Matrix metalloproteinase-9 regulates afferent arteriolar remodeling and function in hypertension-induced kidney disease

This study tested if matrix metalloproteinase (MMP)-9 promoted microvascular pathology that initiates hypertensive (HT) kidney disease in salt-sensitive (SS) Dahl rats. SS rats lacking Mmp9 (Mmp9-/-) and littermate control SS rats were studied after one week on a normotensive 0.3% sodium chloride (Pre-HT SS and Pre-HT Mmp9-/-) or a hypertension-inducing diet containing 4.0% sodium chloride (HT SS and HT Mmp9-/-). Telemetry-monitored blood pressure of both the HT SS and HT Mmp9-/- rats increased and did not differ. Kidney microvessel transforming growth factor-beta 1 (Tgfb1) mRNA did not differ between Pre-HT SS and Pre-HT Mmp9-/- rats, but with hypertension and expression of Mmp9 and Tgfb1 increased in HT SS rats, along with phospho-Smad2 labeling of nuclei of vascular smooth muscle cells, and with peri-arteriolar fibronectin deposition. Loss of MMP-9 prevented hypertension-induced phenotypic transformation of microvascular smooth muscle cells and the expected increased microvascular expression of pro-inflammatory molecules. Loss of MMP-9 in vascular smooth muscle cells in vitro prevented cyclic strain–induced production of active TGF-β1 and phospho-Smad2/3 stimulation. Afferent arteriolar autoregulation was impaired in HT SS rats but not in HT Mmp9-/- rats or the HT SS rats treated with doxycycline, an MMP inhibitor. HT SS but not HT Mmp9-/- rats showed decreased glomerular Wilms Tumor 1 protein–positive cells (a marker of podocytes) along with increased urinary podocin and nephrin mRNA excretion, all indicative of glomerular damage. Thus, our findings support an active role for MMP-9 in a hypertension-induced kidney microvascular remodeling process that promotes glomerular epithelial cell injury in SS rats. This study tested if matrix metalloproteinase (MMP)-9 promoted microvascular pathology that initiates hypertensive (HT) kidney disease in salt-sensitive (SS) Dahl rats. SS rats lacking Mmp9 (Mmp9-/-) and littermate control SS rats were studied after one week on a normotensive 0.3% sodium chloride (Pre-HT SS and Pre-HT Mmp9-/-) or a hypertension-inducing diet containing 4.0% sodium chloride (HT SS and HT Mmp9-/-). Telemetry-monitored blood pressure of both the HT SS and HT Mmp9-/- rats increased and did not differ. Kidney microvessel transforming growth factor-beta 1 (Tgfb1) mRNA did not differ between Pre-HT SS and Pre-HT Mmp9-/- rats, but with hypertension and expression of Mmp9 and Tgfb1 increased in HT SS rats, along with phospho-Smad2 labeling of nuclei of vascular smooth muscle cells, and with peri-arteriolar fibronectin deposition. Loss of MMP-9 prevented hypertension-induced phenotypic transformation of microvascular smooth muscle cells and the expected increased microvascular expression of pro-inflammatory molecules. Loss of MMP-9 in vascular smooth muscle cells in vitro prevented cyclic strain–induced production of active TGF-β1 and phospho-Smad2/3 stimulation. Afferent arteriolar autoregulation was impaired in HT SS rats but not in HT Mmp9-/- rats or the HT SS rats treated with doxycycline, an MMP inhibitor. HT SS but not HT Mmp9-/- rats showed decreased glomerular Wilms Tumor 1 protein–positive cells (a marker of podocytes) along with increased urinary podocin and nephrin mRNA excretion, all indicative of glomerular damage. Thus, our findings support an active role for MMP-9 in a hypertension-induced kidney microvascular remodeling process that promotes glomerular epithelial cell injury in SS rats. Translational StatementHypertensive nephropathy involves kidney dysfunction and microvascular pathology. Using salt-sensitive (SS) rats, the present studies discovered a matrix