Polymeric IgA1 from Patients with IgA Nephropathy Upregulates Transforming Growth Factor-β Synthesis and Signal Transduction in Human Mesangial Cells via the Renin-Angiotensin System

Patient Consent and Materials

The study was conducted in accordance with the Declaration of Helsinki and was approved by the institutional ethics committee for studies with human subjects. All subjects (patients and healthy control subjects) gave their written informed consent for serum and tissue collections.

RPMI 1640 medium and FCS were obtained from Life Technologies (Rockville, MD). Jacalin-agarose was obtained from Pierce (Rockford, IL). The standard and antibodies for the TGF-β ELISA were obtained from R & D Systems (Minneapolis, MN). The Superose fast protein liquid chromatography (FPLC) column was obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Consumables for electrophoresis were obtained from Bio-Rad Laboratories (Hercules, CA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Human Mesangial Cell Cultures

Isolation and characterization of human mesangial cells (HMC) were performed as previously described (15). Glomeruli were prepared from the cortex of a human cadaveric kidney judged to be unsuitable for transplantation or from the intact pole of a kidney removed for treatment of a circumscribed tumor. Histologic examinations of the kidney samples revealed no renal pathologic conditions. Glomerular cells were grown in RPMI 1640 medium supplemented with glutamine (2 mM), HEPES (10 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and 12% FCS, in an atmosphere of 5% CO₂/95% air. Mesangial cells have a stellate appearance and grow in clumps. They exhibit a network of intracellular myosin fibrils, and they contract in the presence of 1 nM AngII. Mesangial cells from a single nephrectomy sample at passage 4 to 7 were used in our experiments.

Purification and Characterization of pIgA1 and Monomeric IgA1 with Jacalin-Agarose Affinity Chromatography and FPLC

IgA1 was purified, with a jacalin-agarose affinity column, from five randomly selected patients with mild IgAN (grade 1) in clinical quiescence. The severity of the renal pathologic condition was classified as grade 1, 2, or 3, as described previously (16); grade 1 indicated mesangial proliferation with no crescents, sclerosis, or tubulointerstitial changes. These five patients exhibited normal creatinine clearance values (>80 ml/min per 1.73 m²), with proteinuria of <0.5 g/d. IgA1 was fractionated at room temperature with an FPLC system (Pharmacia, Uppsala, Sweden), as previously described (15). Briefly, monomeric IgA1 (mIgA1) and pIgA1 were separated with FPLC after jacalin-agarose affinity chromatography. The identity of IgA after FPLC was confirmed with immunoblotting and SDS-polyacrylamide gel electrophoresis. Every IgA preparation from each patient was separated into mIgA1 and pIgA1 according to molecular mass. Chromatography fractions with molecular masses of 300 to 1000 kD were considered high-molecular mass IgA or pIgA1, whereas fractions with molecular masses of 50 to 300 kD were considered low-molecular mass IgA or mIgA1. Distinct mIgA1 and pIgA1 fractions were separately prepared from each patient for subsequent incubation studies. The IgG contents in the fractions were measured with an anti-IgG ELISA. The fractions were dialyzed, concentrated to 2 ml with Centriprep cartridges (Amicon, Beverly, MA), and stored at −70°C until use. The purity of the IgA1 fractions was confirmed with Coomassie blue and silver staining after SDS-polyacrylamide gel electrophoresis and ELISA (15). The endotoxin levels in the IgA preparations were determined with the QCL-1000 Limulus amebocyte lysate kit (BioWhittaker, Walkersville, MD). The levels in all preparations were <10 EU/ml.

Treatment of HMC with Different IgA Preparations or RAS Blockers

HMC were grown to logarithmic phase and were harvested with 0.05% trypsin. The trypsin-recovered cells were subcultured onto six-well culture plates (1 × 10⁶ cells/well). The cells were arrested with 24 h of growth in RPMI 1640 medium with 0.5% FCS, and that medium (with 0.5% FCS) was used for all subsequent experiments. The cells were exposed to IgA preparations or RAS blockers at increasing concentrations (0, 0.125, 0.25, 0.5, 1, or 2 mg/ml for IgA and 0, 1, 10, 100, 1000, and 10000 nM for captopril or losartan) for 6 or 24 h at 37°C, in replicate. Preliminary experiments demonstrated that these incubation periods were convenient and were associated with optimal synthesis of mRNA for renin or TGF-β (6 h) and protein for TGF-β (24 h). Cells were harvested for RNA extraction and nuclear extract preparation. Culture supernatants were collected for determination of TGF-β and/or AngII levels.

