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• W2891027264 abstract "Diabetic nephropathy correlates more closely to defective mitochondria and increased oxidative stress in the kidney than to hyperglycemia. A key driving factor of diabetic nephropathy is angiotensin II acting via the G-protein–coupled cell membrane type 1 receptor. The present study aimed to investigate the role of the angiotensin II type 2 receptor (AT2R) at the early stages of diabetic nephropathy. Using receptor binding studies and immunohistochemistry we found that the mitochondria in renal tubules contain high-affinity AT2Rs. Increased renal mitochondrial AT2R density by transgenic overexpression was associated with reduced superoxide production of isolated mitochondria from non-diabetic rats. Streptozotocin-induced diabetes (28 days) caused a drop in the ATP/oxygen ratio and an increase in the superoxide production of isolated renal mitochondria from wild-type diabetic rats. This correlated with changes in the renal expression profile and increased tubular epithelial cell proliferation. AT2R overexpression in tubular epithelial cells inhibited all diabetes-induced renal changes including a drop in mitochondrial bioenergetics efficiency, a rise in mitochondrial superoxide production, metabolic reprogramming, and increased proliferation. Thus, AT2Rs translocate to mitochondria and can contribute to reno-protective effects at early stages of diabetes. Hence, targeted AT2R overexpression in renal cells may open new avenues to develop novel types of drugs preventing diabetic nephropathy. Diabetic nephropathy correlates more closely to defective mitochondria and increased oxidative stress in the kidney than to hyperglycemia. A key driving factor of diabetic nephropathy is angiotensin II acting via the G-protein–coupled cell membrane type 1 receptor. The present study aimed to investigate the role of the angiotensin II type 2 receptor (AT2R) at the early stages of diabetic nephropathy. Using receptor binding studies and immunohistochemistry we found that the mitochondria in renal tubules contain high-affinity AT2Rs. Increased renal mitochondrial AT2R density by transgenic overexpression was associated with reduced superoxide production of isolated mitochondria from non-diabetic rats. Streptozotocin-induced diabetes (28 days) caused a drop in the ATP/oxygen ratio and an increase in the superoxide production of isolated renal mitochondria from wild-type diabetic rats. This correlated with changes in the renal expression profile and increased tubular epithelial cell proliferation. AT2R overexpression in tubular epithelial cells inhibited all diabetes-induced renal changes including a drop in mitochondrial bioenergetics efficiency, a rise in mitochondrial superoxide production, metabolic reprogramming, and increased proliferation. Thus, AT2Rs translocate to mitochondria and can contribute to reno-protective effects at early stages of diabetes. Hence, targeted AT2R overexpression in renal cells may open new avenues to develop novel types of drugs preventing diabetic nephropathy. Diabetic nephropathy (DN) affects approximately 30% of type 1 and 2 diabetic patients and is the leading cause of end-stage renal failure. The incidence and severity of DN are not determined by hyperglycemia, but correlate with the disruption of mitochondrial bioenergetics and increased oxidative stress in the kidney,1Coughlan M.T. Nguyen T.V. Penfold S.A. et al.Mapping time-course mitochondrial adaptations in the kidney in experimental diabetes.Clin Sci (Lond). 2016; 130: 711-720Crossref PubMed Scopus (84) Google Scholar, 2Forbes J.M. Coughlan M.T. Cooper M.E. Oxidative stress as a major culprit in kidney disease in diabetes.Diabetes. 2008; 57: 1446-1454Crossref PubMed Scopus (937) Google Scholar specifically within the proximal tubular epithelial cells (PTECs), which constitute more than 90% of the kidney cortex.3Vallon V. Blantz R.C. Thomson S. Glomerular hyperfiltration and the salt paradox in early [corrected] type 1 diabetes mellitus: a tubulo-centric view.J Am Soc Nephrol. 