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. 2010 Jan;298(1):F49-61.
doi: 10.1152/ajprenal.00032.2009. Epub 2009 Sep 2.

Interplay between Akt and p38 MAPK pathways in the regulation of renal tubular cell apoptosis associated with diabetic nephropathy

Madhavi J Rane  1 , Ye Song, Shunying Jin, Michelle T Barati, Rui Wu, Hina Kausar, Yi Tan, Yuehui Wang, Guihua Zhou, Jon B Klein, Xiaokun Li, Lu Cai
Affiliations

Interplay between Akt and p38 MAPK pathways in the regulation of renal tubular cell apoptosis associated with diabetic nephropathy

Madhavi J Rane et al. Am J Physiol Renal Physiol. 2010 Jan.

Abstract

Hyperglycemia induces p38 MAPK-mediated renal proximal tubular cell (RPTC) apoptosis. The current study hypothesized that alteration of the Akt signaling pathway by hyperglycemia may contribute to p38 MAPK activation and development of diabetic nephropathy. Immunoblot analysis demonstrated a hyperglycemia-induced increase in Akt phosphorylation in diabetic kidneys at 1 mo, peaking at 3 mo, and dropping back to baseline by 6 mo. Immunohistochemical staining with anti-pAkt antisera localized Akt phosphorylation to renal tubules. Maximal p38 MAPK phosphorylation was detected concomitant with increase in terminal uridine deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells and caspase-3 activity in 6-mo diabetic kidneys. Exposure of cultured RPTCs to high glucose (HG; 22.5 mM) significantly increased Akt phosphorylation at 3, 6, and 9 h, and decreased thereafter. In contrast, p38 MAPK phosphorylation was detected between 9 and 48 h of HG treatment. Increased p38 MAPK activation at 24 and 48 h coincided with increased apoptosis, demonstrated by increased caspase-3 activity at 24 h and increased TUNEL-positive cells at 48 h of HG exposure. Blockade of p38 cascade with SB203850 inhibited HG-induced caspase-3 activation and TUNEL-positive cells. Overexpression of constitutively active Akt abrogated HG-induced p38 MAPK phosphorylation and RPTC apoptosis. In addition, blockade of the phosphatidylinositol-3 kinase/Akt pathway with LY294002 and silencing of Akt expression with Akt small interfering RNA induced p38 MAPK phosphorylation in the absence of HG. These results collectively suggest that downregulation of Akt activation during long-term hyperglycemia contributes to enhanced p38 MAPK activation and RPTC apoptosis. Mechanism of downregulation of Akt activation in 6-mo streptozotocin diabetic kidneys was attributed to decreased Akt-heat shock protein (Hsp) 25, Akt-p38 interaction, and decreased PTEN activity. Thus PTEN or Hsp25 could serve as potential therapeutic targets to modulate Akt activation and control p38 MAPK-mediated diabetic complications.

