Renal Tubulointerstitial Damage Caused by Persistent Proteinuria Is Attenuated in AT1-Deficient Mice
2001; Elsevier BV; Volume: 159; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63036-2
ISSN1525-2191
AutoresYusuke Suzuki, Óscar López-Franco, Dulcenombre Gómez‐Garré, Nuria Tejera, Carmen Gómez‐Guerrero, Takeshi Sugaya, Rosa Bernal, Julia Blanco, Luís Ortega, J. Egido,
Tópico(s)Receptor Mechanisms and Signaling
ResumoUsing angiotensin II (AngII) type 1A receptor-deficient mice [AT1(−/−)], in which we induced protein overload nephropathy, we explored the potential implication of AngII and endothelin-1 (ET-1) in the tubulointerstitial damage because of persistent proteinuria. At day 7, AT1(−/−) showed marked proteinuria to a similar extent to that of wild-type mice (WT). However, at day14, AT1(−/−) had significantly less proteinuria, renal damage, transforming growth factor-β, and matrix mRNA expression and mortality. AT1(−/−) also showed a significant diminution in the activation of the transcriptional factors nuclear factor-κB and AP-1. Unexpectedly, AT1(−/−) had a higher interstitial infiltration than WT. The administration of the angiotensin-converting enzyme inhibitor quinapril to WT caused a marked improvement in proteinuria and renal lesions, resembling that seen in untreated AT1(−/−). However, the interstitial infiltration persisted in AT1(−/−) when treated with quinapril. Because ET-1 may participate in the recruitment of mononuclear cells, we also studied the implication of this peptide. AT1(−/−) had a significantly higher ET-1 expression in tubular epithelial cells than WT. The administration of the dual ETA/ETB antagonist bosentan to AT1(−/−) considerably reduced the interstitial infiltrates. Bosentan also exerted a beneficial effect on proteinuria, renal lesions, and mortality in WT. These data show that in overload nephropathy, proteinuria and renal lesions are, to a large extent, AngII-dependent. The up-regulation of ET-1 in tubular epithelial cells in AT1(−/−), associated with interstitial infiltrates, suggests that the combination of drugs interfering with both vasopeptides may be of therapeutic interest in renal diseases with severe proteinuria and tubulointerstitial damage. Using angiotensin II (AngII) type 1A receptor-deficient mice [AT1(−/−)], in which we induced protein overload nephropathy, we explored the potential implication of AngII and endothelin-1 (ET-1) in the tubulointerstitial damage because of persistent proteinuria. At day 7, AT1(−/−) showed marked proteinuria to a similar extent to that of wild-type mice (WT). However, at day14, AT1(−/−) had significantly less proteinuria, renal damage, transforming growth factor-β, and matrix mRNA expression and mortality. AT1(−/−) also showed a significant diminution in the activation of the transcriptional factors nuclear factor-κB and AP-1. Unexpectedly, AT1(−/−) had a higher interstitial infiltration than WT. The administration of the angiotensin-converting enzyme inhibitor quinapril to WT caused a marked improvement in proteinuria and renal lesions, resembling that seen in untreated AT1(−/−). However, the interstitial infiltration persisted in AT1(−/−) when treated with quinapril. Because ET-1 may participate in the recruitment of mononuclear cells, we also studied the implication of this peptide. AT1(−/−) had a significantly higher ET-1 expression in tubular epithelial cells than WT. The administration of the dual ETA/ETB antagonist bosentan to AT1(−/−) considerably reduced the interstitial infiltrates. Bosentan also exerted a beneficial effect on proteinuria, renal lesions, and mortality in WT. These data show that in overload nephropathy, proteinuria and renal lesions are, to a large extent, AngII-dependent. The up-regulation of