Activation of the renin–angiotensin system within podocytes in diabetes
2007; Elsevier BV; Volume: 71; Issue: 10 Linguagem: Inglês
10.1038/sj.ki.5002195
ISSN1523-1755
AutoresTae‐Hyun Yoo, J.-J. Li, Jae‐Jin Kim, Dong-Il Jung, S.-J. Kwak, Dong‐Ryeol Ryu, Hoon Young Choi, Jin Sung Kim, H.J. Kim, Seung Hyeok Han, Jung Eun Lee, D. S. Han, Shin‐Wook Kang,
Tópico(s)Chronic Kidney Disease and Diabetes
ResumoThe autocrine and paracrine activation of the renin–angiotensin system (RAS) within cells of the kidney plays a role in the overall pathophysiology of the renal disease due to diabetes. In this study, we focus on components of the RAS in the podocyte as these cells are important in the pathogenesis of glomerulosclerosis and proteinuria. Immortalized mouse podocytes were exposed to media containing normal glucose (NG) or high glucose (HG) for in vitro studies. In vivo studies utilized kidney tissue obtained from rats treated for 3 months with streptozotocin to induce diabetes. Angiotensinogen (AGT) and the angiotensin II (AII) type 1 receptor mRNA and protein were significantly increased in the podocytes cultured under the high glucose conditions. Both angiotensins I and II levels were significantly higher in cell lysates and the conditioned media of cells grown in high glucose. There were no differences in renin activity, angiotensin-converting enzyme level, or AII type 2 receptor level. Glomerular AGT and AII type 1 receptor assessed by means of immunohistochemistry were increased in diabetic rats compared with the control rats. Other measured components of the RAS within the glomeruli were not different. We suggest that increased AGT, an attendant increase in AII and increased AII type 1 receptor in podocytes experiencing diabetic conditions play an important role in the pathogenesis of diabetic nephropathy. The autocrine and paracrine activation of the renin–angiotensin system (RAS) within cells of the kidney plays a role in the overall pathophysiology of the renal disease due to diabetes. In this study, we focus on components of the RAS in the podocyte as these cells are important in the pathogenesis of glomerulosclerosis and proteinuria. Immortalized mouse podocytes were exposed to media containing normal glucose (NG) or high glucose (HG) for in vitro studies. In vivo studies utilized kidney tissue obtained from rats treated for 3 months with streptozotocin to induce diabetes. Angiotensinogen (AGT) and the angiotensin II (AII) type 1 receptor mRNA and protein were significantly increased in the podocytes cultured under the high glucose conditions. Both angiotensins I and II levels were significantly higher in cell lysates and the conditioned media of cells grown in high glucose. There were no differences in renin activity, angiotensin-converting enzyme level, or AII type 2 receptor level. Glomerular AGT and AII type 1 receptor assessed by means of immunohistochemistry were increased in diabetic rats compared with the control rats. Other measured components of the RAS within the glomeruli were not different. We suggest that increased AGT, an attendant increase in AII and increased AII type 1 receptor in podocytes experiencing diabetic conditions play an important role in the pathogenesis of diabetic nephropathy. Diabetic nephropathy, the leading cause of end-stage renal disease in the US,1.US Renal Data System USRDS 2004 annual data report.Am J Kidney Dis. 2005; 45: 8-280Abstract Full Text Full Text PDF Google Scholar is characterized pathologically by cellular hypertrophy and increased extracellular matrix accumulation and clinically by proteinuria.2.Adler S. Structure–function relationships associated with extracellular matrix alterations in diabetic glomerulopathy.J Am Soc Nephrol. 1994; 5: 1165-1172PubMed Google Scholar,3.Mogensen C.E. Christensen C.K. Vittinghus E. The stages in diabetic renal disease. With emphasis on the stage of incipient diabetic nephropathy.Diabetes. 