metalloproteinase-9 (MMP-9)–dependent, hypertension-induced osteogenic phenotypic switch by kidney microvascular smooth muscle cells, with concomitant dysregulation of microvascular function. Kidney dysfunction and podocyte injury followed these changes in SS but not in SS rats that lack Mmp9. The observations provide new opportunities for exploration of early diagnosis of impaired kidney autoregulation in hypertension and for development of novel therapeutic strategies that protect against this common mediator of chronic kidney disease. Hypertensive nephropathy involves kidney dysfunction and microvascular pathology. Using salt-sensitive (SS) rats, the present studies discovered a matrix metalloproteinase-9 (MMP-9)–dependent, hypertension-induced osteogenic phenotypic switch by kidney microvascular smooth muscle cells, with concomitant dysregulation of microvascular function. Kidney dysfunction and podocyte injury followed these changes in SS but not in SS rats that lack Mmp9. The observations provide new opportunities for exploration of early diagnosis of impaired kidney autoregulation in hypertension and for development of novel therapeutic strategies that protect against this common mediator of chronic kidney disease. Hypertension-induced nephropathy is a common cause of chronic kidney disease and is the second leading cause of end-stage kidney disease, producing over a quarter of all cases of end-stage kidney disease in the United States.1Centers for Disease Control and Prevention. Health, United States: 2011: Table 51. End-stage renal disease patients, by selected characteristics: United States, selected years 1980–2010.cdc.gov/nchs/data/hus/2011/051.pdfDate accessed: September 25, 2019Google Scholar, 2Ozieh M.N. Gebregziabher M. 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Remodeling of afferent arterioles is a hallmark of human hypertensive nephropathy; accumulation of extracellular matrix and podocyte loss with albuminuria and ultimately glomerulosclerosis and interstitial fibrosis generally accompany the vascular changes.4Seccia T.M. Caroccia B. Calo L.A. Hypertensive nephropathy: moving from classic to emerging pathogenetic mechanisms.J Hypertens. 2017; 35: 205-212Crossref PubMed Scopus (77) Google Scholar,5Fogo A. Breyer J.A. Smith M.C. et al.AASK Pilot Study Investigators. Accuracy of the diagnosis of hypertensive nephrosclerosis in African Americans: a report from the African American Study of Kidney Disease (AASK) trial.Kidney Int. 1997; 51: 244-252Abstract Full Text PDF PubMed Scopus (215) Google Scholar An inherent predisposition to hypertension-induced kidney disease is observed not only in humans but also in rodent models.6Kottgen A. Pattaro C. 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Sanders P.W. l-Arginine abrogates salt-sensitive hypertension in Dahl/Rapp rats.J Clin Invest. 1991; 88: 1559-1567Crossref PubMed Scopus (426) Google Scholar, 15Chen P.Y. St. John P.L. Kirk K.A. et al.Hypertensive nephrosclerosis in the Dahl/Rapp rat: initial sites of injury and effect of dietary l-arginine administration.Lab Invest. 1993; 68: 174-184PubMed Google Scholar, 16Wang P.X. Sanders P.W. Mechanism of hypertensive nephropathy in the Dahl/Rapp rat: a primary disorder of vascular smooth muscle.Am J Physiol Renal Physiol. 2005; 288: F236-F242Crossref PubMed Scopus (18) Google Scholar SS rats uniformly develop hypertension followed by proteinuria and progressive loss of kidney function.15Chen P.Y. St. John P.L. Kirk K.A. et al.Hypertensive nephrosclerosis in the Dahl/Rapp rat: initial sites of injury and effect of dietary l-arginine administration.Lab Invest. 1993; 68: 174-184PubMed Google Scholar,17Chen P.Y. Sanders P.W. Role of nitric oxide synthesis in salt-sensitive hypertension in Dahl/Rapp rats.Hypertension. 