Treatment of HMC with AngII, with or without Losartan

HMC were grown and then arrested with 24 h of growth in RPMI 1640 medium with 0.5% FCS as described above. The cells were then exposed to AngII at different concentrations (0.01 to 100 nM), in the presence or absence of 100 nM losartan, for 6 h at 37°C, in triplicate. Culture supernatants were collected for determination of TGF-β levels.

RNA Extraction, cDNA Synthesis, and Renin/TGF-β Gene Expression in Cultured HMC

Specific primers for renin, TGF-β, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed on the basis of known GenBank sequences (renin, accession number XM_010626; TGF-β₁, accession number X02812; GAPDH, accession number X01677). The sequences of the primer pairs were as follows: renin, sense primer, 5′-TCATCAAGACTGGTGTCTGG-3′; antisense primer, 5′-GGGCCAAGGCGAAGCCAA-3′; TGF-β, sense primer, 5′-GCCCTGGACACCAACTATTGCT-3′; antisense primer, 5′-AGGCTCCAAATGTAGGGGCAGG-3′; GAPDH, sense primer, 5′-ACCACAGTCCAT-GCCATCAC-3′; antisense primer, 5′-TCCACCACCCTGTTGCTGTA-3′. We performed reverse transcription-PCR as previously described (17), using the following profile: first cycle, 94°C for 3 min, 55°C for 1 min, and 72°C for 1 min; cycles 2 to 30, 95°C for 45 s, 55°C for 40 s, and 72°C for 45 s; final cycle, 94°C for 1 min and 72°C for 10 min. The PCR products from the renin or TGF-β and control (GAPDH) amplicons were mixed, separated on 1.5% (wt/vol) agarose gels, and stained with ethidium bromide, and the gel images were captured and analyzed with the Gel Doc 1000 gel documentation system and Quantity One software (Bio-Rad Laboratories, Hercules, CA). We semiquantitatively assessed the renin and TGF-β mRNA yields as renin/GAPDH and TGF-β/GAPDH ratios. We took all necessary steps to ensure the validity of the results, as described previously (18).

Determination of TGF-β and AngII Levels in Culture Supernatants

A sandwich ELISA (R & D Systems) was used to measure TGF-β levels in the culture supernatants. The ELISA for TGF-β measured both the active and latent forms. The sensitivity limit was 5 pg/ml. All samples were assayed at the same time, to avoid interbatch variations. The intra-assay coefficient of variation was <7.3% (19). AngII levels were measured with an enzyme immunoassay using an anti-AngII Fab’ mAb labeled with acetylcholinesterase (20) (SPI Bio, Massy Cedex, France). The minimal detectable concentration was 1 pg/ml, and the intra-assay coefficient of variation was 7%.

Determination of Effects of IgA Treatment or RAS Blockade on Smad Activation by TGF-β in Cultured HMC

Smad activation in HMC with IgA stimulation, with or without RAS blockade, was examined with an electrophoretic mobility shift assay, performed as described previously (21). Briefly, ³²P-labeling of Smad oligonucleotides (5′-TTTCTCAGACAGTCTGTCTGCC-3′ and 5′-GGCAGACAGACTGTCTGAGAAA-3′) (22) was performed with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [γ-³²P]ATP (3000 Ci/mmol). The single-stranded oligonucleotides were annealed for 2 h while the temperature was decreased from 95°C to 25°C. Labeled DNA was separated from unincorporated radioactivity. Nuclear extracts were prepared with a kit for the isolation of nuclei, according to the instructions provided by the manufacturer (Sigma). Binding reactions were performed by adding 2 μg of nuclear protein to 20 μl of binding buffer containing [γ-³²P]ATP-labeled Smad oligonucleotide probes. Samples were incubated at room temperature for 25 min, fractionated by electrophoresis, transferred to 3 MM paper, dried, and exposed to Hyperfilm-MP x-ray film at −70°C, with an intensifying screen. The results were quantified with a scanning densitometer. To assess the specific binding to nuclear extracts of the Smad probes, competitive assays were performed with a 100-fold molar excess of unlabeled oligonucleotides. Where indicated, supershift assays were performed with antibodies against Smad 2, 3, and 4 (Santa Cruz Biotechnology, Santa Cruz, CA).

