Regulation of Elongation Phase of mRNA Translation in Diabetic Nephropathy
2007; Elsevier BV; Volume: 171; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2007.070412
ISSN1525-2191
AutoresKavithalakshmi Sataranatarajan, Meenalakshmi M. Mariappan, Myung Ja Lee, Denis Féliers, Goutam Ghosh Choudhury, Jeffrey L. Barnes, Balakuntalam S. Kasinath,
Tópico(s)Cancer-related gene regulation
ResumoHigh glucose and high insulin, pathogenic factors in type 2 diabetes, induce rapid synthesis of the matrix protein laminin-β1 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-β1 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-β1 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-β1 protein content, without affecting hyperglycemia. These data show that mTOR is an attractive target for amelioration of diabetes-induced renal injury. High glucose and high insulin, pathogenic factors in type 2 diabetes, induce rapid synthesis of the matrix protein laminin-β1 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-β1 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-β1 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-β1 protein content, without affecting hyperglycemia. These data show that mTOR is an attractive target for amelioration of diabetes-induced renal injury. Renal hypertrophy and matrix accumulation are signal events that occur sequentially in the course of renal disease in diabetes. The relevance of early onset renal hypertrophy to long-term complications has been controversial. Recent findings have suggested that interruption of renal hypertrophy in diabetes may ameliorate some but not all aspects of chronic changes in the renal structure and function.1Wolf G Schanze A Stahl RA Shankland SJ Amann K p27(Kip1) knockout mice are protected from diabetic nephropathy: evidence for p27(Kip1) haplotype insufficiency.Kidney Int. 2005; 68: 1583-1589Crossref PubMed Scopus (78) Google Scholar These data emphasize the need to understand fully the pathogenesis of early changes that occur in the kidney in diabetes. Protein synthesis constitutes the common underlying process for both hypertrophy and matrix accumulation in the diabetic kidney. However, the control mechanisms involved in modulation of protein synthesis in the kidney in diabetes are not well understood. Although transcriptional mechanisms and altered degradation contribute to changes in protein content, the role of mRNA translation as a site of dysregulation has not been investigated in depth in diabetic renal disease. mRNA translation is the process of synthesis of a polypeptide chain in the ribosome in accordance with the sequence of codons present in the mRNA.2Kasinath BS Mariappan MM Sataranatarajan K Lee MJ Feliers D mRNA translation: unexplored territory in renal science.J Am Soc Nephrol. 2006; 17: 3281-3292Crossref PubMed Scopus (52) Google Scholar, 3Proud CG Signalling to translation: how signal transduction pathways control the protein synthetic machinery.Biochem J. 2007; 403: 217-234Crossref PubMed Scopus (403) Google Scholar mRNA translation undergoes both spatial and temporal regulation. The temporal sequence of events in mRNA translation has been classified into initiation, elongation, and termination phases. In the initiation phase, under the control of eukaryotic initiation factors (eIFs), the ribosome attaches to the mRNA and is localized to the first codon, usually for methionine. During the elongation phase, sequential addition of amino acids occurs directed by the codon sequence with the participation of amino acyl tRNA; it is tightly regulated by eukaryotic elongation factors (eEFs).2Kasinath BS Mariappan MM Sataranatarajan K Lee MJ Feliers D mRNA translation: unexplored territory in renal science.J Am Soc Nephrol. 2006; 17: 3281-3292Crossref PubMed Scopus (52) Google Scholar During the termination phase, the peptide is released from the ribosomal complex after faithful synthesis of the full-length peptide. Studies in db/db mice with type 2 diabetes have shown that accumulation of the renal matrix protein laminin-β1 is not associated with increase in its mRNA, suggesting potential regulation by mRNA translation.4Ha TS Barnes JL Stewart JL Ko CW Miner JH Abrahamson DR Sanes JR Kasinath BS Regulation of renal laminin in mice with type II diabetes.J Am Soc Nephrol. 