1993; 22: 812-818Crossref PubMed Scopus (193) Google Scholar Glomerular injury results from a vascular disease process that resembles the human condition of hypertension-induced microvascular remodeling in the kidney.15Chen P.Y. St. John P.L. Kirk K.A. et al.Hypertensive nephrosclerosis in the Dahl/Rapp rat: initial sites of injury and effect of dietary l-arginine administration.Lab Invest. 1993; 68: 174-184PubMed Google Scholar,17Chen P.Y. Sanders P.W. Role of nitric oxide synthesis in salt-sensitive hypertension in Dahl/Rapp rats.Hypertension. 1993; 22: 812-818Crossref PubMed Scopus (193) Google Scholar Studies of SS rats support the concept that the remodeling process was related to an intrinsic disorder of vascular smooth muscle cells (VSMCs).16Wang P.X. Sanders P.W. 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Matrix metalloproteinase-9 (MMP-9) participates in the pathophysiology of chronic kidney disease,20Cabral-Pacheco G.A. Garza-Veloz I. Castruita-De la Rosa C. et al.The roles of matrix metalloproteinases and their inhibitors in human diseases.Int J Mol Sci. 2020; 21: 9739Crossref PubMed Scopus (438) Google Scholar but mechanisms of injury are incompletely understood. MMP-9 activity, but not the activity of the other gelatinase MMP-2, associates with albuminuria in patients and in the Munich Wistar Frömter rat, a model of progressive proteinuria.21Pulido-Olmo H. Garcia-Prieto C.F. Alvarez-Llamas G. et al.Role of matrix metalloproteinase-9 in chronic kidney disease: a new biomarker of resistant albuminuria.Clin Sci (Lond). 2016; 130: 525-538Crossref PubMed Scopus (40) Google Scholar Williams et al.22Williams J.M. Zhang J. North P. et al.Evaluation of metalloprotease inhibitors on hypertension and diabetic nephropathy.Am J Physiol Renal Physiol. 2011; 300: F983-F998Crossref PubMed Scopus (38) Google Scholar used 2 different nonselective metalloprotease inhibitors to show an improvement in glomerulosclerosis and interstitial fibrosis without reducing blood pressure in SS rats. Avian erythroblastosis virus E26 oncogene homolog-1 is a transcription factor that is involved in the regulation of the renovascular response to hypertension in SS rats.18Feng W. Guan Z. Xing D. et al.Avian erythroblastosis virus E26 oncogene homolog-1 (ETS-1) plays a role in renal microvascular pathophysiology in the Dahl salt-sensitive rat.Kidney Int. 2020; 97: 528-537Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar,23Feng W. Chumley P. Prieto M.C. et al.Transcription factor avian erythroblastosis virus E26 oncogene homolog-1 is a novel mediator of renal injury in salt-sensitive hypertension.Hypertension. 2015; 65: 813-820Crossref PubMed Scopus (13) Google Scholar,24Feng W. Chen B. Xing D. et al.Haploinsufficiency of the transcription factor Ets-1 is renoprotective in Dahl salt-sensitive rats.J Am Soc Nephrol. 2017; 28: 3239-3250Crossref PubMed Scopus (10) Google Scholar Importantly, MMP-9 is a downstream target of E26 oncogene homolog-1.25Oda N. Abe M. Sato Y. ETS-1 converts endothelial cells to the angiogenic phenotype by inducing the expression of matrix metalloproteinases and integrin β3.J Cell Physiol. 1999; 178: 121-132Crossref PubMed Scopus (199) Google Scholar A unique model of SS rats that lack Mmp9 (termed SSMmp9−/−) was therefore generated to test the hypothesis that MMP-9 promoted critical microvascular remodeling and hypertension-induced kidney injury. The findings of the present studies supported an important role of MMP-9 in kidney microvascular pathobiology and end-organ kidney damage in hypertension. Studies were conducted using Dahl salt-sensitive rats (SS/JrHsd/Mcw, abbreviated as SS, rats)10Dahl L.K. Heine M. Tassinari L. Effects of chronic excess salt ingestion: evidence that genetic factors play an important role in susceptibility to experimental hypertension.J Exp Med. 1962; 115: 1173-1190Crossref PubMed Scopus (522) Google Scholar,26Zicha J. Dobesova Z. Vokurkova M. et al.Age-dependent salt hypertension in Dahl rats: fifty years of research.Physiol Res. 2012; 61: S35-S87Crossref PubMed Google Scholar, 27Elijovich F. Weinberger M.H. Anderson C.A. et al.Salt sensitivity of blood pressure: a scientific statement from the American Heart Association.Hypertension. 