Tubular Expression of Angiotensin II Receptors and Their Regulation in IgA Nephropathy
Loretta Y.Y. Chan,
Joseph C.K. Leung,
Sydney C.W. Tang,
Cindy B.Y. Choy and
Kar Neng Lai
Division of Nephrology, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong
Address correspondence to: Prof. Kar Neng Lai, Department of Medicine, Room 411, Professorial Block, Queen Mary Hospital, The University of Hong Kong, Pokfulam Road, Hong Kong. Phone: 852-2855-4251; Fax: 852-2816-2863; E-mail: knlai{at}hkucc.hku.hk
Received for publication December 21, 2004.
Accepted for publication April 30, 2005.
Enhanced renal expression for the renin-angiotensin system (RAS)is detected in IgA nephropathy (IgAN). Previous data showedan altered glomerular expression of angiotensin II type 1 receptor(AT1R), suggesting a regulatory response to high intrarenalangiotensin II (Ang II) concentration in IgAN. In this study,the expression and regulation of Ang II receptors were examinedin human proximal tubular epithelial cells (PTEC) in IgAN. Tubularexpression of AT1R and Ang II type 2 receptor (AT2R) was increasedin IgAN. In vitro culture experiment showed that the upregulationof Ang II receptors was not due to the direct effect of IgAbut the indirect effect after IgA deposition on human mesangialcell. When PTEC were cultured with conditioned culture mediumfrom human mesangial cells activated with IgA, Ang II productionwas upregulated, leading to inflammation and apoptosis via theAT1R and AT2R, respectively. Sequential expression of Ang IIreceptors determined the injury of PTEC induced by mediatorsin the conditioned medium. The initial interaction between AngII and AT1R activated both protein kinase C and mitogen-activatedprotein kinase pathways, leading to inflammatory responses.This early AT1R-dependent event was followed by upregulationof AT2R expression and continued Ang II release. The interactionbetween Ang II and AT2R subsequently led to expression of cleavedpoly[ADP-ribose] polymerase through downregulation of the mitogen-activatedprotein kinase pathway. The data suggest that appropriate controlof Ang II receptor activities in PTEC may ameliorate tubulointerstitialinjury in IgAN.
The intrarenal renin-angiotensin system (RAS) is pivotal inthe pathogenesis of chronic renal injury, and angiotensin II(Ang II) plays an important role in regulating cell proliferation,apoptosis, and fibrosis (1–3). The Ang II type 1 receptor(AT1R) distributes widely in kidney cells, including mesangial,glomerular epithelial, endothelial, and vascular smooth musclecells. Ang II can directly induce both cell growth and matrixaccumulation in glomerular cells via the AT1R (4). In contrast,the AT2R is expressed at low levels in the endothelium in normaladult kidney and in cultured glomerular visceral epithelium.This receptor is often upregulated in response to injury andcounterbalances the AT1R by increasing the production of bradykinin,nitric oxide, and cyclic guanosine monophosphate, thus mediatingvasodilation, cell differentiation, and apoptosis (5–7).Pharmacologic blockade of the AT2R in wild-type mice and AT2Rknockout mice shows decreased apoptosis and increased fibrosisin the kidney after injuries (8,9).
In rat proximal tubular epithelial cells (PTEC), Ang II inducescellular hypertrophy and activates relevant downstream signaltransduction pathways (10,11). This Ang II–induced tubularcell hypertrophy is inhibited by losartan but not PD123319 (10–12),suggesting that the AT1R rather than the AT2R is contributoryto the tubular cell hypertrophy. Rats that received Ang II infusionhave an increased number of proliferating cell nuclear antigen–andtransferase-dUTP-nick-end labeling–positive cells in proximaltubules (13). Similar findings have also been reported in culturedrat proximal tubular cells (14), suggesting that Ang II triggersboth proliferation and apoptosis in tubular epithelial cells.
IgA nephropathy (IgAN) runs a variable clinical course witha slowly relentless and progressive renal failure in 30 to 50%of patients within 30 yr after the first clinical presentation(15). A subgroup with severe tubulointerstitial atrophy is oftenassociated with rapid progression to end-stage renal failure(16). We recently reported an altered AT1R expression in humanmesangial cells (HMC) in response to raised intrarenal Ang IIconcentration in IgAN (17). We proposed that an imbalance betweenAT1R and AT2R activities in HMC after binding to IgA could playa significant role in the pathogenesis of IgAN. In this study,we further examined the expression and regulation of Ang IIreceptors in PTEC and explored their role in the developmentof tubulointerstitial injury in IgAN.
Experimental Design
The study was conducted in accordance with the Declaration ofHelsinki and was approved by the institutional ethics committee.All participants gave written informed consent for serum andtissue collection. Twenty milliliters of blood was collectedfrom each control subject and from patient at clinical quiescence.The serum was frozen at –20°C until isolation of IgA.Every IgA preparation was obtained separately from each individualpatient or healthy control subject.