1999; 10: 1931-1939Crossref PubMed Google Scholar This possibility was directly studied in an in vitro model of type 2 diabetes. Incubation of renal proximal tubular epithelial cells (MCT cells) with high glucose or high insulin, two important pathogenic factors in type 2 diabetes, resulted in stimulation of laminin-β1 synthesis within minutes.5Mariappan MM Feliers D Mummidi S Choudhury GG Kasinath BS High glucose, high insulin, and their combination rapidly induce laminin-beta1 synthesis by regulation of mRNA translation in renal epithelial cells.Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (68) Google Scholar The rapid phase synthesis of laminin-β1 was not associated with changes in mRNA level; it could be blocked by cycloheximide but not by actinomycin D, suggesting regulation by mRNA translation.5Mariappan MM Feliers D Mummidi S Choudhury GG Kasinath BS High glucose, high insulin, and their combination rapidly induce laminin-beta1 synthesis by regulation of mRNA translation in renal epithelial cells.Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (68) Google Scholar Studies showed involvement of initiation phase of mRNA translation under the control of PI3 kinase-Akt-mTOR axis in high glucose and high insulin regulation of laminin-β1 translation.5Mariappan MM Feliers D Mummidi S Choudhury GG Kasinath BS High glucose, high insulin, and their combination rapidly induce laminin-beta1 synthesis by regulation of mRNA translation in renal epithelial cells.Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (68) Google Scholar In the current study we tested the hypothesis that the elongation phase of translation is also regulated by high glucose and high insulin in rapid phase synthesis of laminin-β1 in MCT cells. We characterized the signaling pathways that regulate elongation phase. Because the mammalian target of rapamycin (mTOR) plays a central role in regulation of elongation phase, we evaluated whether administration of rapamycin, a selective inhibitor of mTOR, would ameliorate pathological processes in the kidney in type 2 diabetes that require augmented protein synthesis. In contrast to the initiation phase of translation, the role of elongation phase has not been well studied, particularly, in in vivo models of disease. SV-40 immortalized murine proximal tubular epithelial (MCT) cells (kindly provided by Dr. Eric Neilson, Vanderbilt University, Nashville, TN) were grown in Dulbecco's modified Eagle's medium (Gibco-Invitrogen, Carlsbad, CA) containing 7% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT), 5 mmol/L glucose with no added insulin, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mmol/L glutamine. MCT cells express in vivo properties of proximal tubular epithelial cells.6Haverty TP Kelly CJ Hines WH Amenta PS Watanabe M Harper RA Kefalides NA Neilson EG Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis.J Cell Biol. 1988; 107: 1359-1368Crossref PubMed Scopus (276) Google Scholar The cells were grown to 90% confluence and then growth-arrested for 18 hours in serum-free Dulbecco's modified Eagle's medium before experiments that used 30 mmol/L glucose or 1 nmol/L insulin to represent high glucose and high insulin levels encountered in mice with type 2 diabetes.4Ha TS Barnes JL Stewart JL Ko CW Miner JH Abrahamson DR Sanes JR Kasinath BS Regulation of renal laminin in mice with type II diabetes.J Am Soc Nephrol. 1999; 10: 1931-1939Crossref PubMed Google Scholar, 5Mariappan MM Feliers D Mummidi S Choudhury GG Kasinath BS High glucose, high insulin, and their combination rapidly induce laminin-beta1 synthesis by regulation of mRNA translation in renal epithelial cells.Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (68) Google Scholar Five mmol/L glucose + twenty-five mmol/L mannitol were used as osmotic control for glucose. C57BLKsJ lepr−/− db/db mice and their lean littermate controls (db/m) were purchased from the Jackson Laboratory, Bar Harbor, ME. The db/db mice develop kidney disease related to type 2 diabetes resembling human disease, ie, hypertrophy, extracellular matrix accumulation, and albuminuria.4Ha TS Barnes JL Stewart JL Ko CW Miner JH Abrahamson DR Sanes JR Kasinath BS Regulation of renal laminin in mice with type II diabetes.J Am Soc Nephrol. 1999; 10: 1931-1939Crossref PubMed Google Scholar, 7Sharma K McCue P Dunn SR Diabetic kidney disease in the db/db mouse.Am J Physiol. 2003; 284: F1138-F1144Crossref PubMed Scopus (377) Google Scholar Experiments were initiated at 2 weeks after the appearance of hyperglycemia in db/db mice. Blood glucose measured by an Accucheck instrument (Bayer Diagnostics, Tarrytown, NY). The db/m and db/db mice were each divided into two groups. Control and diabetes groups received either vehicle of 0.2% of methylcellulose and 0.25% of polysorbate-80 in water or rapamycin (2 mg/kg/day) (LC Laboratories, Woburn, MA)8Shioi T McMullen JR Tarnavski O Converso K Sherwood MC Manning WJ Izumo S Rapamycin attenuates load-induced cardiac hypertrophy in mice.Circulation. 