2016; 68: e7-e46Crossref PubMed Scopus (300) Google Scholar, 28Cowley Jr., A.W. Stoll M. Greene A.S. et al.Genetically defined risk of salt sensitivity in an intercross of Brown Norway and Dahl S rats.Physiol Genomics. 2000; 2: 107-115Crossref PubMed Scopus (68) Google Scholar and Dahl SS/JrHsd/Mcw rats that have a deletion mutation in Mmp9 (SSMmp9−/− rats). The original SS and SSMmp9−/− strains of rats were generous gifts from Dr. Aron Geurts, Medical College of Wisconsin. SSMmp9−/− rats were genetically identical to littermate SS rats, except for homozygous mutation of Mmp9, introduced using genome engineering with the CRISPR/Cas9 system, which targeted exon 4 of the rat Mmp9 gene and resulting in a frameshift mutation, confirmed by genotyping with TaqMan probe real-time polymerase chain reaction (PCR; Transnetyx; Figure 1). Littermate SS rats served as controls in these experiments. Rat breeders and weanlings were fed a purified rodent diet containing 0.3% NaCl (Dyet#100077, AIN-76A Purified Rodent Diet with 0.3% NaCl, Dyets Inc.) and tap water ad libitum until study. In some experiments, doxycycline (Dox, D9891, MilliporeSigma), 30 mg/kg/d, in drinking water was given to SS rats 3 days before the termination of the study. For genotyping, TaqMan probes that recognized the 8-bp deletion sequence (mutant probe) or wild-type sequence (wild-type probe) were designed. The wild-type TaqMan probe was 5ʹ- ACGGGTATCCCTTCGACG-3ʹ, and the TaqMan probe to detect the mutation in Mmp9 was 5ʹ-CCCCGGGTATCGAC-3ʹ. The genotypes were determined as wild-type (homozygous SS) or homozygous SSMmp9−/− for the mutation, depending on whether the samples were positive with only wild-type probes or positive with only mutant probes, respectively, as previously described.24Feng W. Chen B. Xing D. et al.Haploinsufficiency of the transcription factor Ets-1 is renoprotective in Dahl salt-sensitive rats.J Am Soc Nephrol. 2017; 28: 3239-3250Crossref PubMed Scopus (10) Google Scholar DNA samples of rats in the study were amplified with real-time PCR and used to determine genotype (Transnetyx). The forward primer sequence was CCGCCTCTGCAGAGCA, and the reverse sequence was GTGTGCCAGTAGACCATCCTT. Experiments were performed using 9- to 12-week-old age-matched male SSMmp9−/− rats and littermates (SS). Male rats were used in these studies, because female SS rats may have different pathogenetic processes that generate and modify hypertension and end-organ injury.29Moreno C. Dumas P. Kaldunski M.L. et al.Genomic map of cardiovascular phenotypes of hypertension in female Dahl S rats.Physiol Genomics. 2003; 15: 243-257Crossref PubMed Scopus (87) Google Scholar Because of the need to monitor blood pressure in awake unrestrained animals, continuous monitoring of blood pressure was performed using radiotelemetry.30Drueke T.B. Devuyst O. Blood pressure measurement in mice: tail-cuff or telemetry?.Kidney Int. 2019; 96: 36Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar,31Luft F.C. Men, mice, and blood pressure: telemetry?.Kidney Int. 2019; 96: 31-33Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Rats were anesthetized using 2% isoflurane, and an implantable radiotelemetry transmitter (HD-S10, Data Sciences International [DSI]) was inserted into the left femoral artery to continuously record awake unrestrained blood pressure.32Feng W. Ying W.Z. Aaron K.J. et al.Transforming growth factor-β mediates endothelial dysfunction in rats during high salt intake.Am J Physiol Renal Physiol. 