ET-1 in tubular epithelial cells in AT1(−/−), associated with interstitial infiltrates, suggests that the combination of drugs interfering with both vasopeptides may be of therapeutic interest in renal diseases with severe proteinuria and tubulointerstitial damage. Throughout the last few decades, renoprotective effects of renin-angiotensin system (RAS) blockade with angiotensin-converting enzyme (ACE) inhibitor or angiotensin II type 1 receptor (AT1) antagonists have been confirmed by extensive experimental and clinical studies.1Anderson S Rennke HG Brenner BM Therapeutic advantage of converting enzyme inhibitors in arresting progressive renal disease associated with systemic hypertension in the rat.J Clin Invest. 1986; 77: 1993-2000Crossref PubMed Scopus (1048) Google Scholar, 2Remuzzi A Perico N Amuchastegui CS Malanchini B Mazerska M Battaglia C Bertani T Remuzzi G Short- and long-term effect of angiotensin II receptor blockade in rats with experimental diabetes.J Am Soc Nephrol. 1993; 4: 40-49PubMed Google Scholar, 3Remuzzi A Fassi A Sangalli F Malanchini B Mohamed EI Bertani T Remuzzi G Prevention of renal injury in diabetic MWF rats by angiotensin II antagonism.Exp Nephrol. 1998; 6: 28-38Crossref PubMed Scopus (46) Google Scholar, 4Kasiske BL Kalil RS Ma JZ Liao M Keane WF Effect of antihypertensive therapy on the kidney in patients with diabetes: a meta-regression analysis.Ann Intern Med. 1993; 118: 129-138Crossref PubMed Scopus (610) Google Scholar, 5The Gruppo Italiano di Studi Epidemiologici in Nefrologia Group Randomised placebo-controlled trial of effect of ramipril on decline in glomerular filtration rate and risk of terminal renal failure in proteinuric, non-diabetic nephropathy.Lancet. 1997; 349: 1857-1863Abstract Full Text Full Text PDF PubMed Scopus (1770) Google Scholar, 6Salvetti A Mattei P Sudano I Renal protection and antihypertensive drugs: current status.Drugs. 1999; 57: 665-693Crossref PubMed Scopus (49) Google Scholar, 7Burnier M Brunner HR Angiotensin II receptor antagonists.Lancet. 2000; 355: 637-645Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar Early evidence suggested that the beneficial effect was because of the control of glomerular hypertension.1Anderson S Rennke HG Brenner BM Therapeutic advantage of converting enzyme inhibitors in arresting progressive renal disease associated with systemic hypertension in the rat.J Clin Invest. 1986; 77: 1993-2000Crossref PubMed Scopus (1048) Google Scholar, 8Anderson S Diamond JR Karnovsky MJ Brenner BM Mechanisms underlying transition from acute glomerular injury to late glomerular sclerosis in a rat model of nephrotic syndrome.J Clin Invest. 1988; 82: 1757-1768Crossref PubMed Scopus (155) Google Scholar, 9Meyer TW Anderson S Rennke HG Brenner BM Reversing glomerular hypertension stabilizes established glomerular injury.Kidney Int. 1987; 31: 752-759Crossref PubMed Scopus (289) Google Scholar In addition, recent studies have emphasized that locally generated angiotensin II (AngII) modifies resident cell growth and possesses proinflammatory properties, and may therefore participate directly in the pathogenesis of renal injury beyond its hemodynamic effect.10Egido J Vasoactive hormones and renal sclerosis.Kidney Int. 1996; 49: 578-597Crossref PubMed Scopus (209) Google Scholar, 11Suzuki Y Ruiz-Ortega M Egido J Angiotensin II: a double-edged sword in inflammation.J Nephrol. 2000; 13: S101-S110PubMed Google Scholar The prognosis of chronic renal injury, regardless its etiology, is closely correlated to the severity of tubulointerstitial damage.12Eddy AA Molecular insights into renal interstitial fibrosis.J Am Soc Nephrol. 1996; 7: 2495-2508Crossref PubMed Google Scholar, 13Becker GJ Hewitson TD The role of tubulointerstitial injury in chronic renal failure.Curr Opin Nephrol Hypertens. 