We first examined the tubular expression of AT1R and AT2R inpatients with IgAN. Renal tissues were obtained from 13 normotensivepatients who had mild IgAN (grade 1) and were admitted consecutivelyfor renal biopsy with the presentation of microhematuria. Theseverity of renal pathology was classified into grade 1, 2,or 3 as described previously (18). These patients had normalcreatinine clearance (>80 ml/min per 1.73 m2) with proteinuriaranging from 0.8 to 1.7 g/d. They had not previously receivedangiotensin-converting enzyme inhibitor (ACEI) or AT1R antagonist.Control renal tissues were obtained from the intact pole ofkidneys that were removed for circumscribed tumor in eight normotensiveindividuals (comparable in age, gender, and race). For studyingthe dose- and time-response profile of IgA-HMC–conditionedmedium from patients with IgAN on the expression of Ang II receptorsin cultured PTEC, total IgA1 were isolated from sera of fiveof these patients selected randomly. The same IgA preparationswere used in subsequent studies of the expression of Ang IIreceptor modified by different antagonists and agonists afterprolonged exposure to conditioned medium from HMC that wereincubated with IgA.
For comparison of the immediate regulatory effect of IgA onthe expression of Ang II receptors and Ang II release in culturedPTEC, IgA1 was prepared from sera of another 22 patients whohad IgAN and were randomly recruited from the clinic. Theirdiagnosis was made at least 18 mo before the study, and theirserum creatinine remained stable over the previous 12 mo. Theirproteinuria ranged from 0.4 to 2.9 g/d. Their mean endogenouscreatinine clearance was 72.8 ± 23.2 ml/min per 1.73m2. These patients varied in the histologic severity with grade1 in 12, grade 2 in eight, and grade 3 in two. None of thesepatients with IgAN received ACEI or AT1R antagonist for BP controlduring the study period. Control subjects were age- and gender-matchedhealthy volunteers.
Morphometric Studies
Renal tissues were processed by standard methods, and the tubularexpression of AT1R or AT2R was detected by immunohistochemistryusing specific polyclonal antibodies for individual Ang II receptor(Santa Cruz Biotechnology, Santa Cruz, CA) as described previously(17). Two renal pathologists without previous knowledge of clinicalor laboratory data evaluated the expression of AT receptor stainingusing an arbitrary 0 to 5+ scale. Ten consecutive cortical cross-sectionswere examined at high-power field. The preset grading criteriafor tubular AT receptor staining were as follows: 0 if no tubularstaining, 1+ when <10% cortical tubular cells were positive;2+ when 10 to <20% cortical tubular cells were positive;3+ when 20 to <40% cells were positive; 4+ when 40 to <60%cells were positive, and 5+ when 60% or more cells were positive.
Cell Culture and Isolation of IgA1
Culture medium and FBS were obtained from Life Technologies(Rockville, MD). All other chemicals were obtained from Sigma(St. Louis, MO).
Isolation, characterization, and culture of human umbilicalvein endothelial cells (HUVEC), HMC, and PTEC were performedas described previously (19,20). Human podocyte cell line wasprovided by Professor P.W. Mathieson (University of Bristol).In all experiments, cells that were grown to confluence weregrowth arrested in a six-well culture plate (0.5 x 106 cells/well)with culture medium that contained 0.5% FBS for 48 h beforecommencement of experiments. Total IgA was isolated by affinitychromatography with jacalin agarose (Pierce, Rockford, IL) (17).The endotoxin levels for all IgA preparations were <1 pg/mgprotein determined by a Limulus amebocyte lysate assay (BioWhittaker,Walkersville, MD).
Preparation of Conditioned Medium from HMC Activated by IgA
HMC, podocytes, and HUVEC were cultured with medium that containedIgA (final concentration 50 µg/ml) isolated from patientswith IgAN or healthy control subjects for 48 h. The concentrationof IgA preparation used was selected on the basis of our previousdata that 50 µg/ml IgA was able to increase significantlythe macrophage migration inhibitory factor (MIF) by culturedHMC (21). The conditioned media (IgA-HMC–conditioned medium,IgA-podocyte–conditioned medium, and IgA-HUVEC–conditionedmedium) were collected and stored at –70°C until used.Conditioned medium from HMC that were cultured without the additionof IgA (control medium) was used as control. For all subsequentexperiments that examined the effects of IgA-HMC or other conditionedmedium on PTEC, conditioned medium first was diluted 10-foldwith DMEM/F12 that contained 0.5% FBS before use for culturingPTEC. TNF- in the IgA-HMC medium was determined using ELISA(R&D Systems, Minneapolis, MN).
Effect of IgA, Cytokines, or Conditioned Medium on Ang II Release
PTEC, podocytes, HUVEC, or HMC were cultured with an IgA preparation(50 µg/ml) for 96 h. In parallel experiments, PTEC werecultured with IgA-HMC–, IgA-podocyte–, and IgA-HUVEC–conditionedmedia or various cytokines (100 pg/ml TNF-, 1 ng/ml TGF-, or10 ng/ml IL-6) for 96 h. After culture, the supernatants werestored at –70°C before assay for Ang II. Ang II wasmeasured by an enzyme immunoassay using an anti–Ang IImAb labeled with acetylcholinesterase (SPI bio, Massy Cedex,France). The detection limit was 1 pg/ml, and the intra-assaycoefficient of variation was 7% (17).
Effect of IgA-HMC Medium on Ang II Release and Ang II Receptor Expression by PTEC
PTEC were cultured with IgA-HMC medium or IgA preparation (50µg/ml) for either 24 (for mRNA) or 48 h (for protein)unless otherwise stated. The cells then were collected for totalRNA isolation or for cell lysate preparation, whereas the supernatantswere stored at –70°C before assay for Ang II. In parallelexperiments, PTEC were cultured with IgA-HMC medium for varioustime points (12 h to 6 d). The cell lysate was prepared forAT receptors and cleaved poly-(ADP-ribose)-polymerase (PARP)determination, and the supernatants were collected for Ang IIand IL-6 assay using a sandwich ELISA kit (R&D Systems).
