Expression of ACE and ACE2 in Patients with Hypertensive Nephrosclerosis

Background: The interplay between intrarenal angiotensinconverting enzyme (ACE) and type 2 ACE (ACE2) might play important roles in the pathogenesis of hypertensive nephrosclerosis (HTN), but human data are limited. Methods: Renal biopsy specimens of 41 patients with HTN and 10 transplant donors as controls (CTL) were studied. The glomerular and tubulointerstitial mRNA expression of ACE and ACE2 was measured by laser microdissection and real-time quantitative polymerase chain reaction. The corresponding protein level was determined by immunohistochemistry. Results: Neither the glomerular nor tubulointerstitial mRNA expression of ACE or ACE2 correlated with the corresponding protein level by immunohistochemistry. The tubulointerstitial levels of ACE and ACE2 were significantly lower in HTN than CTL, while the glomerular ACE and ACE2 levels were similar between the groups. The tubulointersitial ACE and ACE2 levels significantly correlated with the estimated glomerular filtration rate (GFR) and inversely with the degree of histological damage. The glomerular ACE and ACE2 levels significantly correlated with the rate of GFR decline. The ratio of glomerular ACE and ACE2 level correlated with the estimated GFR and the degree of glomerulosclerosis. Conclusion: Received: August 18, 2010 Accepted: January 17, 2011 Published online: February 24, 2011 Dr. C.C. Szeto Department of Medicine and Therapeutics Prince of Wales Hospital, The Chinese University of Hong Kong Shatin, NT, Hong Kong, SAR (China) Tel. +852 2632 3126, E-Mail ccszeto @ cuhk.edu.hk © 2011 S. Karger AG, Basel 1420–4096/11/0343–0141$38.00/0 Accessible online at: www.karger.com/kbr D ow nl oa de d by : 54 .1 91 .4 0. 80 9 /1 6/ 20 17 8 :4 9: 14 P M Wang /Lai /Kwan /Lai /Chow /Li /Szeto Kidney Blood Press Res 2011;34:141–149 142 angiotensin I to angiotensin 1–9 and angiotensin II to angiotensin 1–7, counterbalancing the detrimental effect of angiotensin II [10, 11] . It is highly possible that the intrarenal activity of angiotensin II is regulated by the interplay between ACE and ACE2. In the present study, we explore the intrarenal mRNA expression and protein level of ACE and ACE2 in patients with biopsy-proven HTN. Subjects and Methods Subjects We studied 41 consecutive patients in our hospital who had biopsy-proved HTN and no feature of other coexisting renal diseases [12] . Renal biopsy tissue was retrieved for gene expression, immunohistochemistry and morphometric analysis (see below). Clinical data including blood pressure, serum creatinine, urea, electrolyte, albumin, liver enzymes and 24 h urine protein were reviewed. The glomerular filtration rate (GFR) was estimated by a standard equation [13] . We also studied 10 kidney transplant donors as controls (CTL). Laser Microdissection The method of laser microdissection has been described in our previous study [14] . In essence, immediately after kidney biopsy, part of the tissue was embedded by OCT compound (Sakura, USA) and kept at –80 ° C under a RNase-free condition for intrarenal gene expression. We then performed laser microdissection to determine the glomerular mRNA expression of ACE and ACE2. Briefly, 8to 10 m thick cryosections were prepared on a cryostat (Leica Microsystems, Wetzlar, Germany) using disposable microtome blades (Leica Microsystems) under RNase-free conditions. Sections were then mounted on glass slides covered with a polyethylene naphthalate membrane (PALM Microlaser Technologies, Bernried, Germany) and were treated with UV irradiation and RNase Away (Invitrogen, Life Technologies, Pa., USA) prior to use. After being fixed in 70% ethanol for 2 min, the sections were stained according to the following protocol: DEPC water 1 min, Mayer’s hemotoxylin (Sigma, USA) solution 30 s, RNase-free water 1 min, 50% ethanol 1 min, 70% ethanol 1 min, 100% ethanol 1 min twice, and air-dried 10 min. Next, PALM Microlaser Systems (PALM, Wolfratshausen, Germany), which is equipped with a pulsed high-quality laser beam, computer-controlled microscope stage and micromanipulator was used to perform microdissection of the prepared slides. Under direct visual control, 20 8 5 glomerular areas of each case were isolated by the focused nitrogen laser beam and then catapulted into different microcentrifuge caps, which were filled with guanidine thiocyanate containing lysis buffer (buffer RLT; Qiagen, Inc., Canada). The tissue lysate of isolated glomerulus was then stored at –80 ° C until RNA extraction was performed. Measurement of Intra-Renal Gene Expression RNeasy Micro kits were used to extract total RNA from microdissected tissue according to the manufacturer’s protocol. DNase was used to wipe off probable genomic DNA. For reverse transcription, 5 l total RNA was mixed with 1  l random primers (150 ng), 1  l dNTP mix (10 m M each), 4  l 5 ! first-strand buffer, 2  l DTT (0.1 M ), 1  l (200 U) Superscript II RNase H reverse transcriptase (all from Invitrogen, Life