2003; 14: 530-537Crossref PubMed Scopus (139) Google Scholar To drive tubular reabsorption, PTECs have a high adenosine triphosphate (ATP) demand, which is preferentially generated from oxidative phosphorylation (OXPHOS) and fatty acids.4Czajka A. Malik A.N. Hyperglycemia induced damage to mitochondrial respiration in renal mesangial and tubular cells: implications for diabetic nephropathy.Redox Biol. 2016; 10: 100-107Crossref PubMed Scopus (59) Google Scholar The total energy production in quiescent kidney cells is similar to that produced by proliferating cancer cells.5Godinot C. de Laplanche E. Hervouet E. Simonnet H. Actuality of Warburg’s views in our understanding of renal cancer metabolism.J Bioenerg Biomembr. 2007; 39: 235-241Crossref PubMed Scopus (39) Google Scholar Hence, second to the heart, PTECs are one of the most mitochondria-rich cell types and are thus vulnerable to impairment of mitochondrial bioenergetics.6Malik A.N. Czajka A. Cunningham P. Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences.Mitochondrion. 2016; 29: 59-64Crossref PubMed Scopus (67) Google Scholar Moreover, increased reactive oxygen species (ROS) generated by defective mitochondria may initiate a vicious cycle, aggravating oxidative stress and mitochondrial dysfunction.7Arora M.K. Singh U.K. Oxidative stress: meeting multiple targets in pathogenesis of diabetic nephropathy.Curr Drug Targets. 2014; 15: 531-538Crossref PubMed Scopus (64) Google Scholar, 8Higgins G.C. Coughlan M.T. Mitochondrial dysfunction and mitophagy: the beginning and end to diabetic nephropathy?.Br J Pharmacol. 2014; 171: 1917-1942Crossref PubMed Scopus (179) Google Scholar A major regulator of DN is angiotensin II (Ang-II). Ang-II inhibition is currently the first-in-line therapy for DN. Ang-II exerts its effects by binding to its G-protein–coupled cell membrane receptors angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R). The detrimental role of Ang-II in DN is attributed to AT1R. However, Ang-II itself can counteract these actions by signaling through AT2R, which, together with the angiotensin converting enzyme 2 (ACE2)/angiotensin 1-7 (Ang-1-7)/mas receptor axis, forms the “protective branch” of the renin-angiotensin system (RAS).9Varagic J. Ahmad S. Nagata S. Ferrario C.M. ACE2: angiotensin II/angiotensin-(1-7) balance in cardiac and renal injury.Curr Hypertens Rep. 2014; 16: 420Crossref PubMed Scopus (84) Google Scholar, 10Steckelings U.M. Kloet A. Sumners C. Centrally mediated cardiovascular actions of the angiotensin II type 2 receptor.Trends Endocrinol Metab. 2017; 28: 684-693Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 11Peluso A.A. Santos R.A. Unger T. Steckelings U.M. The angiotensin type 2 receptor and the kidney.Curr Opin Nephrol Hypertens. 2017; 26: 36-42Crossref PubMed Scopus (11) Google Scholar, 12Peters B. Podlich D. Ritter M. et al.A new transgenic rat model overexpressing the angiotensin II type 2 receptor provides evidence for inhibition of cell proliferation in the outer adrenal cortex.Am J Physiol Endocrinol Metab. 2012; 302: E1044-E1054Crossref PubMed Scopus (8) Google Scholar Ang-II was originally discovered as a circulating hemodynamic factor; however, it is now established that independent paracrine- and autocrine-acting RASs exist in several organs including the kidney. Although it is still debated, several lines of evidence suggest a third level—an intracellular functioning RAS13Carey R.M. Functional intracellular renin-angiotensin systems: potential for pathophysiology of disease.Am J Physiol Regul Integr Comp Physiol. 2012; 302: R479-R481Crossref PubMed Scopus (16) Google Scholar with cells along the entire nephron having their own RAS including AT1R and AT2R.14Navar L.G. Kobori H. Prieto M.C. Gonzalez-Villalobos R.A. Intratubular renin-angiotensin system in hypertension.Hypertension. 2011; 57: 355-362Crossref PubMed Scopus (170) Google Scholar, 15Ellis B. Li X.C. Miguel-Qin E. et al.Evidence for a functional intracellular angiotensin system in the proximal tubule of the kidney.Am J Physiol Regul Integr Comp Physiol. 