To study the effect of Ang II receptor blockade on the expressionof cleaved PARP in PTEC, we performed similar experiments withlosartan (100 nM) or PD123319 (10 µM) added 30 min beforestimulating PTEC with conditioned medium. The concentrationof losartan or PD123319 used in the blockade study was selectedon the basis of data from preliminary experiments. At this concentrationof losartan or PD123319, mitogen-activated protein kinase (MAPK)activation (for losartan) or cleaved PARP expression (for PD123319)induced by Ang II (10–7 M) was completely abolished. AfterPTEC were incubated for 96 h, cell lysates were collected fordetection of cleaved PARP by immunoblotting. The time-responseeffects were studied in growth-arrested PTEC (0.5 x 106 perwells of six-well culture plate) that were exposed to IgA-HMC–conditionedmedium that was prepared from patients with IgAN for differenttime intervals (from 12 h to 6 d) in the presence or absenceof AT1R antagonist (losartan, 100 nM) or AT2R antagonist (PD123319,10 µM). Each antagonist was added 30 min before the culture.At each time point, culture supernatants were collected forassay of Ang II, and cell lysates were collected for analysisof cleaved PARP, AT1R, and AT2R.
Effects of Recombinant TNF- on IL-6 Production and Recombinant IL-6 on Ang II Receptor Expression by PTEC
PTEC were cultured with recombinant 5 to 160 pg/ml TNF- (R&DSystems) for 12 h or at a concentration of 40 pg/ml for 3 hto 2 d. The supernatants were collected for assay of IL-6. PTECwere cultured with 0.25 to 2 ng/ml recombinant IL-6 (R&DSystems) for 12 h or at a concentration of 5 ng/ml for 3 h to2 d. The cell lysate preparation was collected for determinationof AT1R and AT2R expression.
Effect of Ang II on the Ang II Receptor Expression and Cleaved PARP Expression in PTEC
PTEC were cultured with Ang II (10–12 to 10–6 M)in the presence or absence of antagonists to AT1R (losartan,100 nM) or AT2R (PD123319, 10 µM). The cells were culturedfor 48 h, and cell lysates were prepared for determination ofAT1R and AT2R protein. To study the effect of AT2R on cleavedPARP expression in PTEC, we performed similar experiments incells that were incubated with an AT2R agonist CGP42112A (10–12to 10–6 M), and the expression of cleaved PARP was analyzed.
Ang II Binding Studies
PTEC (1 x 105) were cultured with medium or IgA-HMC–conditionedmedium for 2 d. After culture, cells were incubated with increasingconcentrations (0.25 to 10 nM) of [3H]Ang II (50 Ci/mmol; AmericanRadiolabeled Chemical, St. Louis, MO) in medium that was supplementedwith 1% BSA at 20°C for 90 min. After incubation, unbound[3H]Ang II then were removed by washing with cold PBS. The cellsthen were lysed with 1 M NaOH plus 0.1% Triton X-100, and theradioactivity of the lysate was counted. Competitive bindingassay was performed on cells that were incubated with 10 nM[3H]Ang II with or without Ang II receptor antagonists (losartan10 µM or PD123319 10 µM). For determining nonspecificbinding of [3H]Ang II, cells were incubated in the presenceof unlabeled Ang II (10–5 M). The specific [3H]Ang IIbinding, expressed as fmol/mg protein, was calculated as thedifference between the total and nonspecific binding. Scatchardplot analyses were performed to calculate the dissociation constant(Kd) and the maximum binding capacity (Bmax). The protein contentwas determined by a protein assay kit from Bio-Rad Laboratories(Hercules, CA).
Role of AT1R and AT2R on Soluble Intercellular Adhesion Molecule-1 Production and Cleaved PARP Expression in PTEC Cultured with IgA-HMC Medium
PTEC were cultured with IgA-HMC–conditioned medium frompatients with IgAN at different time intervals (from 12 h to6 d) in the presence of (1) losartan (100 nM), (2) PD123319(10 µM), or (3) CGP42112A (10–7 M) added 30 minbefore culture or (4) losartan (100 nM) added 30 min beforeculture and followed by addition of PD123319 (10 µM) 2d after culture. At each time point, supernatants and cell lysateswere collected and stored at –70°C for assay of solubleintercellular adhesion molecule-1 (sICAM-1) and cleaved PARP.The sICAM-1 concentration was determined by a commercial ELISA(Bender MedSystems, Vienna, Austria).
Total RNA Extraction and Reverse Transcription–PCR and Immunoblotting
Total cellular RNA was extracted using NucleoSpin RNA II totalRNA extraction kit (Macherey-Nagel, Duren, Germany), and reversetranscription–PCR (RT-PCR) was performed using designedprimers for AT1R, AT2R, and glyceraldehyde-3-phosphate-dehydrogenase(GAPDH) as described previously (17). The gene expression wassemiquantified as the ratio of AT1R or AT2R amplicon to GAPDHamplicon. Immunoblotting was performed as described previously(17). Rabbit polyclonal anti-AT1R and AT2R receptors were obtainedfrom Santa Cruz Biotechnology. Monoclonal anticleaved PARP andpolyclonal rabbit anti–phospho p42/p44 MAPK and anti–phosphopan-protein kinase C (PKC) were obtained from Cell SignalingTechnology (Beverly, MA). Monoclonal anti-actin was obtainedfrom Neomarkers (Fremont, CA). Briefly, 10 µg of totalprotein from the extract was electrophoresed and transferredto a polyvinylidene difluoride membrane before probing withanticleaved PARP (1:500), anti-AT1R (1:1000), anti-AT2R (1:1000),anti-actin (1:1000), anti–phospho p42/44 MAPK (1:5000),or anti–phospho pan-PKC (1:4000) antibody in PBS-Tween.The images were scanned, and the densitometric results werereported as average arbitrary integrated values (units) afternormalization with the average arbitrary integrated values ofthe actin signal.