2000; 9: 133-138Crossref PubMed Scopus (138) Google Scholar Although persistent proteinuria can induce tubulointerstitial fibrosis,14Remuzzi G Ruggenenti P Benigni A Understanding the nature of renal disease progression.Kidney Int. 1997; 51: 2-15Crossref PubMed Scopus (619) Google Scholar, 15Burton C Harris KP The role of proteinuria in the progression of chronic renal failure.Am J Kidney Dis. 1996; 27: 765-775Abstract Full Text PDF PubMed Scopus (310) Google Scholar, 16Eddy AA Interstitial nephritis induced by protein-overload proteinuria.Am J Pathol. 1989; 135: 719-733PubMed Google Scholar underlying mechanisms still remain unclear. Previous studies from our group have shown that RAS was activated in tubular cells in animals with persistent proteinuria.17Largo R Gomez-Garre D Soto K Marron B Blanco J Gazapo RM Plaza JJ Egido J Angiotensin-converting enzyme is upregulated in the proximal tubules of rats with intense proteinuria.Hypertension. 1999; 33: 732-739Crossref PubMed Scopus (93) Google Scholar Furthermore, ACE inhibitors exert beneficial effects on the tubulointerstitial damage in this disease and these effects were attributed, in part, to an attenuation of the nuclear factor (NF)-κB activation.18Gomez-Garre D Largo R Tejera N Fortes J Manzarbeitia F Egido J Activation of NFkB in tubular epithelial cells of rats with intense proteinuria. Role of angiotensin II and endothelin-1.Hypertension. 2001; 37: 1171-1178Crossref PubMed Scopus (146) Google Scholar Protein overload nephropathy is considered an appropriate experimental model to approach the relationship between proteinuria and interstitial damage. The bovine serum albumin (BSA) overload model is frequently used because it is highly reproducible and can induce heterologous as well as autologous proteinuria.16Eddy AA Interstitial nephritis induced by protein-overload proteinuria.Am J Pathol. 1989; 135: 719-733PubMed Google Scholar, 19Weening JJ Van Guldener C Daha MR Klar N van der Wal A Prins FA The pathophysiology of protein-overload proteinuria.Am J Pathol. 1987; 129: 64-73PubMed Google Scholar, 20Zoja C Benigni A Remuzzi G Protein overload activates proximal tubular cells to release vasoactive and inflammatory mediators.Exp Nephrol. 1999; 7: 420-428Crossref PubMed Scopus (61) Google Scholar, 21Largo R Gomez-Garre D Santos S Penaranda C Blanco J Esbrit P Egido J Renal expression of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor in a rat model of tubulointerstitial damage.Kidney Int. 1999; 55: 82-90Crossref PubMed Scopus (47) Google Scholar In the kidney, AngII exerts its biological effects mainly through AT1.22Zhuo J Song K Harris PJ Mendelsohn FA In vitro autoradiography reveals predominantly AT1 angiotensin II receptors in rat kidney.Renal Physiol Biochem. 1992; 15: 231-239PubMed Google Scholar In rodents, AT1 exists in two isoforms, AT1A and AT1B, encoded by two different genes. The murine AT1A is the predominantly expressed isoform in most tissues.23Llorens-Cortes C Greenberg B Huang H Corvol P Tissular expression and regulation of type 1 angiotensin II receptor subtypes by quantitative reverse transcriptase-polymerase chain reaction analysis.Hypertension. 1994; 24: 538-548Crossref PubMed Scopus (178) Google Scholar Therefore, the AT1AR-deficient mouse strain [AT1(−/−)] is a powerful model to analyze the effect of AngII blockade.24Sugaya T Nishimatsu S Tanimoto K Takimoto E Yamagishi T Imamura K Goto S Imaizumi K Hisada Y Otsuka A Uchida H Sugiura M Fukuta K Fukamizu A Murakami K Angiotensin II type 1a receptor-deficient mice with hypotension and hyperreninemia.J Biol Chem. 1995; 270: 18719-18722Crossref PubMed Scopus (356) Google Scholar In the present study, we further investigated the contribution of RAS in the pathogenesis of tubulointerstitial damage by persistent proteinuria, using the BSA-overload model in AT1(−/−). Because altered bradykinin synthesis and its metabolism,25Campbell DJ Kladis A Duncan AM Effects of converting enzyme inhibitors on angiotensin and bradykinin peptides.Hypertension. 