Failure to phosphorylate AKT in podocytes from mice with early diabetic nephropathy promotes cell death
2008; Elsevier BV; Volume: 73; Issue: 12 Linguagem: Inglês
10.1038/ki.2008.109
ISSN1523-1755
AutoresThor Tejada, Paola Catanuto, Adeel Ijaz, Javier Varona Santos, Xuefeng Xia, Pedro Sánchez, Nahir Y. Sanabria, Oliver Lenz, Sharon J. Elliot, Alessia Fornoni,
Tópico(s)Genetic and Kidney Cyst Diseases
ResumoLoss of podocytes by apoptosis characterizes the early stages of diabetic nephropathy. To examine its mechanism we studied glomeruli and podocytes isolated from db/db mice with early diabetic nephropathy and albuminuria. Phosphorylation of AKT (protein kinase B, a key survival protein) was found to be lower in the glomeruli of 12 week old db/db compared to db/+ mice. In vitro, insulin phosphorylated AKT solely in podocytes from db/+ mice. Serum deprivation and exposure to tumor necrosis factor-α significantly compromised cell viability in podocytes from db/db but not from db/+ mice, and this was associated with a significant decrease in AKT phosphorylation. Inhibition of AKT was necessary to achieve the same degree of cell death in db/+ podocytes. Our study shows that podocyte inability to respond to insulin and susceptibility to cell death may partially account for the decreased podocyte number seen in early diabetic nephropathy. Loss of podocytes by apoptosis characterizes the early stages of diabetic nephropathy. To examine its mechanism we studied glomeruli and podocytes isolated from db/db mice with early diabetic nephropathy and albuminuria. Phosphorylation of AKT (protein kinase B, a key survival protein) was found to be lower in the glomeruli of 12 week old db/db compared to db/+ mice. In vitro, insulin phosphorylated AKT solely in podocytes from db/+ mice. Serum deprivation and exposure to tumor necrosis factor-α significantly compromised cell viability in podocytes from db/db but not from db/+ mice, and this was associated with a significant decrease in AKT phosphorylation. Inhibition of AKT was necessary to achieve the same degree of cell death in db/+ podocytes. Our study shows that podocyte inability to respond to insulin and susceptibility to cell death may partially account for the decreased podocyte number seen in early diabetic nephropathy. The pathogenesis of proteinuria in diabetic nephropathy (DN) is not readily explained by the associated mesangial matrix expansion and glomerular basement membrane thickening. Recently, the role of podocytes in DN has become the subject of intense translational research effort.1.Li J.J. Kwak S.J. Jung D.S. et al.Podocyte biology in diabetic nephropathy.Kidney Int Suppl. 2007; 106: S36-S42Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar,2.Wolf G. Chen S. Ziyadeh F.N. From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy.Diabetes. 2005; 54: 1626-1634Crossref PubMed Scopus (481) Google Scholar Podocyte foot process effacement and decreased podocyte number has been reported in both patients with type 1 and type 2 diabetes.3.Dalla Vestra M. Masiero A. Roiter A.M. et al.Is podocyte injury relevant in diabetic nephropathy? Studies in patients with type 2 diabetes.Diabetes. 2003; 52: 1031-1035Crossref PubMed Scopus (248) Google Scholar, 4.Pagtalunan M.E. Miller P.L. Jumping-Eagle S. et al.Podocyte loss and progressive glomerular injury in type II diabetes.J Clin Invest. 1997; 99: 342-348Crossref PubMed Scopus (808) Google Scholar, 5.Steffes M.W. Schmidt D. McCrery R. et al.Glomerular cell number in normal subjects and in type 1 diabetic patients.Kidney Int. 2001; 59: 2104-2113Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 6.White K.E. Bilous R.W. Structural alterations to the podocyte are related to proteinuria in type 2 diabetic patients.Nephrol Dial Transplant. 2004; 19: 1437-1440Crossref PubMed Scopus (107) Google Scholar, 7.White K.E. Bilous R.W. Marshall S.M. et al.Podocyte number in normotensive type 1 diabetic patients with albuminuria.Diabetes. 