Activation of p42/p44 MAPK and PKC Signal Transduction Pathways in PTEC
PTEC were exposed to IgA-HMC–conditioned medium or AngII (10–10 M) for 24 h. In parallel experiments, individualor a combination of antagonists to AT1R (losartan, 100 nM) andAT2R (PD123319, 10 µM) were added 30 min before the additionof stimulants. For studying the effect of AT2R agonist CGP42112Aon the activation of p42/p44 MAPK and PKC pathways in PTEC byIgA-HMC–conditioned medium, PTEC were exposed to IgA-HMC–conditionedmedium in the presence of CGP42112A (10–12 to 10–6M) for 24 h. At the end of experiment, the expression of phospho-p42/p44and phospho-PKC by PTEC was determined by immunoblotting.
Statistical Analyses
All data were expressed as means ± SD unless otherwisespecified. The significance of differences between groups wasdetermined using one-way ANOVA. P < 0.05 was considered statisticallysignificant.
Tubular Expression of Ang II Receptors
Immunoreactive AT1R or AT2R protein was found in tubular epitheliumcells of normal renal tissue(Figure 1). An increase in stainingfor both Ang II receptors in PTEC was observed in biopsies frompatients with IgAN. With the use of a six-point scale, tubularAT1R immunostaining was marginally higher in IgAN (mean score1.75 in IgAN versus 1.37 in control subjects; P = 0.0217; Figure 2A).In contrast, tubular immunostaining for AT2R was significantlyincreased in patients with IgAN (mean score 3.5 in IgAN versus2.2 in control subjects; P = 0.0004; Figure 2B).
Figure 1. Representative immunohistochemical staining (brown) of tubular angiotensin II type 1 receptor (AT1R) and AT2R in normal and in IgA nephropathy (IgAN) kidney biopsies. Increased signals of AT1R and AT2R were observed in tubules of kidney biopsies from patients with IgAN. Magnification, x400.
Figure 2. Semiquantification of tubular AT1R and AT2R expression in kidney biopsy. (A) Tubular expression of AT1R was increased in kidney biopsies from patients with IgAN when compared with normal kidney (P = 0.0217). (B) Tubular expression of AT2R was significantly increased in kidney biopsies from patients with IgAN when compared with normal kidney (P = 0.0004).
Expression of Ang II Receptors in Cultured PTEC
There was no increase in gene and protein expression of AT1Ror AT2R in PTEC that were cultured with IgA that was isolatedfrom patients with IgAN when compared with control subjects(Figure 3, A and B). However, the gene and protein expressionof AT1R or AT2R were upregulated when PTEC were cultured withIgA-HMC–conditioned medium from patients with IgAN (P< 0.05; Figure 3, C and D). There was increased release ofAng II in HMC that were cultured with IgA that was isolatedfrom patients with IgAN (P < 0.05 versus controls; Figure 4A).Similar increased production of Ang II was not observedin PTEC, podocytes, or HUVEC. There was increased release ofAng II in PTEC that were cultured with IgA-HMC–conditionedmedium from patients with IgAN (P < 0.001 versus controls;Figure 4B). Nonetheless, similar increased production of AngII was not observed in PTEC that were cultured with IgA-podocyte–orIgA-HUVEC–conditioned medium or recombinant TGF-, TNF-,or IL-6 (Figure 4B).
Figure 3. Expression of AT1R and AT2R by proximal tubular epithelial cells (PTEC) that were cultured directly with IgA or with IgA–human mesangial cell (HMC)–conditioned medium. There was no difference in gene (A) and protein expression (B) of AT1R or AT2R in PTEC that were cultured with IgA that was purified from patients with IgAN (n = 22) when compared with that from healthy control subjects (n = 15). The gene (C) and protein expressions (D) of AT1R and AT2R were significantly upregulated in PTEC that were cultured with IgA-HMC–conditioned medium from patients with IgAN when compared with that from healthy control subjects. *P < 0.05.
Figure 4. (A) The synthesis of angiotensin II (Ang II) was not upregulated in PTEC, podocytes, or human umbilical vein endothelial cell (HUVEC) that were cultured directly with IgA alone but was significantly upregulated in HMC. (B) The synthesis of Ang II was significantly upregulated in PTEC that were cultured with IgA-HMC–conditioned medium that was prepared from patients with IgAN (n = 22) for 96 h when compared with that from healthy control subjects (n = 15). The Ang II concentration in the IgA-HMC–conditioned medium (diluted 10-fold with DMEM/F12 that contained 0.5% FBS before use for culturing PTEC) was 0.45 pg/ml. There was no upregulation of Ang II release by PTEC that were cultured with IgA-podocyte–conditioned medium, IgA-HUVEC–conditionedmedium, or various cytokines (TNF-, TGF-, or IL-6).