2012; 302: R494-R509Crossref PubMed Scopus (46) Google Scholar This intrarenal, intracellular RAS appears to be important in DN. Intrarenal Ang-II levels are increased long before diabetes becomes apparent in rat models of type 2 diabetes.16Fan Y.Y. Kobori H. Nakano D. et al.Aberrant activation of the intrarenal renin-angiotensin system in the developing kidneys of type 2 diabetic rats.Horm Metab Res. 2013; 45: 338-343Crossref PubMed Scopus (14) Google Scholar In vitro, high glucose load activate the intracellular RAS of renal mesangial cells.17Vidotti 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 The role of AT2R in DN is poorly understood; however, a renoprotective effect has been suggested. In models of diabetes, activation of AT2Rs, either directly by compound 21, a selective agonist of the AT2R,18Koulis C. Chow B.S. McKelvey M. et al.AT2R agonist, compound 21, is reno-protective against type 1 diabetic nephropathy.Hypertension. 2015; 65: 1073-1081Crossref PubMed Scopus (52) Google Scholar,19Castoldi G. di Gioia C.R. Bombardi C. et al.Prevention of diabetic nephropathy by compound 21, selective agonist of angiotensin type 2 receptors, in Zucker diabetic fatty rats.Am J Physiol Renal Physiol. 2014; 307: F1123-F1131Crossref PubMed Scopus (43) Google Scholar or indirectly via an ACE2 activator,20Goru S.K. Kadakol A. Malek V. et al.Diminazene aceturate prevents nephropathy by increasing glomerular ACE2 and AT2 receptor expression in a rat model of type 1 diabetes.Br J Pharmacol. 2017; 174: 3118-3130Crossref PubMed Scopus (47) Google Scholar prevented DN by reducing renal oxidative stress, inflammation, and fibrosis. On the contrary, AT2R knockout accelerated experimentally induced DN in mice, at least in part via oxidative stress.21Chang S.Y. Chen Y.W. Chenier I. et al.Angiotensin II type II receptor deficiency accelerates the development of nephropathy in type I diabetes via oxidative stress and ACE2.Exp Diabetes Res. 2011; 2011: 521076Crossref PubMed Scopus (50) Google Scholar The present study aimed to investigate the role of AT2Rs at the early stages of DN. Here we show for the first time that renal mitochondria contain high-affinity AT1Rs and AT2Rs and that mitochondrial AT1R (mtAT1R) density, but not mitochondrial AT2R (mtAT2R) density, increases in rats with DN. Furthermore, we show that AT2R overexpression in transgenic rats (TGRs) leads to increased mtAT2R density and attenuates diabetes-induced renal changes, including a decrease in the efficiency of mitochondrial bioenergetics, an increase in mitochondrial superoxide production, metabolic reprogramming, and increased PTEC proliferation. In TGRs, AT2R is highly overexpressed in the kidney, as we have previously shown by receptor binding studies.12Peters B. Podlich D. Ritter M. et al.A new transgenic rat model overexpressing the angiotensin II type 2 receptor provides evidence for inhibition of cell proliferation in the outer adrenal cortex.Am J Physiol Endocrinol Metab. 2012; 302: E1044-E1054Crossref PubMed Scopus (8) Google Scholar Here we show expression of transgenic AT2R, specifically in renal PTECs by in situ hybridization (Figure 1). We examined whether AT2Rs translocate to renal mitochondria, because an activated intracellular RAS and deficient mitochondria might be crucial in DN. In both TGRs and wild-type (WT) rats, we evidenced renal mtAT2Rs using receptor binding studies and immunohistochemistry (Figure 2, Figure 3). Highly purified mitochondria from TGR kidneys contained a high density (maximum binding capacity [Bmax] = 1.5 pmol/mg of protein) of high-affinity Ang-II receptors (dissociation constant [Kd] = 395 pM), constituting approximately 63% of the Bmax measured in the cell membrane fraction (Figure 2a). Possible cross-contamination with the cell membrane fraction was excluded by Western blotting and electron microscopy (Figure 2b). Furthermore, we demonstrated the presence of high-affinity endogenous AT1Rs and AT2Rs in mitochondria isolated from neonatal Sprague-Dawley (Figure 2c and d) and