphorylation of eEF2 and eEF2 kinase induced by high glucose or high insulin. Renal cortical homogenates from db/db mice in early stage of type 2 diabetes showed decrease in eEF2 phosphorylation and increment in eEF2 kinase phosphorylation in association with renal hypertrophy and glomerular and tubular increase in laminin-beta1 content. Rapamycin, an inhibitor of mTOR, abolished diabetes-induced changes in phosphorylation of eEF2, eEF2 kinase, and p70S6 kinase and ameliorated renal hypertrophy and laminin-beta1 protein content, without affecting hyperglycemia. These data show that mTOR is an attractive target for amelioration of diabetes-induced renal injury.

Figure 1

High glucose and high insulin regulate eEF2 phosphorylation in MCT cells. Serum-deprived MCT cells were incubated with high glucose (30 mmol/L) (A), 5 mmol/L glucose + 25 mmol/L mannitol (B), or high insulin (1 nmol/L) (C) for the indicated period of time. Equal amounts of proteins were used for immunoblotting with Thr56 phospho-specific eEF2 antibody. Bottom panels show immunoblot analysis of the same samples with eEF2 antibody to assess loading. A representative blot from three to four independent experiments is shown and composite data are shown in histograms (^‡P < 0.01, ^†P < 0.05 versus control by analysis of variance).

Figure 2

High glucose and high insulin promote Ser366 phosphorylation of eEF2 kinase in MCT cells. Equal amounts of lysate proteins from cells incubated with high glucose (A) or high insulin (B) were immunoblotted with antibody against Ser366-phosphorylated eEF2 kinase. Bottom panels show assessment of loading by immunoblot analysis of the same samples with eEF2 kinase antibody. Composite data from three to four individual experiments are shown in histograms (^‡P < 0.01, ^†P < 0.05 versus control).

Figure 3

Activation of p70S6 kinase is required for high glucose- or high insulin-induced laminin-β1 synthesis in MCT cells. Equal amounts of lysate proteins from cells treated with high glucose (A) or high insulin (B) were immunoblotted with an antibody against Thr389-phosphorylated p70S6 kinase. Bottom panels show immunoblot analysis of the same lysates with actin antibody to assess loading. Composite data from three to four individual experiments are shown in histograms (^‡P < 0.01, ^†P < 0.05 versus control by analysis of variance). C and D: Lysates from cells incubated with high glucose or high insulin after transfection with empty vector or plasmid carrying DN-p70S6 kinase construct were immunoblotted with antibody against laminin-β1 (top) or actin (middle). Immunoblotting with anti-HA antibody was done to demonstrate the expression of dominant-negative p70S6 kinase (bottom). A–D: Representative blots from three to four experiments are shown. Composite data from three to four individual experiments are shown in histograms (^‡P < 0.01, ^†P < 0.05, *P < 0.001 by analysis of variance).

Figure 4

Activation of p70S6 kinase is required for high glucose- or high insulin-induced phosphorylation of eEF2 and eEF2 kinase. Equal amounts of lysate protein from cells incubated with high glucose or high insulin transfected with control plasmid or plasmid carrying DN-p70S6 kinase construct were immunoblotted with antibody against Thr56-phosphorylated eEF2 (A and B) or Ser366-phosphorylated eEF2 kinase (C and D). Loading was assessed by immunoblotting with antibody against eEF2 (A and B) or eEF2 kinase (C and D). Immunoblotting with anti-HA antibody was done to demonstrate the expression of dominant-negative p70S6 kinase (bottom). A–D: Representative blots from three experiments are shown. Composite data from three individual experiments are shown in histograms (^‡P < 0.01, ^†P < 0.05 by analysis of variance).