Time-Course Study of PTEC Cultured with IgA-HMC–Conditioned Medium Figure 5A shows the time course of the response of PTEC thatwere cultured with IgA-HMC–conditioned medium. AT1R expressionin PTEC was increased significantly on days 1 and 2 after incubationwith IgA-HMC–conditioned medium (P < 0.05) but returnedthereafter to basal level on day 6. AT2R expression in PTECincreased gradually after culture with IgA-HMC–conditionedmedium, and the expression was significantly greater than thebasal level on days 4 and 6. Rapid increase in IL-6 productionby PTEC was evident after 12 h after incubation with IgA-HMC–conditionedmedium (P < 0.05). The IL-6 production peaked after culturingfor 2 d and gradually declined on days 4 and 6. Ang II releasein PTEC was upregulated from day 4 after incubation with IgA-HMC–conditionedmedium (P < 0.05; Figure 5B). In parallel, the expressionof cleaved PARP in PTEC was enhanced from day 4 after incubationwith IgA-HMC–conditioned medium (P < 0.05). Blockadeof AT1R with losartan further amplified this upregulation ofcleaved PARP.
Figure 5. (A) Time-course study of AT1R and AT2R expression in PTEC by immunoblotting. AT1R expression (•) increased significantly in PTEC on days 1 and 2 after incubation with IgA-HMC–conditioned medium. AT1R expression then fell to basal level on day 6. AT2R expression () increased significantly in PTEC after incubation with IgA-HMC–conditioned medium for 4 and 6 d. (B) The synthesis of Ang II (•) was upregulated in PTEC that were incubated with IgA-HMC–conditioned medium after day 4. A rapid increase of IL-6 production () by PTEC was evident after 12 h after incubation with IgA-HMC–conditioned medium (P < 0.05). The IL-6 production peaked on the second day of culture and gradually declined on days 4 and 6. The expression of cleaved poly-(ADP-ribose)-polymerase (PARP) () increased significantly in PTEC that were incubated with IgA-HMC–conditioned medium after day 4. The results represent the mean ± SD of five separate experiments. *P < 0.05 versus data from day 0.
TNF- Increased IL-6 Production by PTEC
Recombinant TNF- significantly increased the IL-6 productionby PTEC at a concentration of 40 pg/ml (P < 0.05 Figure 6A).Incubation of PTEC with 40 pg/ml TNF- significantly increasedIL-6 production by 12 h and thereafter (Figure 6B). It shouldbe noted that the concentration of TNF- in the IgA-HMC–conditionedmedium (41 ± 8.6 pg/ml TNF- used for culturing PTEC)could stimulate the production of IL-6 by PTEC after 12 h ofculture.
Figure 6. (A) Recombinant TNF- significantly increased the IL-6 production by PTEC at a concentration of 40 pg/ml. (B) Incubation of PTEC with 40 pg/ml TNF- significantly increased IL-6 production by the 12 h and thereafter. (C) Recombinant IL-6 significantly increased the AT1R or AT2R expression by PTEC at concentrations of 5 and 40 ng/ml, respectively. (D) Recombinant IL-6 at 5 ng/ml significantly increased the expression of AT1R and AT2R receptors by PTEC after 12 and 48 h of culture, respectively. The results represent the mean ± SD of four separate experiments. *P < 0.05 versus data from day 0 or medium control.
IL-6 Upregulated Ang II Receptor Expression by PTEC
Recombinant IL-6 significantly increased the AT1R or AT2R expressionby PTEC at concentrations 5 and 40 ng/ml, respectively (P <0.05; Figure 6C). Recombinant IL-6 at 5 ng/ml significantlyincreased the expression of AT1R and AT2R by PTEC after 12 and48 h of culture, respectively (Figure 6D).
Binding of Ang II to PTEC Cultured with IgA-HMC–Conditioned Medium
Binding of [3H]Ang II to PTEC was specific and saturable (Figure 7A).The results from Scatchard plot analysis (Figure 7B) showedthat incubation of PTEC with IgA-HMC–conditioned mediumcaused a significant increase in Bmax of [3H]Ang II (medium452 ± 57 fmol/mg protein; IgA-HMC–conditioned medium582 ± 18 fmol/mg protein; n = 4; P < 0.05). Therewas no change in Kd after PTEC was incubated with IgA-HMC–conditionedmedium (medium 2.64 ± 0.12 nM; IgA-HMC–conditionedmedium 2.58 ± 0.6 nM; n = 4; P = 0.88), suggesting thatthere was no alteration in Ang II receptor affinity of PTECafter incubation with IgA-HMC–conditioned medium.
Figure 7. (A) Representative saturation binding curves of [3H]Ang II to PTEC that were cultured with (•) or without () IgA-HMC–conditioned medium. (B) Binding of [3H]Ang II to PTEC was specific and saturable. The results from Scatchard plot analysis showed that incubation of PTEC with IgA-HMC–conditioned medium caused a significant increase in Bmax of [3H]Ang II (P < 0.05). There was no change in Kd after PTEC were incubated with IgA-HMC–conditioned medium, suggesting that there was no alteration in Ang II receptor affinity of PTEC after incubation with IgA-HMC–conditioned medium.
The relative amount of AT receptors on PTEC was determined usingcompetitive displacement assay with [3H]Ang II in the presenceof losartan or PD123319 (Table 1). Losartan displaced 43.72± 8.8% of [3H]Ang II binding to PTEC and PD123319 displaced53.29 ± 6.79% of [3H]Ang II at day 0 of the culture.There was no significant difference in the relative amount ofAT receptors after incubation of PTEC with IgA-HMC–conditionedmedium for 2 d (43.62 ± 6.52% for losartan and 54.45± 7.24% for PD123319).
