[Frontiers in Bioscience S3, 98-104, January 1, 2011]

Protein kinase B/Akt regulation in diabetic kidney disease

Mediha Heljic1, Derek P. Brazil2

1School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Ireland, 2 Centre for Vision and Vascular Science, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Northern Ireland, UK

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Akt Signaling in diabetic nephropathy
4. Concluding remarks
5. References

1. ABSTRACT

Many reviews have been written on protein kinase B/Akt focusing on its history dating back from the isolation of the Akt8 transforming murine leukemia virus by Staal in 1977, to the co-discovery of the Akt1 gene by the three groups in 1991 (reviewed in 7). There are currently over 22,000 publications in the PubMed database with "Akt" as a keyword - these publications describe a wealth of diverse data on the physiological functions of Akt isoforms. Many of these publications describe roles of Akt ranging from its requirement for cellular processes such as glucose uptake, cell survival and angiogenesis to roles in diseases such as cancer and ischaemia (22). This review will focus on the evidence for Akt signaling in different kidney cells during diabetes, or diabetic nephropathy (DN).

2. INTRODUCTION

Akt belongs to subfamily of serine/threonine protein kinases called AGC protein kinases and is involved in numerous signaling circuits. Akt is a downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, where generation of PtdIns-3,4,5-P3 recruits Akt to the plasma membrane (2, 9, 24). Full activation of Akt requires phosphorylation on Thr308 by PDK1 (1) and Ser473 by the TORC2 complex (51), in addition to DNA-dependent protein kinase (DNA-PK) in genotoxic stressed cells (23). Downstream targets of Akt control multiple cellular processes such as glucose uptake, cell proliferation, gene transcription, protein synthesis and cell survival (7, 19, 60, 61).

The mammalian Akt gene family consists of three highly homologous genes coding for the three Akt isoforms termed Akt1, Akt2 and Akt3 (5, 8, 12, 15, 32, 43, 45). The three Akt proteins are very similar in sequence and structure and are thought to be activated by a common mechanism (7). The distinct phenotype of transgenic mice lacking individual Akt isoforms however, suggests that there are well-defined physiological roles for each Akt protein in mammals. Both Akt1 and Akt2 are widely expressed in mammalian tissues (66). Mice lacking Akt1 display growth retardation and increased (~40%) neonatal mortality (11, 65). Akt2 homozygous knockout mice develop a metabolic disorder, exhibiting a mild type 2 diabetes-like phenotype due to insulin resistance (13). Akt3 is predominantly expressed in the brain and was shown to be important in brain development and size (21, 57). More complex double and even triple knockout strategies have been employed to determine the minimal Akt "unit of activity" required for cellular and embryonic survival. Yang and colleagues identified that Akt1-/-; Akt3-/- mice died in utero at E11-12. Interestingly, Akt1-/-; Akt3+/- mice developed severe defects in thymus and heart and died several days after birth, whereas Akt1+/-; Akt3-/- survived normally, suggesting a dominant role for Akt1 (65). These experiments were further extended by the demonstration that Akt2-/-;Akt3-/- mice survived normally, and indeed a single functional allele of Akt1 was all that was required for mouse development and survival (20). Liu and colleagues used Akt1-/-;Akt2-/- mouse embryo fibroblasts (MEFs) to transfect Akt3 siRNA. The data showed that apoptosis of these cells was only increased when approximately 80 % reduction of Akt3 expression was achieved (39). Together, these data suggest that only a small amount of Akt activity is required for normal cellular survival and mammalian development.