1994; 23: 439-449Crossref PubMed Scopus (315) Google Scholar, 26Gansevoort RT de Zeeuw D de Jong PE Is the antiproteinuric effect of ACE inhibition mediated by interference in the renin-angiotensin system?.Kidney Int. 1994; 45: 861-867Crossref PubMed Scopus (315) Google Scholar as well as AT2,27Matsubara H Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal diseases.Circ Res. 1998; 83: 1182-1191Crossref PubMed Scopus (437) Google Scholar, 28Ruiz-Ortega M Lorenzo O Ruperez M Blanco J Egido J Angiotensin II activates nuclear factor-kB via AT1 and AT2 receptors in the kidney.Am J Pathol. 2001; 158: 1743-1756Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar may also be involved in the mechanisms of proteinuria and interstitial cell infiltration, a group of animals was treated with ACE inhibitors. Accumulated evidence has shown that ET-1 also participates in the progression of renal diseases, with or without proteinuria.18Gomez-Garre D Largo R Tejera N Fortes J Manzarbeitia F Egido J Activation of NFkB in tubular epithelial cells of rats with intense proteinuria. Role of angiotensin II and endothelin-1.Hypertension. 2001; 37: 1171-1178Crossref PubMed Scopus (146) Google Scholar, 20Zoja C Benigni A Remuzzi G Protein overload activates proximal tubular cells to release vasoactive and inflammatory mediators.Exp Nephrol. 1999; 7: 420-428Crossref PubMed Scopus (61) Google Scholar, 29Gomez-Garre D Largo R Liu XH Gutierrez S Lopez-Armada MJ Palacios I Egido J An orally active ETA/ETB receptor antagonist ameliorates proteinuria and glomerular lesions in rats with proliferative nephritis.Kidney Int. 1996; 50: 962-972Crossref PubMed Scopus (70) Google Scholar, 30Ruiz-Ortega M Gomez-Garre D Liu XH Blanco J Largo R Egido J Quinapril decreases renal endothelin-1 expression and synthesis in a normotensive model of immune-complex nephritis.J Am Soc Nephrol. 1997; 8: 756-768PubMed Google Scholar Moreover, a recent study in rats harboring human renin and angiotensinogen genes suggests that ET-1 might mediate inflammatory processes in AngII-induced tissue damage.31Muller DN Mervaala EM Schmidt F Park JK Dechend R Genersch E Breu V Loffler BM Ganten D Schneider W Haller H Luft FC Effect of bosentan on NF-kappaB, inflammation, and tissue factor in angiotensin II-induced end-organ damage.Hypertension. 2000; 36: 282-290Crossref PubMed Scopus (136) Google Scholar In this regard, we also examined the potential role of ET-1 in proteinuric AT1(−/−) and the potential interrelationship between AngII and ET-1 in the tubulointerstitial damage. The data presented here further extend the implication of both peptides in the pathogenesis of interstitial injury because of proteinuria and support the idea that the combination of drugs modulating both peptides may be useful in nephropathies with persistent proteinuria. We used AT1(−/−) that were generated with a germline chimera derived from TT2 embryonic stem cells with a targeted mutation of the AT1 A gene as previously described.24Sugaya T Nishimatsu S Tanimoto K Takimoto E Yamagishi T Imamura K Goto S Imaizumi K Hisada Y Otsuka A Uchida H Sugiura M Fukuta K Fukamizu A Murakami K Angiotensin II type 1a receptor-deficient mice with hypotension and hyperreninemia.J Biol Chem. 1995; 270: 18719-18722Crossref PubMed Scopus (356) Google Scholar AT1(−/−) were back-crossed for more than six generations with C57BL/6 mice.32Hisada Y Sugaya T Yamanouchi M Uchida H Fujimura H Sakurai H Fukamizu A Murakami K Angiotensin II plays a pathogenic role in immune-mediated renal injury in mice.J Clin Invest. 