2002; 51: 3083-3089Crossref PubMed Scopus (249) Google Scholar, 8.Verzola D. Gandolfo M.T. Ferrario F. et al.Apoptosis in the kidneys of patients with type II diabetic nephropathy.Kidney Int. 2007; 72: 1262-1272Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Studies on kidney biopsies from patients with type2 diabetes showed that loss of podocytes in DN predicts the progressive course of the disease and is a critical determinant for the development of albuminuria.9.Meyer T.W. Bennett P.H. Nelson R.G. Podocyte number predicts long-term urinary albumin excretion in Pima Indians with type II diabetes and microalbuminuria.Diabetologia. 1999; 42: 1341-1344Crossref PubMed Scopus (349) Google Scholar Decreased podocyte density7.White K.E. Bilous R.W. Marshall S.M. et al.Podocyte number in normotensive type 1 diabetic patients with albuminuria.Diabetes. 2002; 51: 3083-3089Crossref PubMed Scopus (249) Google Scholar as well as podocyte detachment10.Toyoda M. Najafian B. Kim Y. et al.Podocyte detachment and reduced glomerular capillary endothelial fenestration in human type 1 diabetic nephropathy.Diabetes. 2007; 56: 2155-2160Crossref PubMed Scopus (188) Google Scholar occurs in the early stages of DN in humans, and correlates with A1C and systolic blood pressure.8.Verzola D. Gandolfo M.T. Ferrario F. et al.Apoptosis in the kidneys of patients with type II diabetic nephropathy.Kidney Int. 2007; 72: 1262-1272Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar A similar loss of podocyte number has been described in experimental models of DN such as the db/db mouse.11.Susztak K. Raff A.C. Schiffer M. et al.Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy.Diabetes. 2006; 55: 225-233Crossref PubMed Scopus (810) Google Scholar This occurs at an age when microalbuminuria is observed without any significant blood pressure increase.12.Koya D. Haneda M. Nakagawa H. et al.Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes.FASEB J. 2000; 14: 439-447Crossref PubMed Scopus (398) Google Scholar Among several signaling pathways that are functional in podocytes, protein kinase B/AKT activation has been recognized as a key survival factor for podocytes exposed to oxidized low-density lipoproteins.13.Bussolati B. Deregibus M.C. Fonsato V. et al.Statins prevent oxidized LDL-induced injury of glomerular podocytes by activating the phosphatidylinositol 3-kinase/AKT-signaling pathway.J Am Soc Nephrol. 2005; 16: 1936-1947Crossref PubMed Scopus (103) Google Scholar Similarly, darbapoietin protects podocytes from ultraviolet-C irradiation induced apoptosis via regulation of AKT.14.Logar C.M. Brinkkoetter P.T. Krofft R.D. et al.Darbepoetin alfa protects podocytes from apoptosis in vitro and in vivo.Kidney Int. 2007; 72: 489-498Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Thus, podocyte loss in early DN may occur through an inability to phosphorylate AKT in response to physiological stimuli, such as insulin. We hypothesize that podocyte inability to regulate AKT precedes the onset of significant albuminuria in db/db mice and is responsible for their susceptibility to cell death. We showed that AKT phosphorylation is downregulated in glomeruli from 12-week-old db/db mice when compared with db/+ mice. We cultured and characterized podocytes from db/db and db/+ mice of the same age, and found that db/db podocytes are more susceptible to cell death induced by serum deprivation and tumor necrosis factor-α (TNF-α) exposure. AKT phosphorylation was markedly suppressed by stress stimuli in db/db podocytes when compared to db/+. The addition of an AKT inhibitor in db/+ podocytes lead to comparable degree of cell death to the one observed in db/db podocytes, suggesting a role for AKT in this model of DN. Glycemia, body weight, and albumin-to-creatinine ratios were evaluated every 1–3 weeks in six db/db and six db/+ mice from 8 to 19 weeks of age. While glycemia and body weight were already significantly different at 8 weeks of age (Table 1), a significant difference in albumin-to-creatinine ratio was observed only at 12 weeks of age and persisted thereafter. In a second experiment db/+ and db/db mice were sacrificed at 12 weeks of age and kidneys were obtained for histology and microdissection of glomeruli for protein analysis and for podocytes culture. No difference in mesangial expansion was observed at this early stage of the disease when compared to controls (0.4±0.2 vs 0.5±0.4; Figure 1).Table 1Metabolic parameters in db/+ and