3. AKT SIGNALING IN DIABETIC NEPHROPATHY

Diabetic nephropathy (DN) is a progressive fibrotic disease of the kidney as a result of chronic hyperglycaemia during diabetes. The pathogenesis of DN is characterised by progressive loss of kidney function due to glomerulosclerosis (thickening of the glomerulus due to deposition of ECM) and tubulointerstitial fibrosis leading to scarring of the kidney tubules and impaired reabsorbtive capacity (40). The cellular mechanisms of both glomerulosclerosis and tubulointerstitial fibrosis have been extensively reviewed (37, 40). Particular focus has been given to the cellular events driving tubulointerstitial fibrosis, as the degree of fibrosis appears to correlate tightly with the severity of renal disease (47). Epithelial-mesenchymal transition is thought to contribute to renal fibrosis in DN, as cytokines such as TGF-beta1 that drive EMT are elevated in the diabetic kidney (6, 59). However, recent evidence has suggested that the source of myofibroblasts in kidney may arise not from renal epithelial cells undergoing EMT, but rather from pericytes (29). In vitro and in vivo, EMT is characterised by loss of epithelial proteins such as E-cadherin and ZO-1, signifying loss of epithelial tight junctions and barrier integrity, leading to compromised renal tubule function. Additionally, EMT features increased expression of fibroblast proteins such as α-smooth muscle actin and vimentin, which leads to altered cell shape and increased motility (4, 31). TGF-beta1 is the primary cytokine that drives EMT in vitro and in vivo, and utilises both canonical and non-canonical signaling pathways to mediate its effects. TGF-beta1-mediated activation of Smad2/3 phosphorylation leads to a range of gene expression changes such as decreased E-cadherin expression and increased alpha-smooth muscle actin that characterise EMT (58, 59). In addition, TGF-beta1 activates other pathways such as the PI3Kà Akt pathway, and this non-canonical cascade has also been implicated in renal damage during diabetes and other fibrotic conditions. Evidence for the involvement of Akt in diabetic kidney disease and other fibrotic conditions of the kidney will be discussed below.

Akt has been implicated in diabetic nephropathy using a wide range of cell and animal models. Early reports identified that extracellular matrix production, a hallmark of glomerulosclerosis, is regulated by Akt. Krepinsky and colleagues demonstrated that both mechanical stretch and high glucose-induced collagen 1 production in mesangial cells required Akt activity (35). Akt activity may also be required for high-glucose induced increases in TGF-beta1 expression in diabetic tubular epithelial cells (36). One of the earliest events in DN is glomerular thickening as a result of mesangial cell hypertrophy. Several publications have identified that Akt signaling contributes to mesangial cell hypertrophy in diabetic kidney disease. Nagai and colleagues identified that growth arrest specific gene-6 (Gas6) signaling through its Axl receptor drives mesangial cell hypertrophy in type 1 diabetic rodent models in an Akt-dependent manner (44). Consistently, hyperglycaemia decreased the expression of the lipid phosphatase PTEN, an inhibitor of Akt activation, leading to hypertrophy of mesangial cells (42). These authors also showed that TGF-beta1 treatment of mesangial cells also decreased PTEN expression with concomitant increases in Akt activation (42). Importantly, Kato and colleagues showed that TGFbeta1 increased the levels miR-216a and miR-217 in mesangial cells. These miRNAs target PTEN, reducing its expression leading to Akt activation (33) Recent findings from Zhang et al demonstrate that micro RNA-21 (miR-21), which targets PTEN, also protects from glomerular mesangial cell proliferation in DN (68). Thus, the regulation of miRNAs by high glucose and TGF-β1 further support the role of Akt signaling in early cellular hypertrophy associated with diabetic nephropathy.

Diabetic nephropathy is associated with loss of renal cells, particularly glomerular podocytes which form the glomerular filtration barrier (3, 55). Akt signaling has been implicated in this process. Levels of Akt phosphorylation are elevated in the kidney tubules of the Goto Kakizaki type 2 diabetic rat (34) and streptozotocin-treated type 1 diabetic mice (46). Chuang and co-workers identified that advanced glycation end-products generated as a result of chronic hyperglycaemia increased apoptosis in vitro (14). Murine podocytes exposed to AGE-modified BSA were more apoptotic, and also displayed decreased Akt phosphorylation (14). This decrease in Akt activity led to the "liberation" of FOXO4 from the inhibitory constraint mediated by Akt phosphorylation, leading to FOXO4 nuclear translocation and increased expression of the pro-apoptotic Bim protein (14). In vivo, the db/db experimental mouse model of DN displayed early diabetic nephropathy at 12 wk which was accompanied by decreased Akt phosphorylation in the glomeruli (56). Podocytes isolated from db/db mice did not display Akt phosphorylation in response to insulin, whereas db/+ mice did. Podocyte viability was reduced in db/db mice, and this was linked to reduced Akt-mediated survival signaling (56). These two reports implicate changes in Akt signaling as a key event in podocyte loss during early diabetic nephropathy. A recent report from Rane and colleagues focussed on renal tubular epithelial cell apoptosis in response to high glucose, an event regulated by p38MAPK activation (50). Rane et al showed that p38MAPK induced apoptosis of renal proximal tubular epithelial cells (RPTCs) could be inhibited by expression of constitutively active Akt. Consistently, PI3Kinase inhibitors or siRNA targeting Akt led to p38MAPK activation in the absence of high glucose (46). Others have suggested that IGF1à Akt signaling may be important for mesangial cell survival in diabetic nephropathy (53).