1999; 103: 627-635Crossref PubMed Scopus (134) Google Scholar As their wild-type littermates (WT), C57BL/6 mice were purchased from Harlan Interfauna Ibérica, S.A. (Barcelona, Spain). Only female mice ages 8 to 10 weeks and weighing 18 to 23 g were used. To decide the experimental condition for murine protein-overload nephropathy, WT weighing 20 g were intraperitoneally injected with four different daily doses (0.1, 0.2, 0.4, 1.0 g) of low endotoxin BSA (Sigma, St. Louis, MO) and followed until day 7. According to their effects on the outcome (see Results), protocols with 0.2 and 0.4 g of BSA daily were used as moderate and severe overload nephropathy models, respectively. Groups of AT1(−/−) and WT, injected with saline, were used as controls in all models. Groups of mice were sacrificed on days 7, 14, and 28 (moderate) and days 7 and 14 (severe overload nephropathy). Four to 16 mice (BSA-injected WT, AT1(−/−), and their controls) were included at each time point. Urinary protein was determined every day by Knight's method, as previously described.33Suzuki Y Shirato I Okumura K Ravetch JV Takai T Tomino Y Ra C Distinct contribution of Fc receptors and angiotensin II-dependent pathways in anti-GBM glomerulonephritis.Kidney Int. 1998; 54: 1166-1174Crossref PubMed Scopus (146) Google Scholar Kidneys of all animals were perfused with cold saline and removed under general anesthesia. To assess the role of AngII or ET-1 in this disease, we treated animals with moderate and severe nephropathy with the ACE inhibitor quinapril (as powdered hydrochloride salt; Parke-Davis, Barcelona, Spain) or with the dual endothelin type A and B receptor (ETA/B) antagonist bosentan (Ro 47-0203; 4-tert-butyl-N- [6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2′-bipyrimidin-4-yl]-benzene-sulfonamide; Hoffmann-La Roche Ltd., Basel, Switzerland) or their combination. Quinapril (16 mg/kg in distilled water) and bosentan (100 mg/kg in a dissolution of 5% arabic gum)18Gomez-Garre D Largo R Tejera N Fortes J Manzarbeitia F Egido J Activation of NFkB in tubular epithelial cells of rats with intense proteinuria. Role of angiotensin II and endothelin-1.Hypertension. 2001; 37: 1171-1178Crossref PubMed Scopus (146) Google Scholar were given daily by gastric gavage with a cannula from 24 hours before first BSA injection until day 13. Paraffin-embedded sections (4-μm thick) were prepared and stained with hematoxylin and eosin and Masson's trichrome for light microscopy. Semiquantification of morphological lesions were evaluated in regard to glomerular lesions (number of glomeruli with hypercellularity or matrix expansion/30 glomeruli scored 0 to 3), tubular lesions (number of tubuli with dilatation or atrophy/×100 high-power fields scored 0 to 4), protein casts (number of casts/×100 high-power fields scored 0 to 4), and interstitial infiltration (number of focus/kidney scored 0 to 4) by three different pathologists in a blind manner. For the immunohistochemistry of ET-1,29Gomez-Garre D Largo R Liu XH Gutierrez S Lopez-Armada MJ Palacios I Egido J An orally active ETA/ETB receptor antagonist ameliorates proteinuria and glomerular lesions in rats with proliferative nephritis.Kidney Int. 1996; 50: 962-972Crossref PubMed Scopus (70) Google Scholar kidneys were fixed in 4% paraformaldehyde and embedded in paraffin, and 4-μm thick sections were mounted on poly-l-lysine-coated slides. After deparaffinization with graded concentrations of xylene and ethanol, they were quenched in methanol containing 3% H2O2 at 25°C for 30 minutes to block the activity of endogenous peroxidase. These slides were washed and incubated in trypsin (0.1% trypsin CaCl2 wt/v) to expose antigenic sites. They were subsequently incubated with 5% normal swine serum for 30 minutes at room temperature to reduce nonspecific background staining and then incubated overnight at 4°C with rabbit polyclonal anti-ET-1 antibody (Peninsula Laboratories Europe Ltd., Merseyside, UK). Control slides were treated with diluted normal rabbit serum. After being washed, the sections were incubated with biotinylated swine anti-rabbit IgG (DAKO A/S, Glostrup, Denmark), washed again, and incubated with avidin-biotin-peroxidase complex (DAKO A/S) for 30 minutes. The sites of peroxidase activity were visualized with 0.05% 3,3′-diaminobenzidine (DAKO A/S) in 0.01% H2O2 for 10 minutes. Sections were counterstained with Mayer's hematoxylin (Sigma, St. Louis, MO) for 2 minutes and coverslipped. The intensity of ET-1 staining was evaluated by a semiautomatic image analysis system with Microimage version 3.0 for Windows (Olympus, Japan). We quantitatively analyzed ET-1 expression in WT and AT1(−/−) by measuring the integrated optical density (OD) of tubular cells and connective perivascular tissue. OD is a logarithmic function of the light transmitted through the stained section. This formula assumes an exponential decay of light inside the transmitting materials, as follows: OD(x, y) = −log [(intensity(x, y) − black)/(incident − black)]. Intensity(x, y) is the intensity at pixel(x, y), black is the intensity generated when no light goes through the material, and incident is the intensity of the incident light. The system was calibrated in a way that the OD of the negative control was 0. Pieces of renal cortex were homogenized, and total RNA was obtained by the acid guanidinium-phenol-chloroform method.21Largo R Gomez-Garre D Santos S Penaranda C Blanco J Esbrit P Egido J Renal expression of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor in a rat model of tubulointerstitial damage.Kidney Int. 1999; 55: 82-90Crossref PubMed Scopus (47) Google Scholar One μg of RNA was reverse-transcribed and then amplified with a commercial kit (Promega, Buckinghamshire, UK), with the use of 0.5 μCi [α32P]dCTP (3000 Ci/mmol, Amersham) and 20 pmol of specific primers for rat transforming growth factor (TGF)-β1 (sense: 5′-AATACGTCAGACATTCGGGAAGCA-3′; antisense: 5′-GTCAATGTACAGCTGCCGTACACA-3′; fragment: 498 bp), rat/mouse fibronectin (FN) (sense: 5′-TGCCACTGTTCTCCTACGTG-3′; antisense: 5′-ATGCTTTGACCCT-TACACCG-3′; fragment: 312 bp), mouse tumor necrosis factor (TNF)-α (sense: 5′-GCGACGTGGAA-CTGGCAGAAG-3′; antisense: 5′-GGTACAACCCAT-CGGCTGGCA-3′; fragment: 384 bp), and mouse prepro ET-1 (sense: 5′-TGATCTTCTCTCTGCTGTT-3′; antisense: 5′-TTTAAGCTTTTCTGCATGGT-3′; fragment: 408 bp). The amplifications were performed with annealing temperatures of 62°C (TGF-β), 60°C (FN), 63°C (TNF-α), or 61°C (prepro ET-1). The optimum number of amplification cycles used for semi quantitative RT-PCR (25, 25, 31, and 32, respectively) was chosen on the basis of pilot experiments (data not shown). The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. Aliquots of each reaction were run on 4% acrylamide-bisacrylamide gels. The gels were dried and exposed to X-OMAT AS films (Eastman Kodak Company, Rochester, NY). Autoradiograms were quantified by scanning densitometry (Molecular Dynamics). The density of each gene was compared after the individual correction by density of GAPDH. Nuclear extracts were obtained as previously described18Gomez-Garre D Largo R Tejera N Fortes J Manzarbeitia F Egido J Activation of NFkB in tubular epithelial cells of rats with intense proteinuria. Role of angiotensin II and endothelin-1.Hypertension. 