1983; 32: 64-78Crossref PubMed Google Scholar The molecular and cellular mechanisms responsible for these disease characteristics remain incompletely resolved. Although the diabetic milieu per se, hemodynamic changes, and local growth factors such as angiotensin II (AII) are considered to be mediators in the pathogenesis of diabetic nephropathy,4.Leehey D.J. Singh A.K. Alavi N. et al.Role of angiotensin II in diabetic nephropathy.Kidney Int. 2000; 77: S93-S98Abstract Full Text Full Text PDF Scopus (222) Google Scholar the underlying pathways mediating these processes are not well understood. Previous clinical and experimental studies in diabetic nephropathy have demonstrated that blockade of the renin–angiotensin system (RAS) by angiotensin-converting enzyme (ACE) inhibitors or AII receptor blockers reduced proteinuria and the progression of nephropathy that cannot be explained merely by their antihypertensive effect.5.Lewis E.J. Hunsicker L.G. Bain R.P. Rohde R.D. The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy.N Engl J Med. 1993; 329: 1456-1462Crossref PubMed Scopus (5022) Google Scholar, 6.Brenner B.M. Cooper M.E. de Zeeuw D. et al.Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy.N Engl J Med. 2001; 345: 861-869Crossref PubMed Scopus (6160) Google Scholar, 7.Gross M.L. El-Shakmak A. Szabo A. et al.ACE-inhibitors but not endothelin receptor blockers prevent podocyte loss in early diabetic nephropathy.Diabetologia. 2003; 46: 856-868Crossref PubMed Scopus (121) Google Scholar These findings suggest that RAS blockade may have direct effects on various renal cells. Among them, many investigators have focused on podocytes because they not only serve as a filtration barrier to protein but also play an important role in the pathogenesis of glomerulosclerosis.8.Barisoni L. Mundel P. Podocyte biology and the emerging understanding of podocyte diseases.Am J Nephrol. 2003; 23: 353-360Crossref PubMed Scopus (107) Google Scholar We have previously demonstrated that AII receptor blockers ameliorates high glucose-induced podocytes hypertrophy by inhibiting p27Kip1 mRNA and protein expression.9.Xu Z.G. Yoo T.H. Ryu D.R. et al.Angiotensin II receptor blocker inhibits p27Kip1 expression in glucose-stimulated podocytes and in diabetic glomeruli.Kidney Int. 2005; 67: 944-952Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar In addition, others have observed that blocking the RAS restores reduced nephrin levels and attenuates foot process broadening in diabetic nephropathy.10.Langham R.G. Kelly D.J. Cox A.J. et al.Proteinuria and the expression of the podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effects of angiotensin converting enzyme inhibition.Diabetologia. 2002; 45: 1572-1576Crossref PubMed Scopus (202) Google Scholar,11.Misfud S.A. Allen T.J. Bertram J.F. et al.Podocyte foot process broadening in experimental diabetic nephropathy: amelioration with renin–angiotensin blockade.Diabetologia. 2001; 44: 878-882Crossref PubMed Scopus (128) Google Scholar Taken together, it seems that the inhibition of AII may have a direct beneficial effect on podocytes. AII is the major effector molecule of the RAS. It is a powerful direct vasoconstrictor, promotes vascular smooth muscle contraction and induces systemic hypertension.12.Wolf G. Butzmann U. Wenzel U.O. The renin–angiotensin system and progression of renal disease: from hemodynamics to cell biology.Nephron Physiol. 2003; 93: P3-P13Crossref PubMed Scopus (158) Google Scholar Besides its hemodynamic effects, AII plays an important role in the pathogenesis of various renal diseases, including diabetic nephropathy. It mediates apoptosis and inflammation via the angiotensin II type 1 receptor (AT1R), directly stimulates transforming growth factor-β1 production and signaling pathways such as mitogen-activated protein kinase, lipooxygenase, and phosphatidylinositol 3-kinase, and contributes to the development and maintenance of renal hypertrophy, matrix expansion, and glomerulosclerosis.13.Zhang S.L. To C. Chen X. et al.Essential role(s) of the intrarenal renin–angiotensin system in transforming growth factor-beta1 gene expression and induction of hypertrophy of rat kidney proximal tubular cells in high glucose.J Am Soc Nephrol. 2002; 13: 302-312Crossref PubMed Google Scholar,14.Wolf G. Ziyadeh F.N. The role of angiotensin II in diabetic nephropathy: emphasis on nonhemodynamic mechanisms.Am J Kidney Dis. 1997; 29: 153-163Abstract Full Text PDF PubMed Scopus (215) Google Scholar In diabetes, intrarenal RAS is known to be activated in spite of low plasma renin activity.15.Price D.A. Porter L.E. Gordon M. et al.The paradox of the low-renin state in diabetic nephropathy.J Am Soc Nephrol. 