2015; 309: F1018-F1025Crossref PubMed Scopus (20) Google Scholar Rats were allowed 7 days to recover and were then divided into 4 groups (n = 6–9 rats per group) and fed either a control diet containing 0.3% NaCl (AIN-76A Purified Rodent Diet with 0.3% NaCl, Dyets Inc.; groups termed Pre-HT SS and Pre-HT SSMmp9−/−) or a 4% NaCl diet for 1 week (Dyet#113756, AIN-76A Purified Rodent Diet with 4.0% NaCl, Dyets Inc.) to induce hypertension (groups termed HT SS and HT SSMmp9−/−). Blood pressure was continuously monitored in awake unrestrained rats throughout the 7-day study. On day 6 of high-salt feeding, some rats of the 4 experimental groups were placed in metabolic cages for 24 hours to collect urine to determine concentrations of albumin and creatinine. Urinary albumin concentrations were determined by enzyme-linked immunosorbent assay (catalog no. E110-125, Bethyl) and normalized against urinary creatinine concentration (catalog no. DICT-500, Bioassay Systems). Rats were anesthetized; blood was collected for determination of serum creatinine using liquid chromatography–tandem mass spectrometry (Agilent 6460 C triple quad tandem mass spectrometry system)33Bondarenko A. Panasiuk O. Stepanenko L. et al.Reduced hyperpolarization of endothelial cells following high dietary Na+: effects of enalapril and tempol.Clin Exp Pharmacol Physiol. 2012; 39: 608-613Crossref PubMed Scopus (9) Google Scholar; and the kidneys were harvested for physiology, histology, and molecular analyses. Kidney tissues were homogenized and sonicated in Pierce RIPA Buffer (Catalog No. 89901, Thermo Scientific) with Halt Protease and Phosphatase Inhibitor Cocktail (Catalog No. 1861284, Thermo Scientific). The total soluble protein concentration in lysates was determined using a bicinchoninic acid assay kit (catalog no. 23227, BCA Protein Assay Reagent Kit, Thermo Scientific). Samples were boiled for 3 minutes in Laemmli buffer and separated using 7% to 12% sodium dodecylsulfate–polyacrylamide gel electrophoresis (catalog no. 5671044 Bio-Rad Laboratories) before electrophoretic transfer onto polyvinylidene diflouride membranes. The membranes were blocked in 5% nonfat milk and then probed with an MMP-9 antibody (catalog no. ab76003, Abcam; or catalog no. sc13520, MMP-9 antibody [7-11C], Santa Cruz Biotechnology, Inc.; diluted 1:1000). After washes, the blots were incubated for 1 hour at room temperature with Alexa Fluor 680 or 790 conjugated AffiniPure anti-rabbit secondary antibody (1:10,000 dilution). The bands were detected using the Odyssey CLx Infrared Imaging System (LI-COR Biosciences), and densitometric analysis was performed using Image Studio Software (LI-COR Biosciences). Autoregulatory behavior of afferent arterioles was assessed in experimental groups of animals by using the in vitro blood-perfused juxtamedullary nephron technique, as described previously.34Inscho E.W. Carmines P.K. Navar L.G. Juxtamedullary afferent arteriolar responses to P1 and P2 purinergic stimulation.Hypertension. 1991; 17: 1033-1037Crossref PubMed Google Scholar, 35Guan Z. Singletary S.T. Cook A.K. et al.Sphingosine-1-phosphate evokes unique segment-specific vasoconstriction of the renal microvasculature.J Am Soc Nephrol. 2014; 25: 1774-1785Crossref PubMed Scopus (24) Google Scholar, 36Feng W. Remedies C.E. Obi I.E. et al.Restoration of afferent arteriolar autoregulatory behavior in ischemia-reperfusion injury in rat kidneys.Am J Physiol Renal Physiol. 