db/db mice between 9 and 19 weeks of age8 weeks10 weeks12 weeks16 weeks19 weeksWeight (g)db/+18.98±0.520.28±0.619.84±1.821.72±1.121.98±0.4db/db34.02±0.6*P<0.0539.44±0.18*P<0.0546.22±0.4*P<0.0551.2±1.8**P<0.01.53.66±1.6**P<0.01.Fasting blood glucose (mg per 100 ml)db/+124.5±5.07137.6±10148.8±16.498.8±1197.2±6.2db/db318.7±26.8*P<0.05207±13*P<0.05302±18.7*P<0.05260±29.4**P<0.01.406±67.8**P<0.01.Albumin/creatinine (μg mg-1)db/+17.20±1.7411.33±12.1016.02±7.4116.3±2716.5±2db/db14.80±3.65244.51±38.3767.12±16.6*P<0.05120±50*P<0.05140±40*P<0.05Weight in grams, fasting blood glucose concentrations (mg per 100 ml) and urinary albumin-to-creatinine ratio on a spot morning urine collection (μg mg-1) are reported. Six db/+ and six db/db at different weeks of age (8, 10, 12, 16, 19) were studied. Mean, standard deviations, and significant differences between db/+ and db/db at each time point are shown* P<0.05** P<0.01. Open table in a new tab Weight in grams, fasting blood glucose concentrations (mg per 100 ml) and urinary albumin-to-creatinine ratio on a spot morning urine collection (μg mg-1) are reported. Six db/+ and six db/db at different weeks of age (8, 10, 12, 16, 19) were studied. Mean, standard deviations, and significant differences between db/+ and db/db at each time point are shown Phosphorylated and total AKT were analyzed on microdissected glomeruli by Luminex technology. db/db glomeruli were characterized by decreased phosphorylated AKT/total AKT when compared with db/+ (Figure 2a). The same assay was performed on whole cortices, where no difference in phosphorylated AKT/total AKT was observed (Figure 2b). We then performed immunofluorescence staining for phosphorylated AKT and synaptopodin in db/+ kidney sections, and found that they partially colocalized (Figure 2c). Initial outgrowth of podocytes from microdissected glomeruli was immortalized as described in methods. We specifically decided not to use the SV40 large T-antigen construct for transfection experiments, since large T-antigen expression interferes with insulin signaling in other cell types.15.DeAngelis T. Chen J. Wu A. et al.Transformation by the simian virus 40 T antigen is regulated by IGF-I receptor and IRS-1 signaling.Oncogene. 2006; 25: 32-42Crossref PubMed Scopus (50) Google Scholar, 16.Lassak A. Del Valle L. Peruzzi F. et al.Insulin receptor substrate 1 translocation to the nucleus by the human JC virus T-antigen.J Biol Chem. 2002; 277: 17231-17238Crossref PubMed Scopus (106) Google Scholar, 17.Valverde A.M. Lorenzo M. Teruel T. et al.Alterations in the insulin signaling pathway induced by immortalization and H-ras transformation of brown adipocytes.Endocrinology. 1997; 138: 3195-3206Crossref PubMed Scopus (18) Google Scholar Podocytes remained in a proliferative state, but were positive for nephrin by western blotting and immunofluorescence (Figure 3a and b). Western blot analysis also confirmed weak positivity for synaptopodin, although synaptopodin detection was possible only when 70 μg of total protein were analyzed (Figure 3b). In addition, nephrin and WT-1 expressions were confirmed by standard reverse transcriptase-polymerase chain reaction (Figure 3c). When podocytes were cultured in regular media, no baseline difference in phosphorylated AKT/total AKT was observed. However, when exposed to increasing concentration of insulin, db/+ podocytes had a dose-dependent increase in phosphorylated AKT/total AKT, whereas db/db podocytes showed no response at insulin concentrations between 0.3 and 30 nM (Figure 4a). This impaired ability of insulin to phosphorylate AKT was most likely mediated by the insulin receptor, since insulin-like growth factor-1 (IGF-1) (100 ng ml-1) resulted in AKT phosphorylation in both db/+ and db/db podocytes (data not shown). In fact, both phosphorylated and total IGF-1 receptor and insulin receptor substrate (IRS)-1 did not differ between diabetic and non-diabetic podocytes (P=NS, data not shown). Because the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been shown to suppress insulin signaling through AKT,18.Tang X. Powelka A.M. Soriano N.A. et al.PTEN, but not SHIP2, suppresses insulin signaling through the phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 