2003; 107: 1664-1670Crossref PubMed Scopus (394) Google Scholar administered intraperitoneally daily for 2 weeks. Kidney weight and blood glucose levels were measured at the time of sacrifice. The Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio approved these animal studies. At termination of the experiment, kidneys were excised, sliced, and fixed in neutral buffered formalin for routine paraffin embedding and subsequent sectioning and staining with hematoxylin and eosin. Glomerular area in stained sections was measured by computer-assisted image analysis as previously described.9Danda RS Habiba NM Rincon-Choles H Bhandari BK Barnes JL Abboud HE Pergola PE Kidney involvement in a nongenetic rat model of type 2 diabetes.Kidney Int. 2005; 68: 2562-2571Crossref PubMed Scopus (123) Google Scholar Photographic images were taken from 25 random glomeruli from each mouse using an Olympus AX70 research microscope equipped with a ×20 objective and DP70 digital camera (Olympus America Inc., Melville, NY). In each digital image, the circumference of the glomerulus was outlined and glomerular area calculated using the polygonal tracing tool of Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD). All images were calibrated to a stage micrometer. Image analysis was also used to measure glomerular and tubular basement membrane expression of laminin-β1 in each of the groups. Indirect immunoperoxidase histochemistry was performed on fresh frozen sections using antibody to laminin-β1 (Biomeda, Foster City, CA) and detected by the ABC technique (Vector Laboratories, Burlingame, CA) using diaminobenzidine as substrate according to previously reported methods.10Faulkner JL Szcykalski LM Springer F Barnes JL Origin of interstitial fibroblasts in an accelerated model of angiotensin II (Ang II)-induced renal fibrosis.Am J Pathol. 2005; 167: 1193-1205Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar The area occupying diaminobenzidine reaction product was measured in digital images by selecting a lower and upper range of gray scale within the limits of background and the highest intensity of diaminobenzidine staining. For glomerular staining, the circumference of each glomerulus was outlined, as above; the image of the reaction product identified by pseudo-coloring and area of specific staining calculated as a percentage of total glomerular area.10Faulkner JL Szcykalski LM Springer F Barnes JL Origin of interstitial fibroblasts in an accelerated model of angiotensin II (Ang II)-induced renal fibrosis.Am J Pathol. 2005; 167: 1193-1205Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Likewise, reaction product in the tubular basement membrane was measured as a percentage of a predetermined area of renal parenchyma excluding glomeruli. Total RNA was isolated from kidney cortex using TRI reagent (Sigma, St. Louis, MO) as previously described.5Mariappan MM Feliers D Mummidi S Choudhury GG Kasinath BS High glucose, high insulin, and their combination rapidly induce laminin-beta1 synthesis by regulation of mRNA translation in renal epithelial cells.Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (68) Google Scholar Two μg of total RNA from each sample was reverse-transcribed with random hexamers using a commercially available kit from Invitrogen (Carlsbad, CA). Mouse laminin-beta1 (Primer Bank ID no. 21595540a2) and mouse GAPDH (Primer Bank ID no. 6679937a1) were amplified using PCR primer sequences obtained from Primer Bank, a public resource for PCR primers.11Wang X Seed B A PCR primer bank for quantitative gene expression analysis.Nucleic Acids Res. 2003; 31: e154Crossref PubMed Scopus (682) Google Scholar Two μl of cDNA was amplified using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) containing 100 nmol/L forward and reverse primers. PCR amplification was performed using 7900HT sequence detection system (Applied Biosystems). Dissociation curve analysis was performed after PCR amplification to confirm the specificity of the primers. Relative mRNA expression was calculated using the ΔΔCt method. Western blotting was performed as described.12Senthil D Choudhury GG McLaurin C Kasinath BS Vascular endothelial growth factor induces protein synthesis in renal epithelial cells: a potential role in