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Figures

Fig. 1.
Fig. 1.
Development of diabetic nephropathy in streptozotocin (STZ)-diabetic mice. In kidneys from control and diabetic mice 6 mo after STZ treatment, urine volume (A), urine albumin for 24 h (B), and blood glucose (C) were measured before mice were killed. Values are means ± SD (n = 6). *P < 0.05 vs. control.
Fig. 2.
Fig. 2.
Renal fibrosis in diabetic mice. Periodic acid-Schiff (PAS) staining was performed on kidney sections of control and diabetic mice 1, 3, and 6 mo after STZ treatment. The images indicate glomeruli with advanced mesangial expansion. A significant increase in PAS-positive material was detected in the diabetic glomeruli at 6 mo after STZ treatment indicative of increased fibrosis. Magnification: ×10 (left) and ×40 (right).
Fig. 3.
Fig. 3.
Diabetes-induced transcriptional upregulation of profibrotic markers in the kidney. Renal tissues from mice in each group (5 mice/group) were individually processed for RNA isolation with TRIzol reagent and purified with RNeasy columns. A gene array was run using the Atlas custom array specific cDNA synthesis primer mix (560 genes) and purified with Nucleospin columns. The membranes were prehybridized with Expresshyb and then exposed to a Molecular Dynamics Phosphoimage Screen. The images were quantified densitomertrically by using Atlas Images v2.0 software for the selected profibrotic markers. Gene expression intensities were first corrected for the external background and then globally normalized with the sum of all genes on the array. *P < 0.05 vs. corresponding controls (A). Kidney slices from 3- and 6-mo FVB and STZ-diabetic mice were subjected to immunohistochemistry (IHC) with anti-transforming growth factor (TGF)-β antibody (B).
Fig. 4.
Fig. 4.
Akt and p38 MAPK phosphorylation profiles during the development of diabetic nephropathy in STZ-induced diabetic mice. Kidney lysates from control and diabetic mice 1, 3, and 6 mo after STZ treatment were subjected to Western blotting with anti-Akt and anti-phosho-Ser473-Akt antibodies (A and B) or anti-p38 and ant-phospho-Thr 180/Tyr 182 p38 antibodies (D). Six mice were used for the above study. Values are means ± SD. *P < 0.05 1-mo vs. 3-mo STZ. #P < 0.05 3- vs. 6-mo STZ kidney lysates. Kidney sections from 3- and 6-mo FVB and STZ-diabetic mice were subjected to immunohistochemistry with anti-phosphoSer473-Akt antibody (C) and phospho-p38 MAPK antibody (E). Three-month STZ-diabetic kidney slices were also immunostained with isotype control antibody (C). Renal tubular lysates from n = 3 each untreated FVB, 10-day STZ-treated, and 10-day STZ- and insulin-treated mice were subjected to phosphoSer473-Akt, phosphop38 MAPK, and GAPDH immunoblotting (IB; F).
Fig. 5.
Fig. 5.
Diabetes-induced transcriptional upregulation of inflammatory markers. Procedures including tissue harvest and RNA isolation for detecting the transcriptional levels of inflammatory makers such as plasminogen activator inhibitor-1 (PAI-1; A) and TNF-α (C) were performed as described in Fig. 3. B: kidney sections from 3- and 6-mo FVB and STZ-diabetic mice were subjected to immunohistochemistry with anti-PAI-1 antibody. Differences in the inflammatory markers between control and diabetes groups were significant. *P < 0.05 vs. corresponding controls.
Fig. 6.
Fig. 6.
Activation of caspase-3 in 6-mo STZ-diabetic mice kidneys. A: kidney lysates from control and diabetic mice 1, 3, and 6 mo after STZ treatment were subjected to Western blotting with anti-actin and anti-active caspase-3 antisera. The activated form of caspase-3 was detected in 6-mo diabetic kidney lysates by Western blotting. Loading was normalized by β-actin immunoblotting. B: caspase-3 activation in 6-mo STZ-diabetic kidney lysates was confirmed by performing a terminal uridine deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay on 6-mo STZ-diabetic and age-matched control kidneys. Results demonstrate increased TUNEL positivity in 6-mo STZ-diabetic kidney sections compared with control kidneys. *P < 0.05 by Student's t-test.
Fig. 7.
Fig. 7.