1999; 10: 2382-2391PubMed Google Scholar Recent studies have demonstrated that local RAS is activated in mesangial cells16.Singh R. Singh A.K. Alavi N. Leehey D.J. Mechanism of increased angiotensin II levels in glomerular mesangial cells cultured in high glucose.J Am Soc Nephrol. 2003; 14: 873-880Crossref PubMed Scopus (130) Google Scholar,17.Vidotti D.B. Casarini D.E. Cristovam P.C. et al.High glucose concentration stimulates intracellular renin activity and angiotensin II generation in rat mesangial cells.Am J Physiol Renal Physiol. 2004; 286: F1039-F1045Crossref PubMed Scopus (182) Google Scholar and proximal tubular cells13.Zhang S.L. To C. Chen X. et al.Essential role(s) of the intrarenal renin–angiotensin system in transforming growth factor-beta1 gene expression and induction of hypertrophy of rat kidney proximal tubular cells in high glucose.J Am Soc Nephrol. 2002; 13: 302-312Crossref PubMed Google Scholar by hyperglycemia and in podocytes by mechanical strain.18.Durvasula R.V. Petermann A.T. Hiromura K. et al.Activation of a local tissue angiotensin system in podocytes by mechanical strain.Kidney Int. 2004; 65: 30-39Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar Moreover, we have also observed previously an increase in AII levels in podocyte lysates as well as in their conditioned culture media by high glucose.9.Xu Z.G. Yoo T.H. Ryu D.R. et al.Angiotensin II receptor blocker inhibits p27Kip1 expression in glucose-stimulated podocytes and in diabetic glomeruli.Kidney Int. 2005; 67: 944-952Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar However, the mechanisms by which high glucose increases AII in podocytes are not yet clear. To clarify the precise mechanism of high glucose-induced AII production, we investigated the changes of RAS components, including angiotensinogen (AGT), renin, AI, ACE, AII, AT1R, and AT2R in high glucose-stimulated podocytes. High glucose-induced changes in AII production were further determined with specific inhibitors of ACE, chymase, or protein kinase C (PKC) to confirm the underlying pathway involved. In addition, we examined the changes of RAS components in diabetic glomeruli to verify the results of the in vitro experiments. We first determined the changes in AGT mRNA and protein expression in podocytes under diabetic conditions. The AGT/18S mRNA ratio was 1.9-fold higher in podocytes exposed to high glucose (HG) than in those exposed to normal glucose (NG), and this HG-induced increment in AGT mRNA expression was ameliorated by 87.5 and 90.9% with the pretreatment of calphostin C and GF 109203X, respectively (Figure 1). The expression of AGT protein showed a similar pattern to its mRNA expression (Figure 2). On the other hand, there was no significant difference in AGT/18S mRNA ratio between the NG and NG+M (mannitol) groups.Figure 2Western blot of AGT in podocytes exposed to NG, NG+M, or HG medium with or without PKC inhibitor, 10−7 M calphostin C (CC) or 10−6 M GF 109203X (GFX) (representative of five blots). AGT protein expression was significantly higher in podocytes exposed to HG than in those exposed to NG. PKC inhibitors nearly normalized the increases in AGT protein expression, whereas mannitol had no effect on AGT protein expression.View Large Image Figure ViewerDownload (PPT) We next evaluated the changes in renin activities in podocytes and their conditioned media by measuring AI levels in the presence of excess exogenous AGT (1 μM). AI levels from AGT-treated conditions were increased about two- to six-fold compared with AI levels without AGT exposure. In cell lysates, the increases in AI concentrations in the presence of AGT from the baseline value were as follows: 4.99±0.91 pg/μg protein in the NG group, 5.24±0.83 pg/μg protein in the NG+M group, and 5.56±0.64 pg/μg protein in the HG group (P>0.05). In the conditioned media, the results were similar to those of cell lysates (Δ increases in AI levels were NG, 2.86±0.14 pg/μl; NG+M, 2.82±0.35 pg/μl; and HG, 2.99±0.23 pg/μl, P>0.05). PKC inhibitors