Table 1. Losartan- and PD123319-displaceable [3H]Ang II binding to PTEC after incubation with IgA-HMC–conditioned mediuma
Effect of Ang II on Ang II Receptor Expression and Cleaved PARP Expression in PTEC
The expression of AT1R, AT2R, and cleaved PARP was studied inPTEC that were incubated with increasing concentrations of AngII with or without antagonists to AT1R or AT2R. Ang II at aconcentration of 10–9 M significantly increased the AT1Rexpression (P < 0.05), and the enhanced AT1R expression wasblocked by losartan but not PD123319 (Figure 8A). Ang II atconcentration of 10–10 M significantly increased the AT2Rexpression (P < 0.05; Figure 8B). The increased expressionof AT2R by Ang II was blocked by PD123319 but not losartan.Furthermore, Ang II at a concentration of 10–11 M increasedthe expression of cleaved PARP (P < 0.05; Figure 8C). Theincreased apoptosis induced by Ang II as determined by the expressionof cleaved PARP was readily suppressed by PD123319 but not losartan.The role of AT2R in inducing cleaved PARP expression of PTECwas further supported by finding that exposure to increasingconcentrations of an AT2R agonist, CGP42112A, significantlyincreased the expression of cleaved PARP at concentrations of10–9 M (Figure 9).
Figure 8. Immunoblot analysis of the expression of AT1R, AT2R, and cleaved PARP in PTEC that were incubated with culture medium that contained increasing concentration of Ang II with or without addition of losartan (100 nM) or PD123319 (10 µM). (A) Ang II at concentrations of 10–9 M significantly increased the AT1R expression. The increased expression of AT1R induced by Ang II was blocked by losartan but not PD123319. (B) Ang II at concentrations of 10–10 M significantly increased the AT2R expression. The increased expression of AT2R induced by Ang II was blocked by PD123319 but not losartan. (C) Ang II at concentrations of 10–11 M significantly increased the cleaved PARP expression. The increased expression of cleaved PARP induced by Ang II was blocked by PD123319 but not losartan. The result represents the mean ± SD of five separate experiments.
Figure 9. Immunoblot analysis of cleaved PARP expression in PTEC that were incubated with culture medium that contained increasing concentrations of CGP42112A. CGP42112A at concentrations of 10–9 M significantly increased the expression of cleaved PARP. The results represent the mean ± SD of five separate experiments. *P < 0.05 versus medium control.
Signal Transduction Pathways Involved in the Activation of PTEC by IgA-HMC–Conditioned Medium Figure 10A depicts the detection of phospho-p42 and phospho-p44subunits of MAPK or phospho-PKC in lysates from PTEC that wereincubated previously with IgA-HMC–conditioned medium preparedfrom patients with IgAN or Ang II (10–10 M). Activationof either signal transduction pathway was reduced by AT1R blockadebut was enhanced after AT2R blockade. Activation of the p42/p44MAPK but not PKC pathway in PTEC that were preincubated withIgA-HMC–conditioned medium was blunted with addition ofCGP42112A at a concentration of 10–9 M (Figure 10B).
Figure 10. Detection of phospho-p42 and phospho-p44 subunits of mitogen-activated protein kinase (MAPK) or phospho-PKC in PTEC lysate after incubation with plain medium alone, IgA-HMC–conditioned medium prepared from patient with IgAN, or Ang II (10–10 M). (A) Activation of the p42/p44 MAPK and protein kinase C (PKC) pathways was detected in PTEC that were incubated with IgA-HMC–conditioned medium or Ang II. The activation was reduced by losartan but was enhanced by PD123319. (B) After incubation with IgA-HMC–conditioned medium that was prepared from patients with IgAN for 24 h in the presence of increasing concentrations of CGP42112A, activation of the p42/p44 MAPK but not the PKC pathway was blunted in PTEC that were incubated with IgA-HMC–conditioned medium in the presence of CGP42112A at concentrations of 10–9 M.
Role of AT1R and AT2R on sICAM-1 Synthesis and Cleaved PARP Expression in PTEC Cultured with IgA-HMC–Conditioned Medium
For studying the role played by individual Ang II receptor inthe inflammatory response and apoptosis of PTEC induced by IgA-HMC–conditionedmedium, PTEC were cultured with IgA-HMC–conditioned mediumin the presence of (1) losartan, (2) PD123319, (3) CGP42112A,or (4) first losartan then addition of PD123319 on day 2 afterculture. IgA-HMC–conditioned medium induced an inflammatoryresponse in PTEC shown by increased synthesis of sICAM-1 (Figure 11).Addition of PD123319 further amplified the synthesis ofsICAM-1. CGP421123A decreased the sICAM-1 synthesis inducedby IgA-HMC–conditioned medium after 2-d incubation. ThesICAM-1 synthesis induced by IgA-HMC–conditioned mediumwas aborted by either losartan alone or losartan supplementedsequentially with PD123319. PTEC that were incubated with IgA-HMC–conditionedmedium exhibited a gradual increase of cleaved PARP. The expressionof cleaved PARP was amplified further by either losartan orCGP42112A. PD123319 suppressed the expression of cleaved PARP.The increased cleaved PARP expression after early addition oflosartan was subsequently blunted with sequential addition ofPD123319.