Crosstalk between different signal transduction cascades downstream of TGF-beta1 is a feature of diabetic nephropathy. Ghosh Choudray and co-workers showed that TGF-beta1 increased fibronectin production in mesangial cells in an Akt-dependent manner (27). Other reports indicate that that Akt can enhance Smad3-mediated collagen I expression in mesangial cells treated with TGF-beta1 (49), a process that may also involve the small GTP binding protein Rac1 (28). In contrast, two reports demonstrated that Akt inhibited Smad3 mediated transcription by direct binding and sequestration of Smad3 in the cytosol (16, 48). Akt kinase activity was not required for this inhibition, and the association of Akt and Smad3 was stimulated by insulin but inhibited by TGF-beta1 (16, 48). Similarly, Seong and co-workers demonstrated that proteins such as Smad3 could interact with PDK1, (the Thr308 kinase of Akt), an association that was also facilitated by insulin stimulation but inhibited by TGF-beta1 (52). In contrast to Akt, binding of Smad proteins to PDK1 (the Thr308 Akt kinase) increased its kinase activity (52). Akt has also been implicated in actin disassembly and mesangial cell dysfunction in mesangial cells. In response to connective tissue growth factor (CTGF), a secreted mediator of many TGF-beta1 effects, mesangial cells undergo a rapid change in actin cytoskeleton structure, a process regulated by Aktà p27Kip1 signaling (17). Furthermore, in the diabetic milieu, mesangial cell Akt activation in response to CTGF is blunted, suggesting that chronic hyperglycaemia may alter Akt signaling in vivo (25). Furlong and colleagues also noted that incubation of high-glucose treated mesangial cells with an inhibitor of PKCbeta could restore cellular responses to CTGF (25). Work from the Krepinsky laboratory has identified EGFRà PLCgammaà PKCbetaà Akt pathway that is activated by hyperglycaemia to drive extracellular matrix production (62, 63). Thus, complex signal transduction pathways downstream of TGF-beta1 and other factors integrate Akt signaling with other molecules such as Smad3 and p38MAPK to regulate kidney cell function.

Akt has also been implicated in TGF-beta1-induced damage to renal epithelial cells during diabetic nephropathy. Both PI3kinase and Akt activation are required for TGF-beta1 induced epithelial-mesenchymal transition (EMT) in NRK52E tubular epithelial cells (34). Recent evidence from the cancer field suggests that Akt isoforms regulate miRNA production and EMT. Iliopoulos and co-workers identified that knockdown of Akt1, but not Akt2 decreased miR-200 abundance, promoting EMT in MCF10A breast cancer cells (30). Zeng et al showed that biliverdin reductase may serve as an upstream activator of PI3K and Akt to drive EMT in response to hypoxia in HK-2 renal epithelial cells (67). Pharmacological inhibition of integrin-linked kinase (ILK) inhibits Akt phosphorylation and attenuates TGF-beta1-induced EMT in renal tubular epithelial cells (38). Together, these data suggest that Akt signaling is an important regulator of TGF-beta1 mediated cellular events during diabetic nephropathy.

Since inappropriate Akt activation has been reported in many models of diabetic nephropathy, strategies to intervene pharmacologically to alleviate DN in human patients have been explored. In particular, the nutrient sensing mTOR (target of rapamycin) has been the subject of several reports in this area. mTOR is activated by the small GTPase Rheb, which is inhibited by tuberous sclerosis complex1/2 (TSC1/2), which act as a GAP protein to inactivate Rheb (18, 26). TSC1/2 activity is inhibited by Akt, leading to Rheb, and mTOR activation. Song and colleagues present data showing that Akt-mediated inhibition of Smad3 activation requires mTOR activity in prostate epithelium cells (54). Nagai and colleagues identified that Gas6-mediated mesangial cell hypertrophy required both Akt and mTOR activity (Nagai K et al, 1999). Treatment of diabetic rats with the mTOR inhibitor rapamycin (siroliumus) for 4 wk decreased mesangial matrix production and attenuated the severity of DN (41, 64). Other pharmacological strategies that affect Akt activity have also been shown to reduce indices of damage in DN in various models. Troglitazone (an insulin-sensitizer) was shown to attenuate high-glucose induced EMT in renal proximal tubule cells, a process that involved Akt signaling (36). Bussolati and colleagues showed that statins could rescue ox-LDL induced podocyte apoptosis, a process requiring statin-mediated Akt activation (10). Thus, inhibition of Akt and its downstream targets such as mTOR may provide future therapeutic benefit for the treatment of diabetic nephropathy.