had no effect on the changes in AI concentrations in the presence of excess exogenous AGT (Figure 3). AI levels were significantly higher in HG-treated cell lysates compared with NG cell lysates (HG, 1.84±0.45 pg/μl; NG, 0.85±0.16 pg/μg protein, P<0.05) and in the HG-conditioned media (HG, 2.60±0.68 pg/μl; NG, 0.88±0.23 pg/μl, P<0.01), and PKC inhibitors nearly normalized these HG-induced increases in AI concentrations in cell lysates and in the conditioned media. On the other hand, there were no significant differences in AI levels in cell lysates or in the media between the NG and NG+M groups (Figure 4). The ACE/18S mRNA ratio was not statistically different between the NG and HG-treated cells (data not shown). As seen in Figure 5, there was also no difference in ACE protein expression among the groups. ACE levels determined by enzyme-linked immunosorbent assay (ELISA) revealed that ACE levels did not change in podocytes by HG (NG, 2.87±0.42 pg/μg protein; HG, 2.92±0.37 pg/μg protein). Mannitol and PKC inhibitors had no effect on the ACE mRNA and protein expression, and ACE levels. AII levels were significantly higher in HG-treated cell lysates (0.31±0.04 pg/μg protein) and in HG-conditioned media (2.54±0.33 pg/μl) compared with NG-treated cells (0.18±0.02 pg/μg protein, P<0.05) and conditioned media (1.13±0.12 pg/μl, P<0.01) (Figure 6a and b). Captopril, chymostatin, calphostin C, and GF 109203X inhibited these HG-induced increments in AII concentrations in cell lysates by 53.8, 69.2, 88.5, and 96.2%, respectively, and in HG-conditioned media by 75.0, 82.1, 96.6, and 89.4%, respectively. Captopril and chymostatin also reduced AII levels in NG cells and NG-conditioned media, but did not reach statistical significance (data not shown). Conversely, there was no significant difference in AII concentrations between the NG and NG+M treatment groups. The AT1R/18S mRNA ratio in podocytes exposed to HG was 288% higher than in NG-treated cells (P<0.005) (Figure 7). Western blot analysis further supported the real-time polymerase chain reaction results (Figure 8). Desitometric measurement revealed a 1.7-fold increase in AT1R protein expression in HG-treated podocytes compared with NG cells. PKC inhibitors, calphostin C, and GF 109203X, nearly normalized the increases in AT1R mRNA and protein expression, whereas mannitol had no effect on AT1R expression. In contrast, there were no differences in AT2R mRNA and protein expression among the groups.Figure 8Western blot of AT1R and AT2R in podocytes exposed to NG, NG+M, or HG medium with or without PKC inhibitor, 10−7 M calphostin C (CC) or 10−6 M GF 109203X (GFX) (representative of five blots). C3H/10T1/2 cell lysate and Hep G2 cell lysate were loaded in the same gel as positive control for AT1R and AT2R, respectively (POS). AT1R protein expression was significantly higher in podocytes exposed to HG than in those exposed to NG. Densitometric quantitation revealed that there was a 1.7-fold increase in AT1R protein expression in HG-treated podocytes compared with NG cells. PKC inhibitors nearly normalized the increases in AT1R protein expression, whereas mannitol had no effect on AT1R protein expression. In contrast, there was no significant difference in the AT2R protein expression among the groups.View Large Image Figure ViewerDownload (PPT) Body weight increased in both control (C) and diabetes mellitus (DM) groups throughout the 3-months study period, but increased more in the former (510±4 g) than in the latter (266±3 g) (P<0.01). The ratio of kidney weight to body weight at the time of killing was significantly higher in DM (1.64±0.11%) than in C rats (0.56±0.05%) (P<0.01). Compared with the C (0.40±0.06 mg/day), 24-h urinary albumin excretion was significantly higher in the DM group (1.99±0.22 mg/day, P<0.05) (Table 1).Table 1Body weight, kidney weight, and urinary albumin excretion of the ratsParametersCDMBody weight (g)510±4266±3aP<0.01 vs C.Kidney weight/body weight (%)0.56±0.051.64±0.11aP<0.01 vs C.Blood glucose (mg/dl)112.1±8.1495.5±11.2aP<0.01 vs C.Urinary albumin excretion (mg/day)0.40±0.061.99±0.22bP<0.05 vs C.a P<0.01 vs C.b P<0.05 vs C. Open table in a new tab The changes in mRNA and protein expression