Confirmation that pIgA1 from Patients with IgAN Exerts Upregulatory Effects on the RAS in HMC

The stimulatory effects of pIgA1 from patients with IgAN on the RAS in HMC were further studied with 30 Chinese patients (17 male and 13 female patients; age range, 24 and 61 yr; mean age, 39 yr) with clinical and renal immunopathologic diagnoses of primary IgAN. The histologic diagnoses had been made at least 18 mo before the study, and the serum creatinine levels of the patients had remained stable during the previous 12 mo. Proteinuria ranged from 0.1 to 4.9 g/d. Systemic lupus erythematosus, Henoch-Schönlein purpura, and hepatic disease were excluded on the basis of detailed clinical history analyses, examination results, and negative results of laboratory tests for hypocomplementemia, anti-DNA antibody, and hepatitis B virus surface antigen. The mean endogenous creatinine clearance was 75.2 ± 26.3 ml/min per 1.73 m². Twenty milliliters of blood were collected from each patient during clinical quiescence (a period free of macroscopic hematuria and mucosal infection, with urinary erythrocyte counts of <10,000 cells/ml in uncentrifuged urine). The serum was separated and frozen at −20°C until isolation of IgA1 with a jacalin-agarose affinity column. Serum IgA levels were determined with nephelometry.

Thirty healthy subjects (16 male and 14 female patients) matched with respect to age and race, with no microscopic hematuria or proteinuria, were used as healthy control subjects. Patients with Henoch-Schönlein purpura, lupus nephritis, minimal-change nephropathy, or membranous nephropathy were recruited as disease control subjects. Serum samples were similarly collected from those individuals for IgA purification.

Statistical Analyses

All data (for patients or cell culture experiments) were expressed as means ± SD. Intergroup differences for continuous variables were assessed with the unpaired t test, except for concentrations exceeding the in vivo relevance (for pIgA1) or the therapeutic level (for captopril or losartan). The renin/TGF-β mRNA expression and TGF-β protein synthesis in cultured cells after exposure to different concentrations of IgA preparations or AngII blockers were analyzed with multivariate ANOVA for repeated measures. All P values presented are two-tailed, and significance was defined as P < 0.05.

Expression of Renin mRNA in Cultured HMC

Inhibition of ACE after incubation of the HMC with captopril led to increased renin gene expression in a dose-dependent manner, peaking at a concentration of 1000 nM (Figure 1). Blockade of AT₁ receptors also resulted in a stepwise increase in renin gene expression in HMC, but the effect peaked at a lower concentration of 100 nM. At comparable molar concentrations, the stimulatory effect of the ACEI (captopril) was stronger than that of the AT₁ receptor antagonist (losartan) (P < 0.001). More interestingly, pIgA1 from patients with IgAN also significantly upregulated renin gene expression in a dose-dependent manner, reaching 83 and 151% increases at concentrations of 0.5 and 2 mg/ml, respectively (Figure 1). Although mIgA1 from patients with IgAN upregulated renin gene expression in a similar, dose-dependent manner, the magnitude of the effect was smaller; renin gene expression in HMC increased by only 22 and 54% with mIgA1 at concentrations of 0.5 and 2 mg/ml, respectively.

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Figure 1. Upregulation of renin mRNA in cultured human mesangial cells (HMC) incubated with captopril, losartan, or IgA1 preparations from patients with IgA nephropathy (IgAN) (P < 0.0001, multivariate ANOVA). Measurements of renin mRNA levels in HMC incubated with captopril at different concentrations differed significantly (P < 0.01). Measurements of renin mRNA levels in HMC incubated with losartan at different concentrations differed significantly (P < 0.05). Measurements of renin mRNA levels in HMC incubated with polymeric IgA1 (pIgA1) at different concentrations differed significantly (P < 0.01). Measurements of renin mRNA levels in HMC incubated with monomeric IgA1 (mIgA1) at different concentrations differed significantly, except for measurements at 0.5 and 1 mg/ml and those at 1 and 2 mg/ml (P < 0.01). The results represent the mean ± SD of five separate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Expression of TGF-β mRNA and Protein in Cultured HMC