4. CONCLUDING REMARKS

A summary of current knowledge of Akt signaling in the kidney is given in Fig. 1. The complexity of these signaling networks emphasises the diverse control of kidney cell processes, be it podocytes, mesangial cells or tubular epithelial cells, regulated by Akt. Future experiments will provide further fascinating insights into the role of the Akt kinase family in kidney physiology and disease.

5. REFERENCES

1. Alessi, D. R., M. Deak, A. Casamayor, F. B. Caudwell, N. Morrice, D. G. Norman, P. Gaffney, C. B. Reese, C. N. MacDougall, D. Harbison, A. Ashworth & M. Bownes: 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol, 7, 776-89(1997)
doi:10.1016/S0960-9822(06)00336-8

2. Andjelkovic, M., D. R. Alessi, R. Meier, A. Fernandez, N. J. Lamb, M. Frech, P. Cron, P. Cohen, J. M. Lucocq & B. A. Hemmings: Role of translocation in the activation and function of protein kinase B. J Biol Chem, 272, 31515-24(1997)
doi:10.1074/jbc.272.50.31515

3. Appel, D., D. B. Kershaw, B. Smeets, G. Yuan, A. Fuss, B. Frye, M. Elger, W. Kriz, J. Floege & M. J. Moeller: Recruitment of podocytes from glomerular parietal epithelial cells. J Am Soc Nephrol, 20, 333-43(2009)
doi:10.1681/ASN.2008070795

4. Badid, C., A. Desmouliere, D. Babici, A. Hadj-Aissa, B. McGregor, N. Lefrancois, J. L. Touraine & M. Laville: Interstitial expression of alpha-SMA: an early marker of chronic renal allograft dysfunction. Nephrol Dial Transplant, 17, 1993-8(2002)
doi:10.1093/ndt/17.11.1993

5. Bellacosa, A., J. R. Testa, S. P. Staal & P. N. Tsichlis: A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science, 254, 274-7(1991)
doi:10.1126/science.1833819

6. Bottinger, E. P. & M. Bitzer: TGF-beta signaling in renal disease. J Am Soc Nephrol, 13, 2600-10(2002)
doi:10.1097/01.ASN.0000033611.79556.AE

7. Brazil, D. P., Z. Z. Yang & B. A. Hemmings: Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem Sci, 29, 233-42(2004)
doi:10.1016/j.tibs.2004.03.006

8. Brodbeck, D., P. Cron & B. A. Hemmings: A human protein kinase Bgamma with regulatory phosphorylation sites in the activation loop and in the C-terminal hydrophobic domain. J Biol Chem, 274, 9133-6(1999)
doi:10.1074/jbc.274.14.9133

9. Burgering, B. M. & P. J. Coffer: Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature, 376, 599-602(1995)
doi:10.1038/376599a0

10. Bussolati, B., M. C. Deregibus, V. Fonsato, S. Doublier, T. Spatola, S. Procida, F. Di Carlo & G. Camussi: Statins prevent oxidized LDL-induced injury of glomerular podocytes by activating the phosphatidylinositol 3-kinase/AKT-signaling pathway. J Am Soc Nephrol, 16, 1936-47(2005)
doi:10.1681/ASN.2004080629

11. Chen, W. S., P. Z. Xu, K. Gottlob, M. L. Chen, K. Sokol, T. Shiyanova, I. Roninson, W. Weng, R. Suzuki, K. Tobe, T. Kadowaki & N. Hay: Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene. Genes Dev, 15, 2203-8(2001)
doi:10.1101/gad.913901

12. Cheng, J. Q., A. K. Godwin, A. Bellacosa, T. Taguchi, T. F. Franke, T. C. Hamilton, P. N. Tsichlis & J. R. Testa: AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. Proc Natl Acad Sci U S A, 89, 9267-71(1992)
doi:10.1073/pnas.89.19.9267

13. Cho, H., J. L. Thorvaldsen, Q. Chu, F. Feng & M. J. Birnbaum: Akt1/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J Biol Chem, 276, 38349-52(2001)
doi:10.1074/jbc.C100462200