Figure 11. Concentration of soluble intercellular adhesion molecule-1 (sICAM-1) in culture supernatant (A) and the expression of cleaved PARP (B) from PTEC that were incubated with IgA-HMC–conditioned medium that was prepared from patients with IgAN for 12 h to 6 d with or without addition of losartan (100 nM), PD123319 (10 µM), CGP42112A (10–7 M), or losartan (100 nM; 30 min before culture) followed by PD123319 (10 µM, day 2 after culture). The results represent the mean ± SD of five separate experiments. There was no change in sICAM-1 concentration or expression of cleaved PARP in PTEC that were incubated with IgA-HMC–conditioned medium that was prepared from control subjects for 12 h to 6 d (data not shown).
The RAS is involved in the development of progressive renalfibrosis in IgAN (22,23). Local Ang II hyperactivity has beendemonstrated in patients with IgAN (24). We demonstrated previouslythat IgA from patients with IgAN is capable of upregulatingthe TGF- production via increased Ang II release by HMC afterbinding to polymeric IgA (pIgA) (25). We have also demonstratedan altered AT1R expression in HMC in response to raised intrarenalAng II in IgAN (17). Although there is an immediate downregulationof mesangial AT1R expression in IgAN after an acute exposureto pIgA, this adaptive mechanism is lost with chronic exposureto Ang II released by pIgA in IgAN. Furthermore, there is adefective counterbalance of AT1R by AT2R as the intrarenal AngII concentration is insufficient to upregulate the mesangialAT2R expression in IgAN. Hence, the imbalance of AT1R and AT2Ractivities in HMC after exposure to pIgA may play a significantpathogenetic role in the glomerular inflammation in IgAN. Theseverity of tubulointerstitial damage in IgAN correlates closelywith the declining renal function and the long-term clinicaloutcome (26). Most recently, we demonstrated no binding of IgAto and absence of IgA receptors in PTEC (19). Other than thetubulotoxic effect of proteinuria, the tubulointerstitial damagein IgAN is an indirect sequel as a result of a novel glomerulotubularcross-talk mediated through soluble factors released by HMCafter IgA deposition (19).
In this study, we demonstrated that there is constitutive expressionof AT1R and AT2R in renal tubules with increased expressionin IgAN. The glomerular AT1R was reduced in IgAN, whereas therewas no change in the expression of glomerular AT2R (data notshown), confirming our previous results (17). In vitro studiesshowed no change in Ang II receptor expression when PTEC werecultured with IgA, yet these receptors were upregulated afterincubation with IgA-HMC–conditioned medium prepared fromthe same patient with IgAN. These findings further support thenotion that disturbance of tubular RAS in IgAN is an indirectevent through signals released by HMC after IgA deposition.Dissociation in tubular expression of Ang II receptors is evidentby the time course of in vitro experiments of PTEC that werecultured with IgA-HMC–conditioned medium that was preparedfrom patients with IgAN. AT1R expression was upregulated inthe first 2 d and decreased gradually to the basal level towardday 6. In contrast, significant increase in the AT2R expressionwas detected only after day 2 and thereafter. Results from the[3H]Ang II binding assay demonstrate that IgA-HMC–conditionedmedium increased the Ang II receptor density without affectingthe affinity or the relative proportion of AT1R and AT2R onPTEC after being cultured with IgA-HMC–conditioned mediumfor 2 d. Simultaneously, there was also increase in IL-6 productionas early as 12 h after culture. The Ang II release and the expressionof cleaved PARP were increased from day 2, associated with increasedAT2R expression induced by IgA-HMC–conditioned medium.Our data suggest that although the initial Ang II level in PTECthat were cultured with IgA-HMC–conditioned medium wasnot high enough to upregulate AT receptor expression immediately,the concentration of TNF- in the IgA-HMC–conditioned mediumcould induce a rapid and early increase of IL-6 level that inturn upregulates AT1R and AT2R expression in "primed PTEC."Increased Ang II production through increased Ang II and itsreceptor binding could further upregulate the production ofAng II. We also demonstrated that IgA-HMC–conditionedmedium induced cleaved PARP expression in PTEC as measured byan apoptotic marker, the cleaved PARP. We found that cleavedPARP was amplified by AT1R antagonist but abolished by AT2Rantagonist. Normally, apoptosis mediated by AT2R counterbalancesgrowth-stimulatory effects of Ang II through AT1R in renal proximaltubular cells (27,28). Pharmacologic blockade of the RAS, eitherby ACEI or AT1R antagonist, may retard the progression to glomerulosclerosis(29). AT2R modulates the actions of chemokine RANTES (30), matrixprotein osteopontin (13), and nitric oxide (31), and blockadeof the AT2R may reduce kidney injury. Our data revealed thatAT2R antagonist could block the cleaved PARP expression in PTECafter incubation with IgA-HMC–conditioned medium. Therole of AT2R in inducing apoptosis in PTEC was strengthenedfurther by our observation that expression of cleaved PARP wasincreased with an AT2R agonist (CGP42112A).
The MAPK pathway is of pivotal importance in cell mitogenesisand hypertrophy. Ang II increases the tyrosine kinase and phosphataseactivities (32). In addition to MAPK, phospholipase C- (PLC-)can be phosphorylated and results in the stimulation of PKCby Ang II (33). Our study showed that IgA-HMC–conditionedmedium from patients with IgAN or exogenous Ang II alone activatedp42/p44 MAPK and PKC pathways in PTEC. The expression of phospho-p42and phospho-p44 subunits of MAPK as well as phospho-PKC wasenhanced by the AT2R antagonist but was reduced by the AT1Rantagonist under an identical experimental setting. However,adding an AT2R agonist to PTEC that were preincubated with IgA-HMC–conditionedmedium blunted the activation of phospho-p42 and phospho-p44subunits of MAPK but not phospho-PKC, indicating that AT2R onlycounteracted the AT1R-mediated signal transduction pathway ofMAPK but not PKC.