adipocytes.J Biol Chem. 2005; 280: 22523-22529Crossref PubMed Scopus (88) Google Scholar we investigated phosphorylated PTEN/total PTEN in our model. PTEN was not affected by increasing insulin concentration in either db/+ or db/db podocytes (Figure 4b). However, db/db podocytes were characterized by decreased phosphorylation of the insulin receptor β-subunit (IR) both at baseline and after insulin exposure (Figure 4c). No difference in total IR was observed between diabetic and non-diabetic podocytes. We defined such inability of insulin to activate downstream signaling events as cellular insulin resistance. Since AKT has been described as an important survival factor, we tested whether db/db podocytes are more susceptible to cell death than db/+ podocytes. We found that serum starvation and addition of TNF-α (2000 U ml-1) to the culture media resulted in a much higher rate of cell death in db/db podocytes when compared to db/+ (Figure 5). When comparing treated and untreated podocytes, serum starvation induced significant increase in cell death in db/db podocytes (P=0.029), but not in db/+ podocytes (P=0.101). The observed cell death was not mediated by activation of caspases, since flow cytometric analysis of fluorescein isothiocyanate (FITC)-conjugated zVAD-labeled cells was not different between cells and between stimuli (data not shown). To assess the role of AKT as a key factor responsible for the observed difference in the susceptibility to cell death, we tested the ratio of phosphorylated to total AKT in cells exposed to either 0.1% fetal bovine serum (FBS) or 0.1% FBS+TNF-α (2000 U ml-1). We found that such stimuli lead to profound downregulation of AKT in db/db podocytes, but not in db/+ podocytes (Figure 6a). Interestingly, addition of an AKT inhibitor to db/+ podocytes resulted in cell death rate resembling the one of db/db podocytes exposed to 0.1% FBS+TNF-α (Figure 6b). Addition of an AKT inhibitor to db/db podocytes exposed to 0.1% FBS+TNF-α did not result in further worsening of cell death. Both p-38 (Figure 7a) and IκB (Figure 7b) were markedly activated by TNF-α, but not by serum starvation, to a degree that did not differ between db/db and db/+ podocytes, suggesting that neither p-38 nor nuclear factor-κB (NF-κB) pathway were responsible for the observed difference in podocytes susceptibility to cell death.Figure 7TNF-α similarly activates p-38 and IκB in db/db and db/+ podocytes. Bar graph representations of four independent experiments showing the effect of 0.1%FBS and 0.1%FBS+TNF-α to p-38 phosphorylation (a) and IκB phosphorylation (b) in db/+ and db/db cells. 0.1% FBS did not affect p-38 and IκB activity alone, whereas addition of TNF-α stimulated both p-38 and IκB equally in both cell types. Data are expressed as percentage of phosphorylated/total p-38 and IκB when compared with the control (untreated) cells; ***P<0.001, **P<0.01, *P<0.05.View Large Image Figure ViewerDownload (PPT) Podocytopenia has become one of the hallmarks of early DN.3.Dalla Vestra M. Masiero A. Roiter A.M. et al.Is podocyte injury relevant in diabetic nephropathy? Studies in patients with type 2 diabetes.Diabetes. 2003; 52: 1031-1035Crossref PubMed Scopus (248) Google Scholar, 4.Pagtalunan M.E. Miller P.L. Jumping-Eagle S. et al.Podocyte loss and progressive glomerular injury in type II diabetes.J Clin Invest. 1997; 99: 342-348Crossref PubMed Scopus (808) Google Scholar, 5.Steffes M.W. Schmidt D. McCrery R. et al.Glomerular cell number in normal subjects and in type 1 diabetic patients.Kidney Int. 2001; 59: 2104-2113Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 6.White K.E. Bilous R.W. Structural alterations to the podocyte are related to proteinuria in type 2 diabetic patients.Nephrol Dial Transplant. 2004; 19: 1437-1440Crossref PubMed Scopus (107) Google Scholar, 7.White K.E. Bilous R.W. Marshall S.M. et al.Podocyte number in normotensive type 1 diabetic patients with albuminuria.Diabetes. 2002; 51: 3083-3089Crossref PubMed Scopus (249) Google Scholar, 8.Verzola D. Gandolfo M.T. Ferrario F. et al.Apoptosis in the kidneys of patients with type II diabetic nephropathy.Kidney Int. 2007; 72: 1262-1272Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 9.Meyer T.W. Bennett P.H. Nelson R.G. Podocyte number predicts long-term urinary albumin excretion in Pima Indians with type II diabetes and microalbuminuria.Diabetologia. 