Figure 5

High glucose- or high insulin-induced changes in phosphorylation of p70S6 kinase, eEF2 and eEF2 kinase, and laminin-β1 synthesis are mTOR-dependent. Equal amounts of lysate protein from cells incubated with high glucose or high insulin with or without preincubation with 22 nmol/L rapamycin were immunoblotted with antibody against Thr389-phosphorylated p70S6 kinase, Thr56-phosphorylated eEF2, Ser366-phosphorylated eEF2 kinase, or laminin-β1. Bottom panels show immunoblotting done with antibody against actin, eEF2 to assess loading. A–F: Representative blots from three experiments are shown. Composite data from three individual experiments are shown in histograms (^‡P < 0.01, ^†P < 0.05, *P < 0.001 by analysis of variance).

Figure 6

Activation of PI-3 kinase and Akt is required for high glucose- and high insulin-induced dephosphorylation of eEF2. A and B: MCT cells were infected with Ad-GFP or Ad-DN-Akt (100 MOI) for 24 hours before the addition of high glucose or high insulin. C and D: In separate experiments, cells were preincubated with or without LY294002, a PI3 kinase inhibitor, before incubation with or without high glucose or high insulin. Equal amounts of lysate protein from cells were immunoblotted with antibody against Thr56-phosphorylated eEF2. Middle panels in A and B, and bottom panels in C and D show immunoblotting with eEF2 to assess loading. Bottom panels (A and B) show immunoblotting with HA to demonstrate successful infection of Ad-DN-Akt. Representative blots from three experiments are shown. Composite data from three individual experiments are shown in histograms (^‡P < 0.01, ^†P < 0.05 by analysis of variance).

Figure 7

Rapamycin inhibits renal hypertrophy in diabetic mice. A: Groups of db/m control and db/db type 2 diabetic mice received either vehicle or rapamycin for 14 days after the onset of hyperglycemia and kidney weights were measured at sacrifice. Composite mean ± SE data from 12 mice in each group are shown in a histogram (^‡P < 0.01, db/db versus control (con), *P < 0.001, db/db versus db/db + rapa). B: Kidney sections were fixed and glomerular area was measured. Representative micrographs from six mice in each of the four groups are shown. Composite mean ± SE data from six mice in each group are shown in a histogram (^‡P < 0.01, db/db versus control, NS, not significant, db/db versus db/db + rapa). Scale bar = 25 μm.

Figure 8

Rapamycin inhibits laminin-β1 accumulation in diabetic mice. A and B: Expression of laminin-β1 in glomeruli (A) and tubules (B) was assessed by immunoperoxidase histochemistry in all of the four groups. Representative micrographs from six mice in each of the four groups are shown. Composite mean ± SE data from six mice in each group are shown in a histogram (*P < 0.001 db/db versus control, db/db versus db/db + rapa). C: Equal amounts of renal cortical lysates were immunoblotted with an antibody against laminin-β1; loading was assessed by immunoblotting for actin as shown in the bottom panel. Each lane represents data from a single animal. Composite values from six mice in each group are shown in a histogram (*P < 0.001, db/db versus control; *P < 0.001, db/db versus db/db + rapa). D: Real-time RT-PCR was performed on renal cortical preparations to explore changes in laminin-β1 mRNA; GAPDH was used as a control. Composite mean ± SE data from six mice in each control (con) and diabetic (db/db) groups are shown in a histogram (NS, not significant, control versus db/db by Student’s t-test). Scale bars = 50 μm.

Figure 9

Effect of rapamycin on elongation phase of mRNA translation in db/db mice. Equal amounts of renal cortical lysates were immunoblotted with antibody against Thr56-phosphorylated eEF2 (A) or Ser366-phosphorylated eEF2 kinase (B) or Thr389-phosphorylated p70S6 kinase (C); loading was assessed by immunoblotting for actin as shown in the bottom panels. Each lane represents data from an individual animal. Composite mean ± SE data from four to six mice in each group are shown in histograms (^†P < 0.05, ^‡P < 0.01, *P < 0.001, control versus db/db; ^†P < 0.05, ^‡P < 0.01, db/db versus db/db + rapa).

Figure 10

A schematic of signaling pathways that control elongation phase of mRNA translation in the kidney in type 2 diabetes.