2001; 37: 1171-1178Crossref PubMed Scopus (146) Google Scholar, 28Ruiz-Ortega M Lorenzo O Ruperez M Blanco J Egido J Angiotensin II activates nuclear factor-kB via AT1 and AT2 receptors in the kidney.Am J Pathol. 2001; 158: 1743-1756Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar and the activity of transcription factors was evaluated by electrophoretic mobility shift assay. Briefly, frozen kidney pieces were pulverized in a metallic chamber and resuspended in a cold extraction buffer [20 mmol/L Hepes-NaOH, pH 7.6, 20% (v/v) glycerol, 0.35 mol/L NaCl, 5 mmol/L MgCl2, 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 1 μg/ml pepstatin A]. The homogenate was vigorously shaken and the insoluble materials precipitated by centrifugation at 40,000 × g for 30 minutes at 4°C. Supernatants were dialyzed overnight against a binding buffer containing 20 mmol/L Hepes-NaOH (pH 7.6), 20% (v/v) glycerol, 0.1 mmol/L NaCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 0.5 mmol/L phenylmethyl sulfonyl fluoride. These dialysates were cleared by centrifugation at 10,000 × g for 15 minutes at 4°C and stored in aliquots at −80°C until use. Protein concentration was quantified by the bicinchoninic acid method (Bio-Rad Laboratories, Richmond, CA). NF-κB and AP-1 consensus oligonucleotides (5′-AGTTGAGGGGACTTT-CCCAGGC-3′ and 5′-CGCTTGATGAGTCAGCCGGAA-3′, respectively) were [32P]-end-labeled by incubation for 10 minutes at 37°C with 10 U T4 polynucleotide kinase (Promega) in a reaction containing 10 μCi of [γ-32P]ATP (3000 Ci/mmol; Amersham, Arlington Heights, IL), 70 mmol/L Tris-HCl, 10 mmol/L MgCl2, and 5 mmol/L dithiothreitol. The reaction was stopped by the addition of EDTA to a final concentration of 0.05 mol/L. Nuclear proteins (20 μg NF-κB and 30 μg AP-1) were equilibrated for 10 minutes in a binding buffer containing 4% glycerol, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 50 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 7.5, and 50 μg/ml poly (dI-dC). When competition assays were performed, the cold probe was added to this buffer 10 minutes before the addition of the labeled probe. Labeled probe (0.035 pmol) was added to the reaction and incubated for 20 minutes at room temperature. The reaction was stopped by the addition of gel-loading buffer (250 mmol/L Tris-HCl, 0.2% bromophenol blue, 0.2% xylene cyanol, and 40% glycerol) and run on a nondenaturing, 4% acrylamide gel at 100 V at room temperature in 89 mmol/L Tris-borate, 2 mmol/L EDTA, pH 8.0 (TBE).18Gomez-Garre D Largo R Tejera N Fortes J Manzarbeitia F Egido J Activation of NFkB in tubular epithelial cells of rats with intense proteinuria. Role of angiotensin II and endothelin-1.Hypertension. 2001; 37: 1171-1178Crossref PubMed Scopus (146) Google Scholar, 21Largo R Gomez-Garre D Santos S Penaranda C Blanco J Esbrit P Egido J Renal expression of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor in a rat model of tubulointerstitial damage.Kidney Int. 1999; 55: 82-90Crossref PubMed Scopus (47) Google Scholar The gel was dried and exposed to X-OMAT AS films (Eastman Kodak Company). Autoradiograms were quantified by scanning densitometry (Molecular Dynamics). Results are expressed as mean ± SD. Comparisons between groups were made using unpaired Student's t-test. For statistical analysis of mortality (survival rate), chi-square test using EPI INFO Ver. 6.04 (Centers for Disease Control, Atlanta, GA) was used. Differences were considered as significant if the P value was <0.05. The protein overload nephropathy model is well established in rats, but not in mice. Initially, we tested the appropriate amounts of BSA for the murine protein overload model. We used four different daily intraperitoneal doses of BSA and followed urinary protein level and mortality until day 7 (Figure 1). Mice weighing 20 g injected with 1 g of BSA daily showed