With decreased binding of AngII to its receptors on HMC, produced with either reduction of AngII synthesis with captopril or blockade of AT₁ receptors with losartan, TGF-β gene expression was decreased in a dose-dependent manner, rapidly reaching a trough at concentrations between 1 and 10 nM (Figure 2a). In contrast, pIgA1 from patients with IgAN significantly upregulated TGF-β gene expression in a dose-dependent manner, with 138 and 213% increases at concentrations of 0.5 and 2 mg/ml, respectively (Figure 2a). Again, mIgA1 from patients with IgAN upregulated TGF-β gene expression in a similar, dose-dependent manner, but to a lesser extent. TGF-β gene expression in HMC was increased by only 43 and 73% with mIgA1 at concentrations of 0.5 and 2 mg/ml, respectively.

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Figure 2. (a) Effects on TGF-β mRNA expression in cultured HMC incubated with captopril, losartan, or IgA1 preparations from patients with IgAN (P < 0.0001, multivariate ANOVA). Measurements of TGF-β mRNA levels in HMC incubated with captopril at 0 and 1 nM differed significantly from measurements at higher concentrations (P < 0.01). Measurements of TGF-β mRNA levels in HMC incubated with losartan at different concentrations were significantly lower than the baseline measurements without losartan (P < 0.01). Measurements of TGF-β mRNA levels in HMC incubated with pIgA1 at different concentrations differed significantly (P < 0.01). Measurements of TGF-β mRNA levels in HMC incubated with mIgA1 at different concentrations differed significantly, except for measurements at 0 and 0.125 mg/ml and those at 0.25 and 0.5 mg/ml (P < 0.05). The results represent the mean ± SD of five separate experiments. (b) Effects on TGF-β protein synthesis in cultured HMC incubated with captopril, losartan, or IgA1 preparations from patients with IgAN (P < 0.0001, multivariate ANOVA). Measurements of TGF-β levels in supernatants from HMC incubated with captopril at 0 and 1 nM differed significantly from measurements at higher concentrations (P < 0.01). Measurements of TGF-β levels in supernatants from HMC incubated with losartan at different concentrations were significantly lower than the baseline measurements without losartan (P < 0.01). Measurements of TGF-β levels in supernatants from HMC incubated with pIgA1 at different concentrations differed significantly, except for measurements at 0 and 0.125 mg/ml and those at 0.125 and 0.25 mg/ml (P < 0.01). Measurements of TGF-β levels in supernatants from HMC incubated with mIgA1 at different concentrations differed significantly, except for measurements at 1 and 2 mg/ml (P < 0.01). The results represent the mean ± SD of five separate experiments.

The findings regarding TGF-β protein levels in supernatants from cultured HMC paralleled those regarding TGF-β mRNA levels. Incubation with captopril or losartan decreased TGF-β protein synthesis by HMC in a dose-dependent manner, rapidly reaching a trough at concentrations between 1 and 10 nM (Figure 2b). Again, pIgA1 from patients with IgAN significantly upregulated TGF-β protein synthesis in a dose-dependent manner, reaching 51 and 122% increases at concentrations of 0.5 and 2 mg/ml, respectively. mIgA1 from patients with IgAN upregulated TGF-β protein synthesis to a lesser extent; TGF-β gene expression in HMC was increased by only 26 and 44%, respectively.

Exposure of cultured HMC to AngII increased TGF-β synthesis in a dose-dependent manner (Figure 3). The TGF-β synthesis induced by preincubation with AngII was significantly decreased in the presence of losartan (100 nM), reaching 63 and 75% reductions at concentrations of 0.1 and 0.01 nM, respectively. A similar reduction of TGF-β synthesis induced by AngII was not observed in HMC exposed to captopril (data not shown).

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Figure 3. Enhanced TGF-β synthesis in cultured HMC incubated with increasing concentrations of angiotensin II (AngII) (P < 0.0001, multivariate ANOVA). Incubation with losartan (100 nM) resulted in a marked reduction in the synthesis of TGF-β. The results represent the mean ± SD of three separate experiments.