diabetic nephropathy.Kidney Int. 2003; 64: 468-479Crossref PubMed Scopus (78) Google Scholar In brief, equal amounts of cell extract protein (10 to 50 μg) were mixed with sample loading buffer and separated under reducing conditions on 7.5% gel. Proteins were electrotransferred onto a nitrocellulose membrane. The membrane was probed with primary antibody overnight and blocked for an hour in 5% nonfat dry milk. After washes in Tris-buffered saline containing 0.1% Tween-20, the membrane was incubated with antibodies against phospho Thr389-p70S6kinase, phospho-Thr56-eEF2, total eEF2, phospho-Ser366-eEF2 kinase, total eEF2 kinase (Cell Signaling Technology, Beverly, MA), actin (Sigma Aldrich, St. Louis, MO), and laminin-β1 (US Biologicals, Swampscott, MA) at 1:1000 for 3 hours. The membranes were then washed and incubated with secondary antibodies linked to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The reactive bands were detected by chemiluminescence using the ECL system (Pierce Biotechnology, Rockford, IL). Signal intensity was assessed by densitometric analysis. MCT cells were transiently transfected with either control plasmid without a cDNA insert (pRK7) or a plasmid containing K100R mutation in p70S6 kinase (kinase dead; kindly provided by Dr. John Blenis, Ph.D., Addgene, Cambridge, MA). Transfection was performed using lipofectamine and Lipo-plus reagent (Invitrogen) as described.13Senthil D Faulkner JL Choudhury GG Abboud HE Kasinath BS Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells.Biochem J. 2001; 360: 87-95Crossref PubMed Scopus (26) Google Scholar MCT cells were infected with adenovirus vector expressing dominant-negative HA-tagged Akt (Ad-DN-Akt).14Choudhury GG Akt serine threonine kinase regulates platelet-derived growth factor-induced DNA synthesis in glomerular mesangial cells: regulation of c-fos and p27(kip1) gene expression.J Biol Chem. 2001; 276: 35636-35643Crossref PubMed Scopus (81) Google Scholar Adenovirus containing green fluorescence protein (Ad-GFP) was used as control. Values are expressed as mean ± SE from a minimum of three experiments. Statistical comparisons between multiple groups were performed by analysis of variance with post testing correction by the Newman-Keuls method. Student's t-test was used for comparing two groups. Results were considered statistically different at P < 0.05. We have previously shown that synthesis of laminin-β1 was stimulated within 5 minutes in MCT cells incubated with high glucose or high insulin.5Mariappan MM Feliers D Mummidi S Choudhury GG Kasinath BS High glucose, high insulin, and their combination rapidly induce laminin-beta1 synthesis by regulation of mRNA translation in renal epithelial cells.Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (68) Google Scholar The rapid phase stimulation of laminin-β1 synthesis involved activation of initiation phase of mRNA translation under mTOR regulation.5Mariappan MM Feliers D Mummidi S Choudhury GG Kasinath BS High glucose, high insulin, and their combination rapidly induce laminin-beta1 synthesis by regulation of mRNA translation in renal epithelial cells.Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (68) Google Scholar Because both initiation and elongation phases of mRNA translation are under the control of mTOR,5Mariappan MM Feliers D Mummidi S Choudhury GG Kasinath BS High glucose, high insulin, and their combination rapidly induce laminin-beta1 synthesis by regulation of mRNA translation in renal epithelial cells.Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (68) Google Scholar we embarked on analysis of elongation phase regulation by high glucose and high insulin in the current study. During the elongation phase of translation, the shift of amino acyl tRNA from the aminoacyl (A) site to the peptidyl (P) site on the ribosome is regulated by eEF2, which is active when dephosphorylated on Thr56.15Redpath NT Price NT Severinov KV Proud CG Regulation of elongation factor-2 by multisite phosphorylation.Eur J Biochem. 