14. Chuang, P. Y., Q. Yu, W. Fang, J. Uribarri & J. C. He: Advanced glycation endproducts induce podocyte apoptosis by activation of the FOXO4 transcription factor. Kidney Int, 72, 965-76(2007)
doi:10.1038/sj.ki.5002456

15. Coffer, P. J. & J. R. Woodgett: Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. Eur J Biochem, 201, 475-81(1991)
doi:10.1111/j.1432-1033.1991.tb16305.x

16. Conery, A. R., Y. Cao, E. A. Thompson, C. M. Townsend, Jr., T. C. Ko & K. Luo: Akt interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis. Nat Cell Biol, 6, 366-72(2004)
doi:10.1038/ncb1117

17. Crean, J. K., F. Furlong, D. Mitchell, E. McArdle, C. Godson & F. Martin: Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B-mediated phosphorylation and cytoplasmic translocation of p27(Kip-1). Faseb J, 20, 1712-4(2006)
doi:10.1096/fj.05-5010fje

18. Di Paolo, S., A. Teutonico, D. Leogrande, C. Capobianco & P. F. Schena: Chronic inhibition of mammalian target of rapamycin signaling downregulates insulin receptor substrates 1 and 2 and AKT activation: A crossroad between cancer and diabetes? J Am Soc Nephrol, 17, 2236-44(2006)
doi:10.1681/ASN.2006030196

19. Dummler, B. & B. A. Hemmings: Physiological roles of PKB/Akt isoforms in development and disease. Biochem Soc Trans, 35, 231-5(2007)
doi:10.1042/BST0350231

20. Dummler, B., O. Tschopp, D. Hynx, Z. Z. Yang, S. Dirnhofer & B. A. Hemmings: Life with a single isoform of Akt: mice lacking Akt2 and Akt3 are viable but display impaired glucose homeostasis and growth deficiencies. Mol Cell Biol, 26, 8042-51(2006)
doi:10.1128/MCB.00722-06

21. Easton, R. M., H. Cho, K. Roovers, D. W. Shineman, M. Mizrahi, M. S. Forman, V. M. Lee, M. Szabolcs, R. de Jong, T. Oltersdorf, T. Ludwig, A. Efstratiadis & M. J. Birnbaum: Role for Akt3/protein kinase Bgamma in attainment of normal brain size. Mol Cell Biol, 25, 1869-78(2005)
doi:10.1128/MCB.25.5.1869-1878.2005

22. Fayard, E., L. A. Tintignac, A. Baudry & B. A. Hemmings: Protein kinase B/Akt at a glance. J Cell Sci, 118, 5675-8(2005)
doi:10.1242/jcs.02724

23. Feng, J., J. Park, P. Cron, D. Hess & B. A. Hemmings: Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J Biol Chem, 279, 41189-96(2004)
doi:10.1074/jbc.M406731200

24. Franke, T. F., S. I. Yang, T. O. Chan, K. Datta, A. Kazlauskas, D. K. Morrison, D. R. Kaplan & P. N. Tsichlis: The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell, 81, 727-36(1995)
doi:10.1016/0092-8674(95)90534-0

25. Furlong, F., J. Crean, L. Thornton, R. O'Leary, M. Murphy & F. Martin: Dysregulated intracellular signaling impairs CTGF-stimulated responses in human mesangial cells exposed to high extracellular glucose. Am J Physiol Renal Physiol, 292, F1691-700(2007)
doi:10.1152/ajprenal.00342.2006

26. Garami, A., F. J. Zwartkruis, T. Nobukuni, M. Joaquin, M. Roccio, H. Stocker, S. C. Kozma, E. Hafen, J. L. Bos & G. Thomas: Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol Cell, 11, 1457-66(2003)
doi:10.1016/S1097-2765(03)00220-X

27. Ghosh Choudhury, G. & H. E. Abboud: Tyrosine phosphorylation-dependent PI 3 kinase/Akt signal transduction regulates TGFbeta-induced fibronectin expression in mesangial cells. Cell Signal, 16, 31-41(2004)
doi:10.1016/S0898-6568(03)00094-9

28. Hubchak, S. C., E. E. Sparks, T. Hayashida & H. W. Schnaper: Rac1 promotes TGF-beta-stimulated mesangial cell type I collagen expression through a PI3K/Akt-dependent mechanism. Am J Physiol Renal Physiol, 297, F1316-23(2009)
doi:10.1152/ajprenal.00345.2009