Stimulatory Effects of IgA on TGF-β Synthesis in Cultured HMC Preincubated with Optimal Doses of an ACEI or a AT₁ Receptor Antagonist

We then investigated the stimulatory effects of IgA (from patients with IgAN) on TGF-β synthesis in cultured HMC that had been preincubated with an optimal dose of captopril or losartan for 4 h. Under baseline conditions, captopril at a concentration of 100 nM significantly reduced TGF-β gene expression (0.243 ± 0.015 versus 0.266 ± 0.019, P < 0.005) and TGF-β synthesis (250.6 ± 10.0 versus 336.1 ± 36.8 pg/ml, P < 0.0001), compared with growth-arrested HMC incubated with culture medium alone (Figure 4). Similarly, losartan at a concentration of 100 nM significantly reduced TGF-β gene expression (0.226 ± 0.013 versus 0.266 ± 0.019, P < 0.0001) and TGF-β synthesis (226.3 ± 13.1 versus 336.1 ± 36.8 pg/ml, P < 0.0001), compared with growth-arrested HMC. However, pIgA1 isolated from patients with IgAN was able to reverse part of the suppressive effect of RAS blockade on TGF-β synthesis, in a dose-dependent manner (Figure 4). The antagonistic effect of pIgA1 on the suppression of TGF-β synthesis with RAS blockade was significantly smaller in HMC that had been pretreated with losartan than in HMC that had been pretreated with captopril. These findings support a better suppressive effect on TGF-β synthesis of losartan, compared with captopril, at comparable molar concentrations.

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Figure 4. (a) Stimulatory effects of IgA1 on TGF-β gene expression in cultured HMC that had been preincubated with an optimal dose of an angiotensin-converting enzyme inhibitor (ACEI) or an AT₁ receptor antagonist. Expression of TGF-β mRNA in cultured HMC was suppressed with either the ACEI or the AT₁ receptor antagonist, but suppression was reversed with pIgA1. pIgA1 (≥0.5 mg/ml) significantly increased TGF-β mRNA levels in HMC that had been preincubated with captopril (100 nM), compared with measurements with lower concentrations of pIgA1 (P < 0.01). The magnitude of TGF-β mRNA induction by mIgA1 in HMC that had been preincubated with captopril (100 nM) or losartan (100 nM) was small. The results represent the mean ± SD of five separate experiments. (b) Stimulatory effects of IgA1 on TGF-β synthesis in cultured HMC that had been preincubated with an optimal dose of an ACEI or an AT₁ receptor antagonist. TGF-β synthesis by cultured HMC was suppressed with either the ACEI or the AT₁ receptor antagonist, but suppression was reversed with pIgA1. pIgA1 significantly increased supernatant TGF-β levels in HMC that had been preincubated with captopril (100 nM) and the measurements differed significantly, except for measurements at 0 and 0.125 mg/ml (P < 0.01). The magnitude of TGF-β synthesis induced by mIgA1 in HMC that had been preincubated with captopril (100 nM) or losartan (100 nM) was small. The results represent the mean ± SD of five separate experiments.

Effects of RAS Blockade in Cultured HMC Preincubated with IgA

Next we preincubated HMC with an optimal dose of IgA for 4 h, to stimulate the RAS. pIgA1 (0.5 mg/ml) produced greater renin gene expression than did mIgA1, at a equivalent concentration, or control medium (P < 0.0001) (Figure 5a). The renin mRNA levels were further increased when HMC that had been preincubated with pIgA1 were treated with captopril (100 nM), reaching levels similar to those in HMC treated with captopril alone (100 nM). However, a further increase in renin mRNA levels was not observed when HMC that had been preincubated with pIgA1 were treated with losartan (100 nM). Parallel to the findings regarding renin gene expression, pIgA induced greater AngII synthesis than did mIgA, at an equivalent concentration, or control medium (P < 0.0001) (Figure 5b). However, AngII synthesis was significantly decreased when HMC that had been preincubated with pIgA1 were treated with captopril or losartan (100 nM). Similarly, the TGF-β synthesis induced by pIgA1 was significantly greater than that induced by mIgA1 or control medium (P < 0.0001) (Figure 5c). The TGF-β levels decreased significantly when HMC that had been preincubated with pIgA1 were treated with captopril (100 nM). However, an additional decrease in TGF-β levels in the supernatant was observed when HMC that had been preincubated with pIgA were treated with a comparable molar concentration of losartan (100 nM). Similarly, TGF-β gene expression induced by pIgA1 was significantly greater than that induced by mIgA1 or control medium (P < 0.0001) (data not shown). Again, the TGF-β mRNA levels were significantly decreased when HMC that had been preincubated with pIgA1 were treated with captopril or losartan (data not shown).