2021; 320: F429-F441Crossref PubMed Google Scholar In the first experiment, 4 groups of rats were included: Pre-HT SS, Pre-HT SSMmp9−/−, HT SS, and HT SSMmp9−/− (n = 5–7 rats examined per group). In a second experiment, we used the MMP inhibitor Dox (D9891), 30 mg/kg/d, in drinking water37Castro M.M. Rizzi E. Figueiredo-Lopes L. et al.Metalloproteinase inhibition ameliorates hypertension and prevents vascular dysfunction and remodeling in renovascular hypertensive rats.Atherosclerosis. 2008; 198: 320-331Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar,38Antonio R.C. Ceron C.S. Rizzi E. et al.Antioxidant effect of doxycycline decreases MMP activity and blood pressure in SHR.Mol Cell Biochem. 2014; 386: 99-105Crossref PubMed Scopus (35) Google Scholar to assess the effect of MMP-9 on autoregulation. Additional 2 groups of rats were included: Pre-HT SS + Dox and HT SS + Dox. Briefly, 2 identical rats were anesthetized with thiobutabarbital i.p. (100 mg/kg body weight) for each juxtamedullary nephron experiment. The right kidney was cannulated and continuously perfused over the course of the kidney dissection with ∼200 to 300 ml of Tyrode’s buffer (Sigma-Aldrich) containing 5.2% bovine serum albumin (Calbiochem). Perfusate blood was collected via a carotid artery cannula from the kidney donor and an identical blood donor and centrifuged to obtain the plasma and erythrocytes for kidney perfusion (hematocrit of ∼33%). The inner cortical surface of the right kidney was exposed, and the ends of the intrarenal arteries and arterial branches were tied with a 10-0 nylon suture to restore kidney perfusion pressure. After completion of the dissection, the kidney was switched to the reconstituted blood at a perfusion pressure of 100 mm Hg. The image of the kidney was displayed on a video monitor via a high-resolution NC-70 Newvicon video camera (Dage-MTI) and recorded on digital video disk for later analysis. After at least 20-minute equilibration period, afferent arteriolar autoregulatory responses were assessed as perfusion pressure was reduced from 100 to 65 mm Hg and then increased to 170 mm Hg in 15-mm Hg increments at 5-minute intervals. The inner arteriole diameter was measured every 12 seconds at a single site using a calibrated image-shearing monitor (model 908, Vista Electronics) and was calculated from the average of all diameter measurements collected during the final 2 minutes of each treatment period. Briefly, rats were anesthetized with thiobutabarbital for retrograde perfusion through the abdominal aorta. The kidneys were perfused with 5 to 10 ml of Tyrode’s buffer to flush out the blood and placed in ice-cold physiological salt solution. Medulla and intrarenal arteries were removed, and cortical tissue was gently pressed through a nylon membrane sieve (100 μm pore size, BioDesign, Inc.). The kidney tissue was transferred into RNAlater stabilization solution (Invitrogen, Thermo Fisher Scientific). Segments of arcuate and interlobular arteries with attached afferent arterioles were identified and collected by microdissection under a stereoscope for mRNA extraction. Kidney microvascular RNA was extracted from dissected kidney microvessels with TRIzol (Invitrogen). Urinary RNA was isolated from 24-hour urinary samples with a kit (Zymo Research). Samples were treated with DNAase I to remove genomic DNA. Protein- and DNA-free RNA was reverse transcribed to cDNA with use of SuperScript IV (Invitrogen). cDNA was amplified by PCR in the LightCycler 480 System (Roche) for 40 cycles using the SYBR Green method (Applied Biosystems) and specific primers (Table 1); relative RNA levels were calculated with the PCR threshold cycle software and a standard equation (Applied Biosystems). mRNA expression was normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample and then standardized to the group