from 8-month-old diabetic ZSF1-ob/ob and nondiabetic ZSF1-lean (Figure 2d) rat kidneys by receptor binding studies. The mtAT1R and mtAT2R densities and the mtAT2R/mtAT1R ratio decrease sharply during aging. Moreover, mtAT1R density increased again in diabetic ZSF1-ob/ob rats with severe DN (Figure 2e), leading to a further decrease in the mtAT1R/mtAT2R ratio. We confirmed the presence of transgenic and endogenous mtAT2R in rats and endogenous mtAT2R in humans by immunohistochemistry. Figure 3 shows colocalization of AT2R with the mitochondrial marker voltage-dependent anion-selective channel 1 (VDAC1) in tubular epithelial cells in healthy (Figure 3a) and early diabetic (Figure 3b) nontransgenic rats and TGRs (Figure 3c) as well as in normal kidney tissue from a human patient with a kidney tumor (Figure 3g). In kidneys from a patient with minimal changed disease, the AT2R level is downregulated and colocalization with mitochondria was rarely seen (Figure 3h). The AT2R colocalization with mitochondria was further confirmed by using a human influenza hemagglutinin (HA) antibody in PTECs transfected with an HA-tagged AT2R (Figure 3d). The specificty of the anti-AT2R antibody was verified on the renal tissue of knockout mice (Figure 3e). Next, we found that AT2R is associated with the outer mitochondrial membrane (Figure 3f). Stepwise removal of the outer membrane, indicated by the loss of the marker porin, strongly correlated with the loss of Ang-II receptor binding. The remaining mitoplasts were intact, as proved by OXPHOS (inner mitochondrial membrane marker) and cyclophilin (matrix marker) expression, which did not differ from intact mitochondria.Figure 2Evidence for mitochondrial angiotensin II type 2 receptors (AT2Rs) in the renal cortex. (a) Transgenic rats (TGRs): maximal binding capacity (Bmax) for angiotensin II (Ang-II) receptors from saturation binding studies of the cytosolic, pure mitochondrial, and microsomal/cell membrane fractions. Bmax was estimated by nonlinear least-squares regression analysis of saturation binding of 125I-[Sar1,Ile8]-Ang-II (left). Representative saturation binding curves for Ang-II receptors of the 3 subcellular fractions obtained (right top) and for binding characteristics (right bottom). Nonspecific binding was determined in the presence of 10 μM Ang-II. Each sample was assayed in triplicate. Data are mean ± SD. **P ≤ 0.01 versus microsomal/cell membrane fraction, ##P ≤ 0.05 versus cytosolic fraction, 3 rats per group. (b) Purity of mitochondrial fractions: Western blot of subcellular preparations probed with antibodies specific for organelle/cell compartment–specific marker proteins: cytosol (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]), nucleus (Histone 3), mitochondria (COX IV [ab16056, Abcam]), and microsome/endoplasmic reticulum (ER)/plasma membrane (KDEL [ab1223, Abcam]; GAPDH, ER chaperone and signaling regulator GRP78, and Histone 3 [ab139415, Abcam]) fractions (left); pure mitochondrial fraction (right), bar = 500 nm. Intact mitochondria are proved by the appearance of bands for a subunit of each of the 5 complexes of oxidative phosphorylation (OXPHOS) that is labile when its complex is not assembled (OXPHOS antibody cocktail [ab110413, Abcam]). Electron microscopy of a pure mitochondrial fraction (right). (c) Wild-type rats (neonatal rats): representative saturation binding curves (left) and Bmax and dissociation constant (Kd) plots (right) for endogenous Ang-II receptors of subcellular fractions demonstrate enriched Ang-II receptor binding in the mitochondrial fractions as compared with the nuclei fraction. (d) Competitive binding studies of isolated highly pure renal mitochondria from WT kidneys using CV 11974 as an AT1R antagonist and PD 123319 as an AT2R antagonist revealed the presence of both AT1Rs and AT2Rs, a striking decrease in mitochondrial Ang-II receptor with aging, and a reversal of AT2R dominance in newborns to AT1R dominance in aged rats. AT1R density is increased in diabetic ZSF1-ob/ob rats compared with ZSF1-lean controls. (e) Diabetic ZSF1-ob/ob rats show severe diabetic nephropathy (albuminuria and structural damage). SD, Sprague-Dawley rat. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Colocalization of angiotensin II type 2 receptors (AT2Rs) and mitochondrial marker. Immunohistochemistry on kidney sections show colocalization of AT2Rs with the mitochondrial marker voltage-dependent anion-selective channel 1 (VDAC1) in rats: (a) healthy wild-type (WT) rats, (b) STZ-induced diabetes (28 days) in WT rats, and (c) transgenic rats (TGRs). (d) Immunohistochemistry on human influenza hemagglutinin (HA)–tagged AT2R-transfected rat proximal tubule cells demonstrates colocalization of AT2R with MitoTracker Deep Red. (e) Specificity of the anti-AT2R antibody was demonstrated by intense signals on kidney sections from TGRs and the lack of signals on kidney sections from AT2R knockout mice. (f) Maximum binding capacity for angiotensin II receptors as determined by receptor binding studies of mitoplasts after digitonin digestion (mitoplast fraction a and b with different purity). Full expression of an oxidative phosphorylation marker on mitoplasts indicates an intact electron transfer chain. (g,h) Immunohistochemistry on human kidney sections show colocalization of AT2Rs with the mitochondrial marker VDAC1 in normal tissue from patients with a kidney tumor (g). In renal tissue from patients with minimal change disease, AT2R is downregulated and colocalizes rarely with mitochondria (h). **P < 0.001 vs. WT rats, ++P < 0.001 vs. neighboring age group. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Colocalization of angiotensin II type 2 receptors (AT2Rs) and mitochondrial marker. Immunohistochemistry on kidney sections show colocalization of AT2Rs with the mitochondrial marker voltage-dependent anion-selective channel 1 (VDAC1) in rats: (a) healthy wild-type (WT) rats, (b) STZ-induced diabetes (28 days) in WT rats, and (c) transgenic rats (TGRs). (d) Immunohistochemistry on human influenza hemagglutinin (HA)–tagged AT2R-transfected rat proximal tubule cells demonstrates colocalization of AT2R with MitoTracker Deep Red. (e) Specificity of the anti-AT2R antibody was demonstrated by intense signals on kidney sections from TGRs and the lack of signals on kidney sections from AT2R knockout mice. (f) Maximum binding capacity for angiotensin II receptors as determined by receptor binding studies of mitoplasts after digitonin digestion (mitoplast fraction a and b with different purity). Full expression of an oxidative phosphorylation marker on mitoplasts indicates an intact electron transfer chain. (g,h) Immunohistochemistry on human kidney sections show colocalization of AT2Rs with the mitochondrial marker VDAC1 in normal tissue from patients with a kidney tumor (g). In renal tissue from patients with minimal change disease, AT2R is downregulated and colocalizes rarely with mitochondria (h). **P < 0.001 vs. WT rats, ++P < 0.001 vs. neighboring age group. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Diabetes was induced by streptozotocin (STZ), and a hyperglycemic state was maintained for 28 days to study the effect of AT2R on the early changes in the kidney. Hyperglycemia was established within 1 week of STZ injection in both genotypes (Table 1). TGRs and WT rats did not differ in their physical and clinical plasma/urine parameters during the experimental period and exhibited the typical features of type 1 diabetes, including diuresis and increased water and food consumption. Although structural damage to the kidney was not yet expected, increased lipid and glycogen deposits were observed within tubules (Figure 4a), suggesting a disturbed cellular metabolism. The lipid and glycogen deposits (Armanni-Ebstein lesions) were found in different parts of the tubules: lipids mainly at the basolateral side of PTECs and glycogen in other tubular segments of the cortex. In response to metabolic changes (Table 1), kidneys were hypertrophic. Cell proliferation increased as indicated by Ki-67 staining (Figure 4a and b). Of note, diabetic TGRs had