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Figure 5. (a) Renin gene expression after renin-angiotensin system (RAS) blockade in cultured HMC that had been preincubated with IgA1 preparations. Incubation with captopril (100 nM) or losartan (100 nM) upregulated the renin gene expression in resting HMC. *P < 0.05, compared with growth-arrested HMC. **P < 0.001, compared with HMC incubated with losartan. Incubation with captopril (100 nM) further upregulated the renin gene expression in HMC that had been preincubated with pIgA1 (0.5 mg/ml). A similar effect was not observed with losartan (100 nM). ^#P < 0.05, compared with pIgA1-treated HMC that were subsequently incubated with captopril. Incubation with captopril (100 nM) or losartan (100 nM) upregulated the renin gene expression in HMC that had been preincubated with mIgA1 (0.5 mg/ml). †P < 0.05, compared with HMC incubated with mIgA1 alone. ‡P < 0.001, compared with mIgA1-treated HMC that were subsequently incubated with captopril. The results represent the mean ± SD of five separate experiments. (b) Effects of RAS blockade on AngII synthesis in cultured HMC that had been preincubated with IgA1 preparations. Incubation with losartan (100 nM) decreased the synthesis of AngII by resting HMC. *P < 0.05, compared with growth-arrested HMC. Incubation with captopril (100 nM) or losartan (100 nM) downregulated the synthesis of AngII in HMC induced by preincubation with pIgA1 (0.5 mg/ml). ^@P < 0.05, compared with HMC incubated with pIgA1 alone. Incubation with losartan (100 nM) downregulated the synthesis of AngII in HMC induced by preincubation with mIgA1 (0.5 mg/ml). †P < 0.05, compared with HMC incubated with mIgA1 alone. The results represent the mean ± SD of five separate experiments. (c) Effects of RAS blockade on TGF-β synthesis in cultured HMC that had been preincubated with IgA1 preparations. Incubation with captopril (100 nM) or losartan (100 nM) downregulated the synthesis of TGF-β in HMC induced by preincubation with pIgA1. ^##P < 0.01, compared with HMC incubated with pIgA1 alone. ^#P < 0.05, compared with pIgA1-treated HMC that were subsequently incubated with captopril. Incubation with captopril (100 nM) or losartan (100 nM) downregulated the synthesis of TGF-β in HMC induced by preincubation with mIgA1 (0.5 mg/ml). †P < 0.05, compared with HMC incubated with mIgA1 alone. The results represent the mean ± SD of five separate experiments.

Identical experiments performed with mIgA1 demonstrated similar patterns for renin gene expression and AngII and TGF-β protein synthesis, but the magnitude of the stimulatory effect was smaller than with pIgA1. For HMC cultured with control medium, the addition of captopril or losartan enhanced renin gene expression (Figure 5a). This was associated with a mild reduction in AngII synthesis (for losartan) and an insignificant decrease in TGF-β expression (Figure 5, b and c).

Confirmation that pIgA1 from Patients with IgAN Exerts Upregulatory Effects on the RAS in HMC

The stimulatory effects of pIgA1 from patients with IgAN on the RAS in HMC were investigated for 30 patients, compared with 30 healthy control subjects comparable with respect to age and gender. The serum IgA levels among patients with IgAN (2.94 ± 1.44 g/L) were significantly higher than those among healthy control subjects (1.88 ± 0.89 g/L, P < 0.001). No IgG or IgM was detected in the mIgA1 fractions and IgG represented 0.1% of protein in total IgA1 fractions, as measured by ELISA. Studies of IgA in the FPLC fractions with IgA ELISA demonstrated that mIgA1 amounted to 90% of total IgA1 from either control subjects or patients (data not shown).