29. Humphreys, B. D., S. L. Lin, A. Kobayashi, T. E. Hudson, B. T. Nowlin, J. V. Bonventre, M. T. Valerius, A. P. McMahon & J. S. Duffield: Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol, 176, 85-97(2010)
doi:10.2353/ajpath.2010.090517

30. Iliopoulos, D., C. Polytarchou, M. Hatziapostolou, F. Kottakis, I. G. Maroulakou, K. Struhl & P. N. Tsichlis: MicroRNAs differentially regulated by Akt isoforms control EMT and stem cell renewal in cancer cells. Sci Signal, 2, ra62(2009)
doi:10.1126/scisignal.2000356

31. Iwano, M., D. Plieth, T. M. Danoff, C. Xue, H. Okada & E. G. Neilson: Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest, 110, 341-50(2002)
No DOI Found

32. Jones, P. F., T. Jakubowicz & B. A. Hemmings: Molecular cloning of a second form of rac protein kinase. Cell Regul, 2, 1001-9(1991)
No DOI Found

33. Kato, M., S. Putta, M. Wang, H. Yuan, L. Lanting, I. Nair, A. Gunn, Y. Nakagawa, H. Shimano, I. Todorov, J. J. Rossi & R. Natarajan: TGF-beta activates Akt kinase through a microRNA-dependent amplifying circuit targeting PTEN. Nat Cell Biol, 11, 881-9(2009)
doi:10.1038/ncb1897

34. Kattla, J. J., R. M. Carew, M. Heljic, C. Godson & D. P. Brazil: Protein kinase B/Akt activity is involved in renal TGF-beta1-driven epithelial-mesenchymal transition in vitro and in vivo. Am J Physiol Renal Physiol, 295, F215-25(2008)
doi:10.1152/ajprenal.00548.2007

35. Krepinsky, J. C., Y. Li, Y. Chang, L. Liu, F. Peng, D. Wu, D. Tang, J. Scholey & A. J. Ingram: Akt mediates mechanical strain-induced collagen production by mesangial cells. J Am Soc Nephrol, 16, 1661-72(2005)
doi:10.1681/ASN.2004100897

36. Lee, Y. J. & H. J. Han: Troglitazone ameliorates high glucose-induced EMT and dysfunction of SGLTs through PI3K/Akt, GSK-3{beta}, Snail1, and {beta}-catenin in renal proximal tubule cells. Am J Physiol Renal Physiol(2009)

No DOI Found

37. Li, M. X. & B. C. Liu: Epithelial to mesenchymal transition in the progression of tubulointerstitial fibrosis. Chin Med J (Engl), 120, 1925-30(2007)

No DOI Found

38. Li, Y., J. Yang, C. Dai, C. Wu & Y. Liu: Role for integrin-linked kinase in mediating tubular epithelial to mesenchymal transition and renal interstitial fibrogenesis. J Clin Invest, 112, 503-16(2003)
No DOI Found

39. Liu, X., Y. Shi, M. J. Birnbaum, K. Ye, R. De Jong, T. Oltersdorf, V. L. Giranda & Y. Luo: Quantitative analysis of anti-apoptotic function of Akt in Akt1 and Akt2 double knock-out mouse embryonic fibroblast cells under normal and stressed conditions. J Biol Chem, 281, 31380-8(2006)
doi:10.1074/jbc.M606603200

40. Liu, Y.: Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol, 15, 1-12(2004)
doi:10.1097/01.ASN.0000106015.29070.E7

41. Lloberas, N., J. M. Cruzado, M. Franquesa, I. Herrero-Fresneda, J. Torras, G. Alperovich, I. Rama, A. Vidal & J. M. Grinyo: Mammalian target of rapamycin pathway blockade slows progression of diabetic kidney disease in rats. J Am Soc Nephrol, 17, 1395-404(2006)
doi:10.1681/ASN.2005050549

42. Mahimainathan, L., F. Das, B. Venkatesan & G. G. Choudhury: Mesangial cell hypertrophy by high glucose is mediated by downregulation of the tumor suppressor PTEN. Diabetes, 55, 2115-25(2006)
doi:10.2337/db05-1326

43. Masure, S., B. Haefner, J. J. Wesselink, E. Hoefnagel, E. Mortier, P. Verhasselt, A. Tuytelaars, R. Gordon & A. Richardson: Molecular cloning, expression and characterization of the human serine/threonine kinase Akt-3. Eur J Biochem, 265, 353-60(1999)
doi:10.1046/j.1432-1327.1999.00774.x