significantly less Ki-67–positive PTECs than did diabetic WT rats, which points to an antiproliferative effect of AT2R. In addition, the outer mitochondrial membrane protein VDAC1 increased in diabetic kidneys as shown by immunohistochemistry and Western blotting (Figure 4a and b, left). VDAC1 is a multifunctional mitochondrial protein regulating cell life and death22Abu-Hamad S. Sivan S. Shoshan-Barmatz V. The expression level of the voltage-dependent anion channel controls life and death of the cell.Proc Natl Acad Sci U S A. 2006; 103: 5787-5792Crossref PubMed Scopus (187) Google Scholar, 23Shoshan-Barmatz V. Krelin Y. Shteinfer-Kuzmine A. VDAC1 functions in Ca2+ homeostasis and cell life and death in health and disease.Cell Calcium. 2018; 69: 81-100Crossref PubMed Scopus (74) Google Scholar as well as the metabolic switch from glucose oxidation to enhanced glycolysis.24Maldonado E.N. VDAC-tubulin, an anti-Warburg pro-oxidant switch.Front Oncol. 2017; 7: 4Crossref PubMed Scopus (50) Google Scholar As this might reflect changes in gene expression pattern, we performed gene expression profiling of the renal cortex.Table 1Physiological and clinical data in plasma and urine of 4-month-old AT2R overexpressing TGRs and WT littermates at baseline and 28 d after induction of diabetes by STZ injection (35 mg/kg, i.v.)ParameterWT controlsTGR controlsWT-STZTGR-STZBody weight (g)470 ± 22504 ± 11330 ± 23aP < 0.01 versus controls.319 ± 22aP < 0.01 versus controls.Kidney/body weight ratio (mg/g)3.0 ± 0.063.1 ± 0.164.9 ± 0.69bP < 0.05 versus controls.5.2 ± 0.10aP < 0.01 versus controls.Water consumption (ml/24 h)23.8 ± 724.3 ± 5215 ± 33aP < 0.01 versus controls.174 ± 23aP < 0.01 versus controls.Urine volume (ml/24 h)14.9 ± 419.9 ± 1201.6 ± 44aP < 0.01 versus controls.169.5 ± 22aP < 0.01 versus controls.Urine urea level (mg/24 h)813 ± 143818 ± 761905 ± 400bP < 0.05 versus controls.1577 ± 185aP < 0.01 versus controls.Urine creatinine level (mg/24 h)15.1 ± 0.717.3 ± 0.39.0 ± 0.4aP < 0.01 versus controls.8.8 ± 2.2aP < 0.01 versus controls.Urine glucose level (mg/24 h)27.9 ± 404.1 ± 2.017,745 ± 4131aP < 0.01 versus controls.14,116 ± 2145aP < 0.01 versus controls.Urine protein/creatinine ratio0.94 ± 0.20.66 ± 0.050.89 ± 0.150.9 ± 0.31Urine albumin/creatinine ratio0.018 ± 0080.018 ± 0.0060.018 ± 0.0030.072 ± 0.09Plasma urea level (mg/dl)33.5 ± 2.039.7 ± 3.1cP < 0.05 versus WT, 3–4 rats per group.53.7 ± 8.1bP < 0.05 versus controls.71.6 ± 5.3aP < 0.01 versus controls.,cP < 0.05 versus WT, 3–4 rats per group.Plasma creatinine level (mg/dl)0.33 ± 0.060.32 ± 0.040.23 ± 0.010.24 ± 0.04bP < 0.05 versus controls.Plasma cholesterol level (mg/dl)76 ± 9.174 ± 3.6104 ± 5.6bP < 0.05 versus controls.101 ± 10.2aP < 0.01 versus controls.Plasma triglyceride level (mg/dl)72.3 ± 24.290 ± 44229.5 ± 37bP < 0.05 versus controls.136 ± 25cP < 0.05 versus WT, 3–4 rats per group.Plasma protein level (mg/dl)55.7 ± 3.855 ± 1.050 ± 2.849 ± 0.9bP < 0.05 versus controls.Plasma glucose level (mg/dl)172 ± 31177 ± 35691 ± 5aP < 0.01 versus controls.799 ± 45aP < 0.01 versus controls.,cP < 0.05 versus WT, 3–4 rats per group.Clearance creatinine level (ml/min per 100 g)0.7 ± 1.40.77 ± 0.120.89 ± 0.110.79 ± 0.09Clearance urea level (ml/min per 100 g)0.37 ± 0.070.28 ± 0.030.75 ± 0.300.48 ± 0.07bP < 0.05 versus controls.AT2R, angiotensin II type 2 receptor; STZ, streptozotocin; TGR, transgenic rat; WT, wild-type.Data are mean ± SD.a P < 0.01 versus controls.b P < 0.05 versus controls.c P < 0.05 versus WT, 3–4 rats per group. Open table in a new tab AT2R, angiotensin II type 2 receptor; STZ, streptozotocin; TGR, transgenic rat; WT, wild-type. Data are mean ± SD. More than 200 mRNAs and 20 pathways were significantly altered in diabetic rats as compared with nondiabetic controls. Diabetic TGRs and WT rats shared changes in 129 genes and 16 pathways. Mainly metabolic pathways were upregulated owing to diabetes, which may reflect the adaptation of the cells to the increased demand for ATP and building blocks required for both increased reabsorption and increased cell