severe proteinuria, and all animals died before day 7. In contrast, 0.1 g of BSA daily was not enough to induce pathological proteinuria. Therefore, we finally used 0.2 and 0.4 g of BSA daily to elicit the moderate and severe overload nephropathy models, respectively. In AT1(−/−) and their WT littermates injected with 0.2 g of BSA, we followed the kinetics of proteinuria for 4 weeks (Figure 2a). Peak proteinuria in AT1(−/−) appeared earlier than in WT, although there was no significant difference between them. In AT1(−/−) proteinuria decreased after day 7, whereas in WT it was still elevated until day 14. Both groups did however reveal moderate proteinuria after 3 weeks. Both groups of animals injected with 0.4 g of BSA daily showed threefold to fourfold higher proteinuria peaks than those of moderate models, but their kinetics were basically similar (Figure 2b). AT1(−/−) showed high proteinuria from day 1, returning to the normal limits at approximately day 7. By contrast, severe proteinuria was persistent in WT for several days with an elevated mortality at approximately day 14 (69% animals). AT1(−/−) had a significantly less mortality (22% animals, P < 0.05) at that time. In both groups of animals with moderate and severe overload nephropathy, we evaluated the renal damage at days 7 and 14. Only data on the severe model are depicted in Figure 3. Morphological lesions were significantly less marked in AT1(−/−) than in WT only at day 14 (Figure 3a). Differences in the moderate nephropathy were less clear between AT1(−/−) and WT (not shown). Semiquantitative RT-PCR also showed that TGF-β and FN mRNA expression was significantly attenuated in AT1(−/−) in relation to WT at day 14 (Figure 3, b and c). At day 7, FN expression in AT1(−/−) was significantly attenuated in both models (moderate model: 1.1 ± 0.2-fold versus 3.4 ± 1.2-fold increase,n = 3, P < 0.05; severe model; Figure 3c). On the other hand, we found a significant diminution of TGF-β expression in AT1(−/−) at day 7 in moderate overload nephropathy (1.4 ± 0.1-fold versus 1.8 ± 0.3-fold increase,n = 4, P < 0.05), but not in severe nephropathy (Figure 3b). As it is well known, AngII is a strong inducer of TGF-β. To search for another potential candidate that could explain the high expression of TGF-β at day 7 in AT1(−/−), we examined the gene expression of TNF-α one of the most important inflammatory cytokines able to elicit TGF-β expression.34Phillips AO Topley N Steadman R Morrisey K Williams JD Induction of TGF-beta 1 synthesis in D-glucose primed human proximal tubular cells by IL-1 beta and TNF alpha.Kidney Int. 1996; 50: 1546-1554Crossref PubMed Scopus (83) Google Scholar, 35Pawluczyk IZ Harris KP Cytokine interactions promote synergistic fibronectin accumulation by mesangial cells.Kidney Int. 1998; 54: 62-70Crossref PubMed Scopus (40) Google Scholar We noted that TNF-α expression in AT1(−/−) with severe nephropathy at day 7 increased as in WT in relation to controls (3.5 ± 0.5-fold versus 3.8 ± 1.4-fold increase, n = 4 to 5, P = 0.76). Several groups have previously demonstrated that overload proteinuria induces NF-κB activation in tubular epithelial cells bothin vivo and in vitro.18Gomez-Garre D Largo R Tejera N Fortes J Manzarbeitia F Egido J Activation of NFkB in tubular epithelial cells of rats with intense proteinuria. Role of angiotensin II and endothelin-1.Hypertension. 2001; 37: 1171-1178Crossref PubMed Scopus (146) Google Scholar, 31Muller DN Mervaala EM Schmidt F Park JK Dechend R Genersch E Breu V Loffler BM Ganten D Schneider W Haller H Luft FC Effect of bosentan on NF-kappaB, inflammation, and tissue factor in angiotensin II-induced end-organ damage.Hypertension. 2000; 36: 282-290Crossref PubMed Scopus (136) Google Schola
Referência(s)