44. Nagai, K., T. Matsubara, A. Mima, E. Sumi, H. Kanamori, N. Iehara, A. Fukatsu, M. Yanagita, T. Nakano, Y. Ishimoto, T. Kita, T. Doi & H. Arai: Gas6 induces Akt/mTOR-mediated mesangial hypertrophy in diabetic nephropathy. Kidney Int, 68, 552-61(2005)
doi:10.1111/j.1523-1755.2005.00433.x

45. Nakatani, K., H. Sakaue, D. A. Thompson, R. J. Weigel & R. A. Roth: Identification of a human Akt3 (protein kinase B gamma) which contains the regulatory serine phosphorylation site. Biochem Biophys Res Commun, 257, 906-10(1999)
doi:10.1006/bbrc.1999.0559

46. Rane, M. J., Y. Song, S. Jin, M. T. Barati, R. Wu, H. Kausar, Y. Tan, Y. Wang, G. Zhou, J. B. Klein, X. Li & L. Cai: Interplay between Akt and p38 MAPK pathways in the regulation of renal tubular cell apoptosis associated with diabetic nephropathy. Am J Physiol Renal Physiol, 298, F49-61(2010)
doi:10.1152/ajprenal.00032.2009

47. Rastaldi, M. P., F. Ferrario, L. Giardino, G. Dell'Antonio, C. Grillo, P. Grillo, F. Strutz, G. A. Muller, G. Colasanti & G. D'Amico: Epithelial-mesenchymal transition of tubular epithelial cells in human renal biopsies. Kidney Int, 62, 137-46(2002)
doi:10.1046/j.1523-1755.2002.00430.x

48. Remy, I., A. Montmarquette & S. W. Michnick: PKB/Akt modulates TGF-beta signalling through a direct interaction with Smad3. Nat Cell Biol, 6, 358-65(2004)
doi:10.1038/ncb1113

49. Runyan, C. E., H. W. Schnaper & A. C. Poncelet: The phosphatidylinositol 3-kinase/Akt pathway enhances Smad3-stimulated mesangial cell collagen I expression in response to transforming growth factor-beta1. J Biol Chem, 279, 2632-9(2004)
doi:10.1074/jbc.M310412200

50. Sakai, N., T. Wada, K. Furuichi, Y. Iwata, K. Yoshimoto, K. Kitagawa, S. Kokubo, M. Kobayashi, A. Hara, J. Yamahana, T. Okumura, K. Takasawa, S. Takeda, M. Yoshimura, H. Kida & H. Yokoyama: Involvement of extracellular signal-regulated kinase and p38 in human diabetic nephropathy. Am J Kidney Dis, 45, 54-65(2005)
doi:10.1053/j.ajkd.2004.08.039

51. Sarbassov, D. D., D. A. Guertin, S. M. Ali & D. M. Sabatini: Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science, 307, 1098-101(2005)
doi:10.1126/science.1106148

52. Seong, H. A., H. Jung, K. T. Kim & H. Ha: 3-Phosphoinositide-dependent PDK1 negatively regulates transforming growth factor-beta-induced signaling in a kinase-dependent manner through physical interaction with Smad proteins. J Biol Chem, 282, 12272-89(2007)
doi:10.1074/jbc.M609279200

53. Singh, L. P., Y. Jiang & D. W. Cheng: Proteomic identification of 14-3-3zeta as an adapter for IGF-1 and Akt/GSK-3beta signaling and survival of renal mesangial cells. Int J Biol Sci, 3, 27-39(2007)
No DOI Found

54. Song, K., H. Wang, T. L. Krebs & D. Danielpour: Novel roles of Akt and mTOR in suppressing TGF-beta/ALK5-mediated Smad3 activation. Embo J, 25, 58-69(2006)
doi:10.1038/sj.emboj.7600917

55. Stitt-Cavanagh, E., L. MacLeod & C. Kennedy: The podocyte in diabetic kidney disease. ScientificWorldJournal, 9, 1127-39(2009)

56. Tejada, T., P. Catanuto, A. Ijaz, J. V. Santos, X. Xia, P. Sanchez, N. Sanabria, O. Lenz, S. J. Elliot & A. Fornoni: Failure to phosphorylate AKT in podocytes from mice with early diabetic nephropathy promotes cell death. Kidney Int, 73, 1385-93(2008)
doi:10.1038/ki.2008.109