of Pre-HT SS as 1.Table 1Primers used in real-time polymerase chain reaction analysesGeneForward primerReverse primerMmp9GAATCACGGAGGAAGCCAATGTGTACACCCACATTTTGCGNphs2 (podocin)GCAGTCTAGCTCATGTGTCCCTGAGTCCAAGGCAACCTTTNphs1 (nephrin)CTGTGGACATAGTCTGCACCCTTTCTCCATGTCGTCCAGGTgfb1CTACTGCTTCAGCTCCACAGAGAACCTTGGGCTTGCGACCFn1 (fibronectin)GCCTTCAACTTCTCCTGTGAGTTGCAAACCTTCAATGGTCRunx2TGACCTTTGTCCCAATGTGGTTTGCTACTGGGTGGGTTTCSpp1 (osteopontin)AGGAGTTTCCCTGTTTCTGGTCTTCCCGTTGCTGTCPostn (periostin)CATAGACGGGGTTCCTGTTGTGCAAGAATTTCTGCAGGGTSelp (P-selectin)AATGAAATCGCTCACCTCTTATTGGGCTCGTTGTCTTLR2GCTCCTGTGAACTCCTGTCCGACACTCCAAGACTGAGGGCICAMCAAACGGGAGATGAATGGTGGCGGTAATAGGTGTAAATGAPDHATTCTTCCACCTTTGATGCTGGTCCAGGGTTTCTTACT18SATTTGACTCAACACGGGAAATCGCTCCACCAACTAAGAAC Open table in a new tab Three-micrometer-thick kidney sections were prepared from paraffin-embedded tissues. After deparaffinization, antigen retrieval was performed with Antigen Unmasking Solution (H-3300, Vector Laboratories). For immunohistochemical staining, an avidin-biotin-peroxidase complex-based immunohistochemical technique was used to detect Wilms’ tumor 1 (WT1) protein, a podocyte marker, in glomeruli. Section peroxidase activity was quenched with BLOXALL Endogenous Blocking Solution (SP-6000, Vector Laboratories); endogenous biotin was blocked with the Avidin/Biotin Blocking Kit (SP-2001, Vector Laboratories); and other nonspecific staining was blocked with normal serum. Sections were incubated with the primary antibody rabbit antibody against WT1 (ab224806, Abcam) overnight, followed by application of a biotinylated goat anti-rabbit antibody for 30 minutes. Slides were then incubated with VECTASTAIN ABC Kit (PK-4001, Vector Laboratories) reagents according to the manufacturer’s instructions and developed with Vector NovaRED Substrate Kit (SK-4800, Vector Laboratories) substrate. Images were acquired using a Leica DM6000 microscope (Leica Microsystems). WT1-positive cells were counted in at least 30 glomeruli in each section of the experimental groups by a blinded observer. Counts were normalized using the glomerular area determined by Image J (National Institutes of Health) and then averaged per kidney. Values were averaged in experimental groups and expressed as mean ± SEM. For immunofluorescence staining, sections were incubated with rabbit primary antibodies to phosphorylated Smad2 (#18338, Cell Signaling) and fibronectin (ab2413, Abcam) at 4 °C overnight. Another primary antibody to smooth muscle actin (Mouse, 1A4, Invitrogen; or Rabbit, ab5694, Abcam) was also used to outline the vessel in kidney sections. Sections were washed and incubated with the respective secondary antibodies conjugated with either Alexa Fluor 488 (green, Invitrogen) or Alexa Fluor 594 (red, Invitrogen). Counterstaining of the nucleus was achieved by mounting sections with hardset mounting media containing 4ʹ,6-diamidino-2-phenylindole (DAPI, blue color; Vector Laboratories). Negative controls by omission of the primary antibody were included in each experiment. Images were acquired using a Leica DM6000 epifluorescence microscope (Leica Microsystems) with a Hamamatsu ORCA ER cooled CCD camera (Hamamatsu Photonics) and SimplePCI software (Compix Inc). Primary cultures of VSMCs were derived from 10-week-old inbred SS or SSMmp9−/− rats. Thoracic aortas were obtained by careful dissection under anesthesia. The adventitia and endothelium were carefully removed from segments of aorta and washed several times with Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific). The segments were cut into ∼0.5-mm squares, placed on 25-mm Petri dishes, cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum at 37 °C and 5% CO2. The cells that grew from the explants had become relatively conflu