1993; 213: 689-699Crossref PubMed Scopus (157) Google Scholar, 16Thornton S Anand N Purcell D Lee J Not just for housekeeping: protein initiation and elongation factors in cell growth and tumorigenesis.J Mol Med. 2003; 81: 536-548Crossref PubMed Scopus (119) Google Scholar Both high glucose and high insulin caused rapid dephosphorylation of eEF2 to ∼50% of its basal level at 5 minutes; whereas the effect of high glucose was biphasic, that of insulin was persistent for up to 60 minutes (Figure 1, A and C). Equimolar mannitol did not affect eEF2 phosphorylation (Figure 1B) or laminin-β1 content (data not shown) suggesting that high glucose regulation was not attributable to its osmotic effect. The temporal profile of eEF2 dephosphorylation induced by high glucose and high insulin was consonant with that of increment in laminin-β1 synthesis reported recently.5Mariappan MM Feliers D Mummidi S Choudhury GG Kasinath BS High glucose, high insulin, and their combination rapidly induce laminin-beta1 synthesis by regulation of mRNA translation in renal epithelial cells.Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (68) Google Scholar Phosphorylation of eEF2 on Thr56 is under the control of eEF2 kinase, a calcium-calmodulin-dependent kinase III.17Nairn AC Palfrey HC Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2.J Biol Chem. 1987; 262: 17299-17303Abstract Full Text PDF PubMed Google Scholar Activity of eEF2 kinase is reduced by phosphorylation on Ser366, which is regulated by p70S6 kinase.18Wang X Li W Williams M Terada N Alessi DR Proud CG Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase.EMBO J. 2001; 20: 4370-4379Crossref PubMed Scopus (637) Google Scholar Both high glucose and high insulin significantly augmented Ser366 phosphorylation of eEF2 kinase, which began at 5 minutes and peaked at 10 minutes (Figure 2, A and B). At 60 minutes, there was discordance between reduction in eEF2 phosphorylation induced by high glucose and high insulin and predicted changes in Ser366 phosphorylation of eEF2 kinase. These data suggest that another factor such as a phosphatase may also be involved in regulation of eEF2 phosphorylation. Immunoblotting with phospho-specific antibodies showed that high glucose and high insulin augmented Thr389 phosphorylation of p70S6 kinase corresponding to changes in phosphorylation of eEF2 and eEF2 kinase (Figure 3, A and B). MCT cells were transiently transfected with control empty plasmid or a plasmid carrying a hemagglutinin (HA)-tagged kinase-dead construct of p70S6 kinase with K100R mutation. High glucose and high insulin stimulated laminin-β1 synthesis in cells expressing the control vector but not in those expressing dominant-negative p70S6 kinase mutant (Figure 3, C and D). Additionally, high glucose and high insulin promptly reduced Thr56 phosphorylation of eEF2 (Figure 4, A and B) and augmented Ser366 phosphorylation of eEF2 kinase (Figure 4, C and D) in control cells transfected with empty plasmid; these changes were abrogated by the expression of kinase dead p70S6 kinase (Figure 4). These data showed the requirement of p70S6 kinase for changes in phosphorylation of eEF2 kinase and eEF2 and laminin-β1 synthesis induced by high glucose or high insulin.Figure 4Activation 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We sought to identify upstream kinases that regulate activation of p70S6 kinase beginning with mTOR. Rapamycin, a specific inhibitor of mTOR, significantly inhibited high glucose- and high insulin-induced increase in Thr389 phosphorylation of p70S6 kinase (Figure 5, A and B) and Ser366 phosphorylation of eEF2 kinase and reduction in Thr56 phosphorylation of eEF2 (Figure 5, C and D). Lack of inhibition of baseline eEF2 Thr56 and eEF2 kinase Ser366 phosphorylation by rapamycin, suggests that a kinase other than mTOR may be involved in these reactions. In addition, rapamycin abolished high glucose- and high insulin-induced laminin-β1 synthesis (Figure 5, E and F). Because Akt is upstream of mTOR in receptor tyrosine kinase signaling pathways,19Hay N Sonenberg N Upstream and downstream of mTOR.Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3484) Google Scholar we tested its role in control of eEF2. High glucose and high insulin promoted eEF2 dephosphorylation in MCT cells expressing the control Ad-GFP but not in cells infected with Ad-DN Akt (Figure 6, A and B). mTOR is under the control of PI3 kinase in MCT cells incubated with high glucose and high insulin.5Mariappan MM Feliers D Mummidi S Choudhury GG Kasinath BS High glucose, high insulin, and their combination rapidly induce laminin-beta1 synthesis by regulation of mRNA translation in renal epithelial cells.Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (68) Google Scholar However, in that study we had not explored if PI3 kinase-mTOR axis-controlled phosphorylation of eEF2. LY294002, a selective PI3 kinase inhibitor, inhibited eEF2 dephosphorylation in cells incubated with either high glucose or high insulin (Figure 6, C and D) suggesting that PI3 kinase was an upstream regulator of eEF2 phosphorylation. Taken together, these data demonstrate that high glucose- and high insulin-induced laminin-β1 synthesis in MCT cells involves elongation phase of translation that is under the control of PI3 kinase-Akt-mTOR-p70S6 kinase axis. We proceeded to verify high glucose and high insulin regulation of eEF2 and eEF2 kinase in renal cortex of diabetic mice during hypertrophy and onset of laminin-β1 synthesis when protein synthesis and elongation phase of translation would be predicted to be stimulated. We used db/db mice with type 2 diabetes and administered rapamycin in early stages when hyperglycemia and hyperinsulinemia are evident. Blood glucose level in the untreated db/db mice was significantly elevated compared to db/m controls (380 ± 46 versus 134 ± 9 mg/dl, mean ± SE, *P < 0.001). Rapamycin given from day 14 of hyperglycemia for 14 days did not significantly affect the blood glucose concentration in either the control mice (147 ± 6 versus 134 ± 9 mg/dl) or the diabetic mice (418 ± 25 versus 380 ± 46 mg/dl). After 4 weeks of hyperglycemia, the kidney weight in db/db mice was significantly increased by 16% (193 ± 9 mg versus 166 ± 5 mg) (P < 0.01 by analysis of variance) (Figure 7A). Rapamycin abolished the renal growth induced by diabetes (148 ± 5 versus 193 ± 9 mg) (Figure, 7A, P < 0.001); however, rapamycin did not affect kidney weight in control mice (172 ± 4 mg versus 166 ± 5 mg). Because glomeruli form a small part of the kidney and changes in kidney weight may mask glomerular changes, we directly assessed rapamycin's effect on glomerular hypertrophy. Glomerular area by morphometry was significantly increased by 27% in the db/db mice compared to control db/m mice (2908 ± 63 μm2 versus 3679 ± 108 μm2, P < 0.01 by analysis of variance), suggesting hypertrophy induced by diabetes (Figure 7B). Rapamycin did not affect glomerular area in db/db mice (3679 ± 108 μm2 versus 3551 ± 145 μm2) or in control mice (2908 ± 63 μm2 versus 2894 ± 182 μm2). These data suggest that under conditions of our experiment, the effect of rapamycin in reversing hypertrophy was not uniformly seen in all segments of the nephron but was selective for the tubular compartment. We studied regulation of the laminin-β1 chain because it is a major component of mesangial and tubulointerstitial matrix20Miner JH Patton BL Lentz SI Gilbert DJ Snider WD Jenkins NA Copeland NG Sanes JR The laminin alpha chains: expression, developmental transitions, and chromosomal locations 8–11, and cloning of a novel alpha3 iso-form.J Cell Biol. 1997; 137: 685-701Crossref PubMed Scopus (584) Google Scholar and its content is increased in renal parenchyma in db/db mice with type 2 diabetes.4Ha TS Barnes JL Stewart JL Ko CW Miner JH Abrahamson DR Sanes JR Kasinath BS Regulation of renal laminin in mice with type II diabetes.J Am Soc Nephrol. 1999; 10: 1931-1939Crossref PubMed Google Scholar A fourfold increment in laminin-β1 expression in glomerular mesangium (P < 0.001) was seen that was almost completely restored to control values in rapamycin-administered db/db mice (P < 0.001, db/db versus db/db + rapa) (Figure 8A). Immunostaining of laminin-β1 was augmented in the tubular basement membranes by nearly 12-fold in diabetic mice compared to control mice (Figure 8B, P < 0.001); this was also significantly ameliorated by administration of rapamycin (P < 0.001). Immunostaining data were validated by immunoblotting for laminin-β1 in renal cortical lysates. Laminin-β1 chain content was significantly elevated in the renal cortex of mice with type 2 diabetes (P < 0.001), which was abolished by rapamycin (P < 0.001, db/db versus db/db + rapa) (Figure 8C). Rapamycin did not affect laminin-β1 content in control mice. Laminin-β1 chain mRNA content was unchanged in renal cortex of db/db mice compared to control db/m mice as measured by quantitative RT-PCR (Figure 8D). These data showed that increment in laminin-β1 chain protein was attributable to nontranscriptional mechanisms, suggesting mRNA translation could be involved. Thr56 phosphorylation of eEF2 was significantly reduced by nearly 35% and phosphorylation of Ser366 on eEF2 kinase was increased by twofold in the renal cortex of db/db mice (P < 0.05 and P < 0.01, respectively, by analysis of variance) (Figure 9, A and B); these changes were prevented by rapamycin (P < 0.05, db/db versus db/db + rapa). Thus, rapamycin normalized important steps in regulation of
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