HMC incubated with equivalent concentrations of pIgA1 from patients or control subjects exhibited significantly greater renin gene expression, compared with cells incubated with culture medium alone (Figure 6a). A smaller but significant stimulatory effect was observed with mIgA1 isolated from patients but not healthy control subjects. Although the IgA was isolated during clinical quiescence, pIgA1 from patients induced significantly greater renin gene expression (23%) than did pIgA1 from healthy control subjects (P < 0.0001). Similarly, pIgA1 from patients induced greater protein synthesis of AngII (56%) and TGF-β (37%) than did pIgA1 from healthy control subjects (P < 0.0001) (Figure 6, b and c). mIgA1 from patients also induced greater AngII and TGF-β protein synthesis than did mIgA1 from healthy control subjects (P < 0.01).

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Figure 6. Significant upregulatory effects of pIgA1 (0.5 mg/ml) from patients with IgAN (n = 30), compared with pIgA1 (0.5 mg/ml) from healthy control subjects (n = 30), on renin gene expression (a), AngII protein synthesis (b), and TGF-β protein synthesis (c) in cultured HMC. The results represent the mean ± SD.

HMC were incubated with pIgA1 from patients in various disease groups, to explore whether IgA preparations from patients with other glomerulonephritides would also upregulate the RAS in HMC. As indicated in Figure 7, there were significant increases in AngII and TGF-β production after culturing of HMC with pIgA1 preparations from patients with quiescent IgAN, compared with healthy control subjects or disease control subjects, including subjects with Henoch-Schönlein purpura, lupus nephritis, minimal-change nephropathy, or membranous nephropathy. In contrast, AngII and TGF-β production after culturing of HMC with pIgA1 preparations from disease control subjects did not differ from the results with preparations from healthy control subjects.

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Figure 7. AngII (a) and TGF-β (b) synthesis in cultured HMC incubated with pIgA1 (0.5 mg/ml) from normal control subjects (n = 15) or subjects with IgAN (n = 15), Henoch-Schönlein purpura (n = 10), lupus nephritis (n = 10), minimal-change nephropathy (n = 10), or membranous nephropathy (n = 10). All patients with IgAN and disease control subjects were in periods of clinical quiescence. pIgA1 from patients with IgAN induced greater production of AngII or TGF-β by HMC than did pIgA1 from healthy or disease control subjects. *P < 0.01, **P < 0.02, compared with HMC incubated with pIgA1 from patients with IgAN. HSP, Henoch-Schönlein purpura, MCN, minimal-change nephropathy; MGN, membranous nephropathy. The results represent the mean ± SD.

Effects of IgA Treatment or RAS Blockade on TGF-β Signal Transduction in Cultured HMC

Lastly, we determined whether IgA treatment or RAS blockade affected transcriptional control by the TGF-β/Smad signaling system in HMC. Treatment with either captopril or losartan led to a significant decrease in Smad activity in HMC (P < 0.001) (Figure 8). Compared with resting HMC, incubation with pIgA1 from patients with IgAN significantly induced the Smad activity by 110% (2127.2 ± 329.2 versus 1014.0 ± 85.9 units, P < 0.001). In contrast, incubation with mIgA1 from patients with IgAN induced an insignificant increase in the Smad activity, compared with resting HMC (1426.2 ± 342.3 versus 1014.0 ± 85.9 units, P = 0.058). Blockade of the RAS with either captopril or losartan at 100 nM effectively reduced the Smad activity in HMC induced by pretreatment with pIgA1 from patients with IgAN (P < 0.001). Supershift experiments confirmed that these Smad proteins contained Smad 2, 3, and 4 (data not shown).

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Figure 8. Electrophoretic mobility shift assay studying the Smad activity in cultured HMC. pIgA1 (0.5 mg/ml, prepared from five patients with IgAN) stimulated the expression of Smad 2, 3, and 4 in cultured HMC. Losartan or captopril (at concentrations of 100 nM) downregulated the Smad activity in HMC. *P < 0.001, compared with HMC incubated only with pIgA1. ^#P < 0.001, compared with HMC incubated only with mIgA1. **P < 0.001, compared with HMC incubated only with culture medium (control). The results represent the mean ± SD.