growth/proliferation (Figure 5a and b). Changes in 15 pathways and 109 genes were specific to diabetic TGRs, most of which were downregulated. As expected, the RAS pathway was clearly upregulated in TGRs owing to AT2R overexpression. Finally, diabetic TGRs and WT rats differed in 21 pathways, including “glycolysis,” “fatty acid degradation,” and “fatty acid metabolism,” which had reduced expression in TGRs. On the contrary, the “OXPHOS” pathway and pathways that are related to a higher cell differentiation state (cell adhesion molecule CAMS, Ras-proximate-1 Rap1, focal adhesion, and regulation of actin cytoskeleton) had significantly increased expression in TGRs. The OXPHOS pathway was further studied by Western blotting. Figure 6 demonstrates increased expression and assembly of most complexes of the mitochondrial respiratory chain in diabetic TGR kidneys compared with diabetic WT and nondiabetic TGR kidneys, supporting the mRNA expression data.Figure 5Gene expression profiling of the renal cortex. (a) Number of significant differently expressed genes and pathways. (b) Top left: Significantly changed pathways in diabetic wild-type (WT) kidneys compared with nondiabetic WT controls. It is also indicated whether these pathways are significantly different from those in diabetic transgenic rat (TGR) kidneys and different between diabetic TGR and nondiabetic TGR kidneys. Top right: Additional pathways, which are significantly different between diabetic TGR and nondiabetic TGR kidneys. Bottom: All pathways, which are differently expressed between diabetic TGR and diabetic WT kidneys. n = 3–4 rats per group. ABC, ATP-binding cassette; C, control; CoA, coenzyme A; D, diabetic; MAPK, mitogen-activated protein kinase; PPAR, peroxisome proliferator–activated receptor; RAS, renin-angiotensin system; STZ, streptozotocin; tRNA, transfer RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Western blot of mitochondrial respiratory chain complexes. All complexes of the respiratory chain, except complex III, are increased in diabetic transgenic rats (TGRs) relative to nondiabetic controls and diabetic wild-type (WT) rats. The antibodies are against a subunit of each complex (CI:NDUFB8, CII:SDHB, CIII:UQCRC2, CIV:MTC01, and CV:ATPVA) that is labile when its complex is not assembled. Data are mean ± SD. *P ≤ 0.05 versus controls, #P ≤ 0.0 versus WT, 3–4 rats per group. STZ, streptozotocin. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we analyzed the respiratory function of mitochondria isolated from the kidney cortex by determining the oxygen consumption rate and the ATP and superoxide production. To collect the quantity of mitochondria required for functional studies, they were harvested by modified differential centrifugation as previously described25Peters J. Kranzlin B. Schaeffer S. et al.Presence of renin within intramitochondrial dense bodies of the rat adrenal cortex.Am J Physiol. 1996; 271: E439-E450PubMed Google Scholar (Figure 7a). At baseline, AT2R overexpression specifically decreased superoxide production in isolated mitochondria (Figure 7b). Mitochondria from diabetic WT kidneys consumed more oxygen for ATP generation and produced more superoxide than did mitochondria from nondiabetic WT kidneys. The presence of transgenic AT2Rs in diabetic TGR mitochondria prevented the increased superoxide production and limited the oxygen consumption. Moreover, the membrane potential decreased in diabetic WT mitochondria compared with nondiabetic controls, but not in diabetic TGR mitochondria. Altogether, in renal mitochondria isolated from healthy and diabetic kidneys, AT2Rs blunted mitochondrial superoxide production and mitigated the decrease in the efficiency of mitochondrial energy production in renal mitochondria during early diabetes (Figure 7c). The major finding of t" @default.
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• W2891027264 title "The angiotensin II type 2 receptors protect renal tubule mitochondria in early stages of diabetes mellitus" @default.
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