57. Tschopp, O., Z. Z. Yang, D. Brodbeck, B. A. Dummler, M. Hemmings-Mieszczak, T. Watanabe, T. Michaelis, J. Frahm & B. A. Hemmings: Essential role of protein kinase B gamma (PKB gamma/Akt3) in postnatal brain development but not in glucose homeostasis. Development, 132, 2943-54(2005)
doi:10.1242/dev.01864

58. Tsuchida, K., B. Cronin & K. Sharma: Novel aspects of transforming growth factor-Beta in diabetic kidney disease. Nephron, 92, 7-21(2002)
doi:10.1159/000064486

59. Valcourt, U., M. Kowanetz, H. Niimi, C. H. Heldin & A. Moustakas: TGF-beta and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition. Mol Biol Cell, 16, 1987-2002(2005)
doi:10.1091/mbc.E04-08-0658
PMid:15689496    PMCid:1073677

60. Whiteman, E. L., H. Cho & M. J. Birnbaum: Role of Akt/protein kinase B in metabolism. Trends Endocrinol Metab, 13, 444-51(2002)
doi:10.1016/S1043-2760(02)00662-8

61. Woodgett, J. R.: Recent advances in the protein kinase B signaling pathway. Curr Opin Cell Biol, 17, 150-7(2005)
doi:10.1016/j.ceb.2005.02.010

62. Wu, D., F. Peng, B. Zhang, A. J. Ingram, D. J. Kelly, R. E. Gilbert, B. Gao & J. C. Krepinsky: PKC-beta1 mediates glucose-induced Akt activation and TGF-beta1 upregulation in mesangial cells. J Am Soc Nephrol, 20, 554-66(2009)
doi:10.1681/ASN.2008040445

63. Wu, D., F. Peng, B. Zhang, A. J. Ingram, D. J. Kelly, R. E. Gilbert, B. Gao, S. Kumar & J. C. Krepinsky: EGFR-PLCgamma1 signaling mediates high glucose-induced PKCbeta1-Akt activation and collagen I upregulation in mesangial cells. Am J Physiol Renal Physiol, 297, F822-34(2009)
doi:10.1152/ajprenal.00054.2009

64. Yang, Y., J. Wang, L. Qin, Z. Shou, J. Zhao, H. Wang, Y. Chen & J. Chen: Rapamycin prevents early steps of the development of diabetic nephropathy in rats. Am J Nephrol, 27, 495-502(2007)
doi:10.1159/000106782

65. Yang, Z. Z., O. Tschopp, N. Di-Poi, E. Bruder, A. Baudry, B. Dummler, W. Wahli & B. A. Hemmings: Dosage-dependent effects of Akt1/protein kinase Balpha (PKBalpha) and Akt3/PKBgamma on thymus, skin, and cardiovascular and nervous system development in mice. Mol Cell Biol, 25, 10407-18(2005)
doi:10.1128/MCB.25.23.10407-10418.2005

66. Yang, Z. Z., O. Tschopp, M. Hemmings-Mieszczak, J. Feng, D. Brodbeck, E. Perentes & B. A. Hemmings: Protein kinase B alpha/Akt1 regulates placental development and fetal growth. J Biol Chem, 278, 32124-31(2003)
doi:10.1074/jbc.M302847200

67. Zeng, R., Y. Yao, M. Han, X. Zhao, X. C. Liu, J. Wei, Y. Luo, J. Zhang, J. Zhou, S. Wang, D. Ma & G. Xu: Biliverdin reductase mediates hypoxia-induced EMT via PI3-kinase and Akt. J Am Soc Nephrol, 19, 380-7(2008)
doi:10.1681/ASN.2006111194

68. Zhang, Z., H. Peng, J. Chen, X. Chen, F. Han, X. Xu, X. He & N. Yan: MicroRNA-21 protects from mesangial cell proliferation induced by diabetic nephropathy in db/db mice. FEBS Lett, 583, 2009-14(2009)
doi:10.1016/j.febslet.2009.05.021

Key Words: Akt, Diabetic Nephropathy, Fibrosis, Epithelial Cell, Review

Send correspondence to: Derek P. Brazil, Centre for Vision and Vascular Science, ICS-A, Queen's University Belfast, Grosvenor Road, Belfast BT12 6BA, Northern Ireland UK, Tel: 0044-28-9063-2572, Fax: 0044-28-9063-2699, E-mail:d.brazil@qub.ac.uk