It seems that the temporal sequence of Ang II receptor expressionwill determine the fate of PTEC after exposure to mediatorsreleased from IgA-activated HMC. On the basis of our recentdata (19) and our study, we propose a hypothetical mechanismof IgA-induced tubulointerstitial injury in IgAN (Figure 12).HMC develop inflammatory injury when chronically exposed topathogenic IgA from IgAN as a result of the loss of an adaptivedownregulation of the AT1R. There is also increased releaseof mediators by HMC that include TNF- and Ang II. In PTEC, HMC-derivedTNF- first upregulates IL-6 production by PTEC. The releasedIL-6 in turn increases the AT1R expression and also enhancesAng II production gradually through a positive feedback loop.The early interaction between Ang II and AT1R activates bothPKC and MAPK pathways, leading to subsequent inflammatory responses.This is supported by our finding of increased release of sICAM-1from PTEC that were incubated with IgA-HMC–conditionedmedium that is readily abolished by losartan. The early phaseof AT1R-dependent inflammation is followed by subsequent upregulationof AT2R expression with continued Ang II release. The interactionbetween Ang II and AT2R then will lead to cleaved PARP expressionthrough downregulation of the MAPK pathway. One of the proposedmechanisms of downregulation of MAPK is the increased activationof MAPK phosphatase-1 (34), which will counterbalance the AT1R-inducedMAPK activation. Initial inflammatory responses in tubular cellsare followed by later stages of tubular lysis or apoptosis,depending on the balance between activation of the MAPK pathwayby AT1R and the suppression of the MAPK by an AT2R-dependentmechanism. If our hypothesis is correct, then sequential suppressionof AT1R and AT2R could be a better way to reduce the IgA-inducedPTEC damage. Indeed, this novel approach is supported by ourpreliminary in vitro data that sequential administration oflosartan followed by PD123319 is effective in reducing earlyinflammatory responses (determined by sICAM-1) and preventinglate onset of apoptosis caused by AT2R overexpression (determinedby cleaved PARP).
Figure 12. Hypothetical model of tubular regulation of Ang II receptors in IgAN. HMC that are exposed to pathogenic IgA from patients with IgAN stimulate the synthesis and release of TNF- and Ang II. An immediate downregulation of mesangial AT1R expression will ameliorate the proliferative and inflammatory changes induced by Ang II released by IgA. However, this adaptive downregulation of AT1R gradually disappears after chronic exposure to pathogenic IgA (@) and is likely to permit the development of proliferative and inflammatory processes in the glomerulus subsequently. In the tubular lumen, TNF- released by HMC first increases the IL-6 production. The IL-6 in turn increases the AT1R expression and then gradually enhances Ang II production. The interaction of Ang II and early expressed AT1R will activate the PKC and MAPK pathways, leading to inflammatory responses. The early phase of AT1R-dependent inflammation is followed by subsequent upregulation of AT2R expression with continued release of Ang II. The interaction between Ang II and AT2R leads to apoptosis through downregulation of the MAPK pathway, and this will counterbalance the AT1R-induced MAPK activation (double arrow denotes interaction).
In conclusion, our in vitro model suggests that dysregulationof the RAS in PTEC is due to the effects of cytokine releasefrom HMC after IgA incubation. This dysregulation of the RASin PTEC is pivotal in the pathogenesis of tubulointerstitialinjury in IgAN. The tubulointerstitial injury could be amelioratedby a novel therapeutic approach of sequential targeting theAT1R and AT2R expression. Further in vivo study is warrantedto test the hypothesis.
Acknowledgments
This study was supported by the Research Grant Committee (HongKong SAR; HKU7403/04) and the Seed Funding for Basic Researchof the University of Hong Kong. L.Y.Y.C. is partly supportedby Fresenius Medical Supplies and J.C.K.L. is supported by theL & T Charitable Fund and INDOCAFE.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication December 21, 2004.
Accepted for publication April 30, 2005.
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Abstract
Introduction
in the IgA-HMC medium was determined using ELISA (R&D Systems, Minneapolis, MN). 
, or 10 ng/ml IL-6) for 96 h. After culture, the supernatants were stored at –70°C before assay for Ang II. Ang II was measured by an enzyme immunoassay using an anti–Ang II mAb labeled with acetylcholinesterase (SPI bio, Massy Cedex, France). The detection limit was 1 pg/ml, and the intra-assay coefficient of variation was 7% (17). 
40 pg/ml (P < 0.05 Figure 6A). Incubation of PTEC with 40 pg/ml TNF-
(PLC-
) increased significantly in PTEC after incubation with IgA-HMC–conditioned medium for 4 and 6 d. (B) The synthesis of Ang II (•) was upregulated in PTEC that were incubated with IgA-HMC–conditioned medium after day 4. A rapid increase of IL-6 production (
) by PTEC was evident after 12 h after incubation with IgA-HMC–conditioned medium (P < 0.05). The IL-6 production peaked on the second day of culture and gradually declined on days 4 and 6. The expression of cleaved poly-(ADP-ribose)-polymerase (PARP) (