of the RAS components in DM glomeruli were similar to those in high glucose-stimulated podocytes. The mRNA expression of AGT and AT1R were increased in DM glomeruli by 63.1 and 81.3%, respectively, compared with C glomeruli (P<0.05) (Figure 9). Western blot analysis also revealed significant increases in AGT and AT1R protein expression in DM glomeruli (Figure 10). The positive-stained cells within glomeruli by immunohistochemistry were podocytes and mesangial cells, with a clear predominance in the former. The semiquantitative staining scores for glomerular AGT and AT1R were significantly higher in DM (AGT, 59.3±9.1; AT1R, 71.2±10.2) than in C rats (AGT, 21.5±3.7; AT1R, 28.7±5.3) (P<0.05) (Figure 11). On the other hand, there were no differences in ACE and AT2R mRNA and protein expression between C and DM glomeruli.Figure 10Western blot of AGT, ACE, AT1R, and AT2R in control and DM glomeruli (representative of six blots). There were significant increases in glomerular AGT and AT1R protein expression in DM vs C rats, whereas ACE and AT2R protein expression of the two groups were comparable.View Large Image Figure ViewerDownload (PPT)Figure 11Immunohistochemical staining for (a and b) AGT, (c and d) ACE, (e and f) AT1R, (g and h) AT2R, (i and j) β-actin, and (k and l) synaptopodin in (a, c, e, g, i, and k) C and (b, d, f, h, j, and l) DM at 3 months after streptozotocin injection. The positive-stained cells within glomeruli were podocytes and mesangial cells, with a clear predominance in the former. There were significant increases in glomerular AGT and AT1R protein expression in DM vs C rats (arrowhead), whereas ACE and AT2R protein expression were not different between C and DM glomeruli. On the other hand, the staining intensity of glomerular β-actin and synaptopodin in C and DM rats were comparable (original magnification × 400).View Large Image Figure ViewerDownload (PPT) In this study, we show for the first time that AII levels are increased concurrently with ATIR mRNA and protein expressions in podocytes exposed to high glucose and PKC pathway is involved in these changes. In addition, high glucose-induced AII generation is attributed to an increase in AGT expression which results in an increase in the AII substrate, rather than more conversion of AGT to AI or increased conversion of AI to AII. The results of these in vitro experiments were further verified by the in vivo findings. Recently, studies have focused on the role of podocytes in the pathophysiology of proteinuria in various glomerular diseases, including diabetic nephropathy. It has been reported that proteinuria is associated with decreased podocyte numbers and/or densities and alterations in glomerular filtration barrier-associated molecules.19.Bains R. Furness P.N. Critchley D.R. A quantitative immunofluorescence study of glomerular cell adhesion proteins in proteinuric states.J Pathol. 1997; 183: 272-280Crossref PubMed Scopus (44) Google Scholar,20.Xu Z.G. Ryu D.R. Yoo T.H. et al.P-cadherin is decreased in diabetic glomeruli and in glucose-stimulated podocytes in vivo and in vitro studies.Nephrol Dial Transplant. 2005; 20: 524-531Crossref PubMed Scopus (26) Google Scholar Glomerular podocyte numbers are known to decrease in type 1 and 2 diabetic patients, even in diabetes of short duration.21.Steffes M.W. Schmidt D. McCrery R. Basgen J.M. 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,22.Pagtalunan M.E. Miller P.L. Jumping-Eagle S. et al.Podocyte loss and progressive glomerular injury in type 2 diabetes.J Clin Invest. 1979; 99: 342-348Crossref Scopus (901) Google Scholar A recent study has also demonstrated a strong inverse correlation between the changes in podocyte numbers and urinary albumin excretion rates in diabetic patients.23.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 (269) Google Scholar Consistent with the observed loss of podocytes, podocytes were more detectable in the urine of diabetic patients with proteinuria.24.Nakamura T. Ushiyama C. Suzuki S. et al.Urinary excretion of podocytes in patients with diabetic nephropathy.Nephrol Dial Transplant. 