1999; 42: 1341-1344Crossref PubMed Scopus (349) Google Scholar, 10.Toyoda M. Najafian B. Kim Y. et al.Podocyte detachment and reduced glomerular capillary endothelial fenestration in human type 1 diabetic nephropathy.Diabetes. 2007; 56: 2155-2160Crossref PubMed Scopus (188) Google Scholar Although mechanisms leading to podocytopenia are not entirely characterized, a role for AKT as a survival signal in podocytes has been described. AKT/PKB is usually phosphorylated in response to insulin, and podocytes are a novel target for insulin and can regulate glucose uptake upon insulin stimulation.19.Coward R.J. Welsh G.I. Yang J. et al.The human glomerular podocyte is a novel target for insulin action.Diabetes. 2005; 54: 3095-3102Crossref PubMed Scopus (212) Google Scholar Interestingly, insulin-receptor signaling is highly compromised in the retina of a model of streptozotocin-induced diabetes in rats,20.Reiter C.E. Wu X. Sandirasegarane L. et al.Diabetes reduces basal retinal insulin receptor signaling: reversal with systemic and local insulin.Diabetes. 2006; 55: 1148-1156Crossref PubMed Scopus (133) Google Scholar suggesting that a disrupted insulin-signaling pathway may be responsible for the development of microvascular complications of diabetes. In the kidney, studies on patients with type2 diabetes have suggested that insulin resistance per se is independently associated with microalbuminuria.21.Parvanova A.I. Trevisan R. Iliev I.P. et al.Insulin resistance and microalbuminuria: a cross-sectional, case–control study of 158 patients with type 2 diabetes and different degrees of urinary albumin excretion.Diabetes. 2006; 55: 1456-1462Crossref PubMed Scopus (136) Google Scholar With this study, we were able to demonstrate that insulin-dependent phosphorylation of AKT is impaired in murine podocytes isolated from mice with diabetes at the onset of albuminuria, and that such dysregulation of AKT phosphorylation is associated with podocytes' susceptibility to cell death. We elected to use the db/db mouse model on a C57BL6 background because it is characterized by pancreatic islets cells hypertrophy rather than islet cell degeneration as seen in the C57BLKS/J background.22.Sharma K. McCue P. Dunn S.R. Diabetic kidney disease in the db/db mouse.Am J Physiol Renal Physiol. 2003; 284: F1138-F1144Crossref PubMed Scopus (334) Google Scholar In addition, since we wanted to study the early phases of DN, use of the C57BLKS/J background would have been unpractical, since albuminuria is already present at 6 weeks of age.22.Sharma K. McCue P. Dunn S.R. Diabetic kidney disease in the db/db mouse.Am J Physiol Renal Physiol. 2003; 284: F1138-F1144Crossref PubMed Scopus (334) Google Scholar,23.Breyer M.D. Bottinger E. Brosius III, F.C. et al.Mouse models of diabetic nephropathy.J Am Soc Nephrol. 2005; 16: 27-45Crossref PubMed Scopus (412) Google Scholar Based on our findings that albuminuria develops at 12 weeks of age on the C57BL6 background (Table 1), prior to significant mesangial expansion (Figure 1), we chose this age for the subsequent analysis of glomeruli and podocyte culture. AKT is a key survival signal in podocytes in the experimental model of darbapoietin cytoprotection and oxidized low-density lipoprotein toxicity.13.Bussolati B. Deregibus M.C. Fonsato V. et al.Statins prevent oxidized LDL-induced injury of glomerular podocytes by activating the phosphatidylinositol 3-kinase/AKT-signaling pathway.J Am Soc Nephrol. 2005; 16: 1936-1947Crossref PubMed Scopus (103) Google Scholar,14.Logar C.M. Brinkkoetter P.T. Krofft R.D. et al.Darbepoetin alfa protects podocytes from apoptosis in vitro and in vivo.Kidney Int. 2007; 72: 489-498Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar On the contrary, increased podocyte apoptosis despite AKT activation has also been reported,11.Susztak K. Raff A.C. Schiffer M. et al.Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy.Diabetes. 2006; 55: 225-233Crossref PubMed Scopus (810) Google Scholar and vascular endothelial growth factor activates AKT in nephrin-deficient podocytes, without rescuing them from apoptosis.24.Foster R.R. Saleem M.A. Mathieson P.W. et al.Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes.Am J Physiol Renal Physiol. 2005; 288: F48-F57Crossref PubMed Scopus (102) Google Scholar More recently, AKT has been reported to mediate proximal tubular cells hypertrophy, which is usually observed in the early phases of DN.25.Chuang T.D. Guh J.Y. Chiou S.J. et al.Phosphoinositide 3-kinase is required for high glucose-induced hypertrophy and p21WAF1 expression in LLC-PK1 cells.Kidney Int. 2007; 71: 867-874Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Activation of AKT was also described in the kidney of db/db mice in the early phases of DN; of note, whole cortices and not glomeruli were used.26.Feliers D. Duraisamy S. Faulkner J.L. et al.Activation of renal signaling pathways in db/db mice with type 2 diabetes.Kidney Int. 2001; 60: 495-504Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar Our data show that glomeruli from 12-week-old db/db mice are characterized by less phosphorylated AKT when compared with db/+ glomeruli, suggesting that loss of AKT activity may be a very early contributor to podocyte damage resulting in albuminuria (Figure 2a). Although leptin regulates insulin sensitivity per se,27.Szanto I. Kahn C.R. Selective interaction between leptin and insulin signaling pathways in a hepatic cell line.Proc Natl Acad Sci USA. 2000; 97: 2355-2360Crossref PubMed Scopus (164) Google Scholar,28.Morton G.J. Gelling R.W. Niswender K.D. et al.Leptin regulates insulin sensitivity via phosphatidylinositol-3-OH kinase signaling in mediobasal hypothalamic neurons.Cell Metab. 2005; 2: 411-420Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar our result showing equal degree of AKT phosphorylation in whole-kidney cortices of db/db and db/+ mice (Figure 2b) suggests that leptin-receptor mutation per se is not responsible for AKT phosphorylation in our model. Since AKT phosphorylation may be important to maintain normal podocyte physiology, we cultured db/+ and db/db podocytes to address this question. We have previously shown that glomerular cells in culture retain their in vivo phenotype when isolated from mice with diabetic nephropathy and/or glomerulosclerosis.29.Fornoni A. Rosenzweig S.A. Lenz O. et al.Low insulin-like growth factor binding protein-2 expression is responsible for increased insulin receptor substrate-1 phosphorylation in mesangial cells from mice susceptible to glomerulosclerosis.Endocrinology. 2006; 147: 3547-3554Crossref PubMed Scopus (13) Google Scholar,30.Fornoni A. Striker L.J. Zheng F. et al.Reversibility of glucose-induced changes in mesangial cell extracellular matrix depends on the genetic background.Diabetes. 2002; 51: 499-505Crossref PubMed Scopus (31) Google Scholar HPV16-transformed podocytes were used because our preliminary experiments showed an equal ability of insulin to phosphorylate AKT in HPV16-transformed and untransformed podocytes (data not shown). Although podocytes cultured as described retain a proliferative phenotype, we were able to detect podocyte-specific markers, confirming that the phenotype was at least partially retained (Figure 3). Among the markers tested, nephrin expression was similar between db/+ and db/db podocytes (Figure 3). This is particularly important, since nephrin has been recently shown to mediate insulin sensitivity in podocytes.31.Coward R.J. Welsh G.I. Koziell A. et al.Nephrin is critical for the action of insulin on human glomerular podocytes.Diabetes. 2007; 56: 1127-1135Crossref PubMed Scopus (124) Google Scholar Although there was a difference in phosphorylated AKT content in db/+ versus db/db glomeruli, db/+ and db/db podocytes exhibited the same level of phosphorylated AKT under standard cell culture conditions. However, insulin stimulated AKT phosphorylation in a dose-dependent manner in db/+ but not in db/db podocytes via a PTEN-independent mechanism (Figure 4a and b), supporting the hypothesis that the insulin-signaling pathway may play an important role in podocyte pathophysiology in diabetes. Although high glucose concentrations failed to have any effect on AKT phosphorylation in murine podocytes,11.Susztak K. Raff A.C. Schiffer M. et al.Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy.Diabetes. 2006; 55: 225-233Crossref PubMed Scopus (810) Google Scholar it is important to note that the latter experiments were performed to study the effect of acute exposure to high glucose concentration (18 h) rather than the specific response to insulin in diabetic podocytes cultured after chronic exposure to a diabetic environment in vivo. Interestingly, preliminary results showed that the ability of IGF-1 to phosphorylate AKT was comparable in both db/db and db/+ podocytes, suggesting that a specific defect in insulin response occurred in diabetic podocytes. Furthermore, a lower ability to phosphorylate the insulin receptor (Figure 4c), but not the IGF-1 receptor or IRS-1, was observed in db/db podocytes compared with db/+. This was unexpected, since increased serine phosphorylation of IRS-1 is described in the peripheral tissue of diabetic patients and experimental models of type2 diabetes.32.Shoelson S.E. Lee J. Goldfine A.B. Inflammation and insulin resistance.J Clin Invest. 2006; 116: 1793-1801Crossref PubMed Scopus (43) Google Scholar,33.White M.F. Insulin signaling in health and disease.Science. 2003; 302: 1710-1711Crossref PubMed Scopus (527) Google Scholar More recently, however, a role for inflammatory mediator in the suppression of the insulin pathway at the level of receptor phosphorylation was described in patients with insulin resistance,34.Ghanim H. Aljada A. Daoud N. et al.Role of inflammatory mediators in the suppression of insulin receptor phosphorylation in circulating mononuclear cells of obese subjects.Diabetologia. 2007; 50: 278-285Crossref PubMed Scopus (83) Google Scholar suggesting that modulation of the insulin pathway at sites different than IRS-1 may occur in diabetes. Our findings from db/db podocytes are rather different than those observed in mesangial cells from db/db mice, where we have reported an autocrine activation of the IGF-1 pathway.35.Karl M. Potier M. Schulman I.H. et al.Autocrine activation of the local insulin-like growth factor I system is upregulated by estrogen receptor (ER)-independent estrogen actions and accounts for decreased ER expression in type 2 diabetic mesangial cells.Endocrinology. 2005; 146: 889-900Crossref PubMed Scopus (36) Google Scholar Such cell-specific modulation of insulin and IGF-1 pathway in different glomerular cells is interesting and further studies are needed to elucidate the underlying mechanisms. We then choose to look at cell death as a biological read out linked to AKT phosphorylation. We observed that db/db podocytes were by far more susceptible to cell death than db/+ podocytes (Figure 5). We choose serum starvation and exposure to TNF-α as stress stimuli, since serum starvation has been linked to podocytes apoptosis in prior studies,36.Foster R.R. Hole R. Anderson K. et al.Functional evidence that vascular endothelial growth factor may act as an autocrine factor on human podocytes.Am J Physiol Renal Physiol. 2003; 284: F1263-F1273Crossref PubMed Scopus (147) Google Scholar and TNF-α is strongly upregulated in the early phases of DN.37.Moriwaki Y. Yamamoto T. Shibutani Y. et al.Elevated levels of interleukin-18 and tumor necrosis factor-alpha in serum of patients with type 2 diabetes mellitus: relationship with diabetic nephropathy.Metabolism. 2003; 52: 605-608Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar,38.Navarro J.F. Mora-Fernandez C. The role of TNF-alpha in diabetic nephropathy: pathogenic and therapeutic implications.Cytokine Growth Factor Rev. 2006; 17: 441-450Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar Interestingly, this was associated with downregulation of AKT phosphorylation in db/db but not db/+ podocytes (Figure 6a). Addition of an AKT inhibitor to db/+ podocytes exposed to 0.1% FBS and TNF-α was necessary to observed a degree of cell death comparable to the one observed in db/db podocytes (Figure 6b), suggesting that cell survival in podocytes is AKT-dependent. Finally, since NF-κB and p-38 are also important mediators of DN that are relevant to cell survival,11.Susztak K. Raff A.C. Schiffer M. et al.Glucose-induced reac
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