Hyperglycemia-induced changes in Akt and p38 MAPK phosphorylation profiles in renal proximal tubular cells (RPTCs). RPTCs were incubated in DMEM/F12 medium containing either 5.5 mM d-glucose/17 mM mannitol (LG) or 22.5 mM d-glucose (HG) for 3–48 h. RPTC lysates were subjected to immunoblot analysis with anti-phoshoSer473Akt or anti-Akt antibodies (A and B) or anti-phosphop38 or anti-p38 antibodies (A and C). HG induced Akt and p38 MAPK phosphorylation in a time-dependent manner. Values are means ± SD obtained from at least 3 separate experiments. *P < 0.05 vs. corresponding LG groups.
Fig. 8.
Fig. 8.
Contrasting Akt kinase and p38 MAPK activities in RPTCs cultured with HG for 3 and 48 h. RPTCs were incubated in DMEM/F12 medium containing either 5.5 mM d-glucose/17 mM mannitol (LG) or 22.5 mM d-glucose (HG) for 3 or 48 h. RPTC lysates were subjected to anti-Akt or anti-p38 MAPK immunoprecipitation. Akt and p38 MAPK immunoprecipitates were subjected to an in vitro kinase assay utilizing histone H2B and Hsp27 as a substrate, respectively. A: similar to the Akt phosphorylation profile, Akt activity was detected after 3 h of HG treatment followed by a decrease in Akt activity at 48 h. B: in contrast, p38 MAPK activity was detected at 48 h of HG treatment.
Fig. 9.
Fig. 9.
The p38 MAPK pathway contributes to hyperglycemia-induced renal proximal tubular apoptosis. The effect of HG on RPTC apoptosis was examined by TUNEL staining (A and C) and caspase-3 activation (B and D). A: RPTCs were incubated in DMEM/F12 medium containing either 5.5 mM d-glucose/17 mM mannitol (LG) or 22.5 mM d-glucose (HG) for 12, 24, and 48 h followed by determination of TUNEL-positive cells. B: RPTCs were exposed to LG and HG for 24 h, and caspase-3 activity was determined. C: RPTCs were exposed to LG and HG in the presence and absence of p38 MAPK inhibitor SB203580 (3 μM final concentration) for 24 h, and cells were subjected to TUNEL assay. D: RPTCs were exposed to LG and HG in the presence and absence of p38 MAPK inhibitor SB203580 (3 μM final concentration) for 24 h, and caspase-3 activity was determined. Values are means ± SD obtained from at least 3 separate experiments with samples in triplicate. aP < 0.05 vs. corresponding LG group. bP < 0.05 vs. HG.
Fig. 10.
Fig. 10.
Blockade of phosphatidylinositol-3 kinase (PI-3K)/Akt pathway stimulates p38 MAPK phosphorylation. A: RPTCs were incubated with LG and HG medium for 24 h in the presence and absence of PI-3K inhibitor LY294002 (LY; 25 μM). RPTC lysates were immunoblotted with anti-phospho-p38 MAPK and anti-β-actin antisera. HG treatment for 24 h induces p38 MAPK phosphorylation in RPTCs. Blockade of the PI-3K pathway, a known upstream regulator of Akt, induces p38 MAPK phosphorylation in the absence of HG. B: RPTCs were transfected with control small interfering RNA (siRNA) or Akt siRNA for 24 h. Cell lysates generated were immunoblotted with anti-Akt antibody to document effective gene silencing. In addition, blots were stripped and reprobed with anti-phospho-p38 MAPK and β-actin antisera.
Fig. 11.
Fig. 11.
Akt overexpression inhibits p38 MAPK activation and RPTC apoptosis. RPTCs were transfected with pUse vector or c-myc-tagged constitutively active Akt (AktCA) construct for 24 h. Transfected cells were then subjected to control (no mannitol) or LG (5 mM d-glucose ± 17.5 mM mannitol) or HG (22 mM d-glucose) medium for 48 h. Cell lysates were subjected to immunoblot analysis with anti-pp38 MAPK, c-myc, and anti-β-actin (A). B: anti-active caspase-3 immunoblot analysis. *P < 0.05 by Student's t-test.
Fig. 12.
Fig. 12.
Diabetes-induced changes in Akt-Hsp25, Akt-p38 MAPK, and Akt-PTEN interactions in control and diabetic kidneys or renal tubules. Three- and 6-mo FVB and STZ-diabetic kidney homogenates were subjected to anti-Akt immunoprecipitation and immunoblotted with anti-Hsp25 and anti-Akt antisera. A: representative immunoblot from 2 independent experiments performed. B: renal tubular lysates from 3- and 6-mo FVB and STZ-diabetic mice were subjected to anti-Akt immunoprecipitation and immunoblotted with anti-p38 MAPK and anti-PTEN antisera. C: renal tubular lysates from 3- and 6-mo STZ-diabetic mice were immunoblotted with phospho-PTEN and GAPDH antisera.
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