2000; 15: 1379-1383Crossref PubMed Scopus (265) Google Scholar Furthermore, the expression of nephrin, a key podoctye-specific molecule, is decreased in human and experimental diabetic nephropathy.25.Doublier S. Salvidio G. Lupia E. et al.Nephrin expression is reduced in human diabetic nephropathy: evidence for a distinct role for glycated albumin and angiotensin II.Diabetes. 2003; 52: 1023-1030Crossref PubMed Scopus (317) Google Scholar,26.Bonnet F. Cooper M.E. Kawachi H. et al.Irbesartan normalises the deficiency in glomerular nephrin expression in a model of diabetes and hypertension.Diabetologia. 2001; 44: 874-877Crossref PubMed Scopus (230) Google Scholar However, both the loss of podocytes and the reduction of nephrin expression are inhibited by RAS blockade, suggesting that AII plays an important role in the pathogenesis of diabetic nephropathy, especially in the development of proteinuria. AII, an octapeptide, is predominantly converted from a decapeptide AI that is synthesized from AGT by renin. On the other hand, the formation of active AII from inactive AI is mediated by ACE and non-ACE enzymes. Under physiologic conditions, ACE is known to be the most important enzyme in the generation of AII.27.Siragy H.M. AT(1) and AT(2) receptors in the kidney: role in disease and treatment.Am J Kidney Dis. 2000; 36: S4-S9Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar In this study, high glucose had no effects on ACE expression and levels in podocytes. These results were in accordance with previous studies with high glucose-stimulated mesangial cells16.Singh R. Singh A.K. Alavi N. Leehey D.J. Mechanism of increased angiotensin II levels in glomerular mesangial cells cultured in high glucose.J Am Soc Nephrol. 2003; 14: 873-880Crossref PubMed Scopus (130) Google Scholar and freshly isolated diabetic glomeruli.28.Singh R. Singh A.K. Leehey D.J. A novel mechanism for angiotensin II formation in streptozotocin-diabetic rat glomeruli.Am J Physiol Renal Physiol. 2005; 288: F1183-F1190Crossref PubMed Scopus (84) Google Scholar However, several investigators have suggested that a non-ACE pathway may play a role in the formation of local AII. Singh et al.16.Singh R. Singh A.K. Alavi N. Leehey D.J. Mechanism of increased angiotensin II levels in glomerular mesangial cells cultured in high glucose.J Am Soc Nephrol. 2003; 14: 873-880Crossref PubMed Scopus (130) Google Scholar,28.Singh R. Singh A.K. Leehey D.J. A novel mechanism for angiotensin II formation in streptozotocin-diabetic rat glomeruli.Am J Physiol Renal Physiol. 2005; 288: F1183-F1190Crossref PubMed Scopus (84) Google Scholar raised the possibility of the existence of an ACE-related carboxypeptidase other than ACE1 that could be inhibited by an ACE inhibitor in mesangial cells and glomeruli. Among non-ACE enzymes, chymase is the best known enzyme to be involved in the conversion of AI to AII. A study using immunohistochemistry showed chymase upregulation in diabetic kidney, especially in mesangial cells and vascular smooth muscle cells. However, chymase expression has not been detected in the podocytes even in diabetic patients with hypertension, in whom the chymase staining was extremely strong.29.Huang X.R. Chen W.Y. Truong L.D. Lan H.Y. Chymase is upregulated in diabetic nephropathy: implications for an alternative pathway of angiotensin II-mediated diabetic renal and vascular disease.J Am Soc Nephrol. 2003; 14: 1738-1747Crossref PubMed Scopus (218) Google Scholar In this study, we demonstrate that even though renin activity and ACE levels and expression do not change under diabetic conditions, AGT expression is significantly increased in high glucose-stimulated podocytes and in podocytes of diabetic glomeruli. Similar to our results, the increase in AGT expression by high glucose has also been demonstrated in proximal tubular cells,30.Wang T.T. Wu X.H. Zhang S.L. Chan J.S. Effect of glucose on the expression of the angiotensinogen gene in opossum kidney cells.Kidney Int. 1998; 53: 312-319Abstract Full Text PDF PubMed Scopus (36) Google Scholar mesangial cells,16.Singh R. Singh A.K. Alavi N. Leehey D.J. Mechanism of increased angiotensin II levels in glomerular mesangial cells cultured in high glucose.J Am Soc Nephrol. 