Technologies) and made up to 20  l with H 2 O. Reverse transcription was performed at 65 ° C for 5 min, 25 ° C for 10 min, 42 ° C for 50 min, and the inactivated reaction at 70 ° C for 10 min. The resulting cDNA was stored at –80 ° C until use. We quantified intrarenal expression of ACE and ACE2 using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif., USA). Commercially available TaqMan primers and probes, including two unlabeled PCR primers and one FAM TM dye-labeled TaqMan MGB probe were used for all target genes (all from Applied Biosystems). The primer and probe set was deliberately designed across the intron-exon boundary so as not to detect probable genomic DNA. For the real-time quantitative polymerase chain reaction, 10  l universal master mix, 1  l primer and probe set, 2 l cDNA, and 7  l H 2 O were mixed to make a 20 l reaction volume. Each sample was run in triplicate. Time quantitative polymerase chain reactions were performed at 50 ° C for 2 min, 95 ° C for 10 min, followed by 40 cycles at 95 ° C for 15 s and 60 ° C for 1 min. 18S rRNA (Applied Biosystems) was used as a housekeeping gene to normalize the mRNA expression level of each target gene. Results were analyzed with Sequence Detection Software version 1.7 (Applied Biosystems). In order to calculate the differences of expression level for each target gene among samples, the C T method for relative quantitation was used according to the manufacturer’s manual [15] . Immunohistochemistry and Morphometric Study of Kidney Biopsy The intrarenal ACE and ACE2 expression at protein level was explored by a standard immunohistochemical technique. Briefly, the renal tissue was fixed in 10% neutral buffered formaldehyde overnight and then dehydrated by alcohol and embedded in paraffin. 4m thick sections of renal biopsy specimens of each patient were prepared on precoated slides and deparaffinized in xylene and rehydrated by graded alcohol. Antigens were unmasked by heat treatment (95 ° C for 6 min) in 10 m M sodium citrate buffer, pH 6.0. Rabbit ImmunoCruz TM staining system (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) was then used for staining. Briefly, endogenous peroxidase activity was quenched by peroxidase block for 5 min at room temperature. Sections were then incubated in a serum block for 20 min at room temperature. Rabbit polyclonal to ACE (catalog No. sc-20791; Santa Cruz Biotechnology, Inc.) and ACE2 (catalog code ab15348; Abcam, Cambridge, UK) were diluted in a serum block at a concentration of 1: 500. Specimens were then incubated with dilute primary antibody at 4 ° C overnight, followed by incubation with biotinylated secondary antibody and HRP-streptavidin complex at room temperature for 30 min each. After incubation with DAB chromogen for 5 min, slides were counterstained with hematoxylin for 2 min. Sections were dehydrated with graded alcohol and xylene, covered with Histomount (VWR, Inc., Poole, UK) and a coverslip. As described in our previous studies [16, 17] , Jones’ silver staining was also performed on 5m thick sections of renal biopsy specimens of each patient to assess renal scarring. We used a computerized image analysis method to quantify the degree of immunohistochemical and silver staining. Briefly, a Leica Twin Pro image analysis system (Leica Microsystems) was connected to a Leica DC500 digital camera on a Leica DMRXA2 microscope working with a 40 ! objective (final calibration: 0.258 D ow nl oa de d by : 54 .1 91 .4 0. 80 9 /1 6/ 20 17 8 :4 9: 14 P M ACE2 in Hypertensive Nephrosclerosis Kidney Blood Press Res 2011;34:141–149 143 mm/pixel) and to a microcomputer for storage of the morphometric measurements and to perform image analysis by using imageanalyzing software (MetaMorph 4.0; Universal Imaging Corp., Downingtown, Pa., USA). Ten glomeruli and ten randomly selected tubular interstitial areas were assessed in each patient and the average protein level and scarring degree in glomerular and tubulointerstitial areas, as represented by the percentage of the area with positive staining, were computed for each patient. Clinical Follow-Up All patients were followed for at least 12 months after renal biopsy. Their treatments were decided by an individual nephrologist and not affected by the study. Disease progression was measured by the rate of GFR decline [18] , which was calculated by the least-squares regression method. Statistical Analysis Statistical analysis was performed by SPSS for Windows version 11.0 (SPSS, Inc., Chicago, Ill., USA). All results were presented as mean 8 SD for normally distributed data and median (lower and upper quartiles) for the others. Since data of gene expression levels were highly skewed, nonparametric statistical methods were used. We used the Mann-Whitney U test to compare gene expression levels between groups and Spearman’s rank-order correlations to test associations between gene expression levels and clinical parameters. p ! 0.05 was considered statistically significant. All probabilities were two-tailed.