2003; 14: 873-880Crossref PubMed Scopus (130) Google Scholar,17.Vidotti D.B. Casarini D.E. Cristovam P.C. et al.High glucose concentration stimulates intracellular renin activity and angiotensin II generation in rat mesangial cells.Am J Physiol Renal Physiol. 2004; 286: F1039-F1045Crossref PubMed Scopus (182) Google Scholar and peritoneal mesothelial cells.31.Noh H. Ha H. Yu M.R. et al.Angiotensin II mediates high glucose-induced TGF-beta1 and fibronectin upregulation in HPMC through reactive oxygen species.Perit Dial Int. 2005; 25: 38-47PubMed Google Scholar Taken together, these findings suggest that the increase of AII generation in podocytes by high glucose is primarily due to an increase in AGT, serving as more substrate for AII, rather than more conversion of AGT to AI or increased conversion of AI to AII. In addition, our results, that pretreatment with ACE inhibitor or chymase inhibitor ameliorates the increase of AII levels in high glucose-stimulated podocytes and their conditioned media, also implies the involvement of both ACE and non-ACE pathways in high glucose-induced AII production in podocytes. AII exerts biological effects through its interaction with the G-protein-coupled AII receptors on cell surface. Based on the availability of nonpeptide receptor ligands, AII receptors are classified into AT1R and AT2R groups. In vivo and in vitro studies have revealed that podocytes express both AT1R and AT2R. Differentiated podocytes predominantly express AT1R (about 75%), with a lesser amount of AT2R (about 25%).32.Wang L. Flannery P.J. Spurney R.F. Characterization of angiotensin II-receptor subtypes in podocytes.J Lab Clin Med. 2003; 142: 313-321Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,33.Liebau M.C. Lang D. Bohm J. et al.Functional expression of the renin angiotensin system in human podocytes.Am J Physiol Renal Physiol. 2006; 27: F710-F719Google Scholar AT1R is more important in mediating most of the physiologic actions of AII. The role of AT1R was emphasized in a recent study with transgenic rats that had overexpression of human ATIR in podocytes.34.Hoffmann S. Podlich D. Hahnel B. et al.Angiotensin II type 1 receptor overexpression in podocytes induces glomerulosclerosis in transgenic rats.J Am Soc Nephrol. 2004; 15: 1475-1487Crossref PubMed Scopus (172) Google Scholar These rats developed albuminuira and showed structural changes in the glomerulus, including pseudocysts formation, foot process effacement, and local detachments, followed by progression to focal segmental glomerulosclerosis. In addition, AT1R has been reported to mediate the processes of enhanced DNA synthesis,32.Wang L. Flannery P.J. Spurney R.F. Characterization of angiotensin II-receptor subtypes in podocytes.J Lab Clin Med. 2003; 142: 313-321Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar depolarization,35.Gloy J. Henger A. Fischer K.G. et al.Angiotensin II modulates cellular functions of podocytes.Kidney Int. 1998; 67: S168-S170Abstract Full Text Full Text PDF Scopus (38) Google Scholar and reorganization of F-actin fibers and redistribution of zonula occludens-1 of podocytes induced by AII,36.Macconi D. Abbate M. Morigi M. et al.Permselective dysfunction of podocyte–podocyte contact upon angiotensin II unravels the molecular target for renoprotective intervention.Am J Pathol. 2006; 168: 1073-1085Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar as well as mechanical strain-induced apoptosis of podocytes,18.Durvasula R.V. Petermann A.T. Hiromura K. et al.Activation of a local tissue angiotensin system in podocytes by mechanical strain.Kidney Int. 2004; 65: 30-39Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar and macrophage infiltration in experimental diabetic nephropathy.37.Lee F.T. Cao Z. Long D.M. et al.Interactions between angiotensin II and NF-kappaB-dependent pathways in modulating macrophage infiltration in experimental diabetic nephropathy.J Am Soc Nephrol. 2004; 15: 2139-2151Crossref PubMed Scopus (136) Google Scholar In contrast, AT2R receptors are highly expressed in fetal tissues, but their expression is markedly downregulated after birth.38.Perlegas D. Xie H. Sinha S. et al.The angiotensin type-2 receptor regulates smooth muscle growth and force generation in late fetal
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