The normal renal cortex of human kidney was obtained immediately after nephrectomy. The glomeruli were treated with collagenase (Gibco BRL, Gaitherburg, USA), and they were plated onto culture dishes in Dulbecco's modified Eagle medium (DMEM, Gibco BRL) containing 10% heat inactivated fetal calf serum (Sigma Chemical, St. Louis, USA). Mesangial cells were cultured in DMEM supplemented with 17% fetal calf serum, 2% penicillin, 2% streptomycin, 1% HEPES, 2 g sodium bicarbonate and 2 mM L-glutamine at 37℃ in 5% CO₂, 95% air. With the use of immunohistochemical staining, the mesangial cells were identified due to their large stellate shape, and the myosin and fibronectin were also stained, whereas the common leukocyte antigen and factor VIII were not stained.

The mesangial cells were incubated for 24 hours in serum-free media, and then they were cultured in the media containing an IgA aggregate (n=4, 100 µg/mL), or an IgG aggregate (n=3, 100 µg/mL). The cell were pretreated with 100 nM of phorbol 12-myristate 13-acetate (PMA) for 24hrs in order to deplete their protein kinase C (PKC), and some of the cells were pretreated with 200 nM calphostin C (a PKC inhibitor).

Aggregation was brought about by heating the IgA or IgG for 30 minutes at 63℃. After cooling it in room temperature, the proteins were centrifuged at 11,000 g for 5 minutes to remove the insoluble aggregate. The supernatants were then used as the heat-aggregated IgA or IgG7).

Glomular microdissection was carried out within 30 minutes after biopsy in a cooled dish containing RNase inhibitor (10 mM of vanadyl ribonucleoside complex, Life technology, Gaithersburg, USA). The glomeruli were separated from the tubules, and they were washed with saline to remove tissue debris and VRC, and then they were pooled at 5 glomeuli per PCR tube with the tube containing 5 mM dithiothreitol and 1.2 U/µL of RNase inhibitor (Boehringer Mannheim Corp., Indianapolis, USA). The glomeruli were permeated immediately before reverse transcription (RT) with a mixture containing 0.9% Triton X-100, 1.2 U/µL of placental RNase inhibitor, and 5 mM dithiothreitol. The five glomeruli in the pools were reverse transcribed with oligo dT, and the products were then combined after RT to inhibit errors that could be derived from the variation in glomerular size. RT was performed using a cDNA synthesis kit (Boehringer Mannheim) as described previously8).

The mesangial cells were collected using a cell scraper, and they were resuspended in Trizol reagent (Invitrogen, Carlsbad, CA) containing RNase inhibitor. A cDNA synthesis kit was used for RT, and a random primer (6mer) was used to prime the mRNA RT. The RT reaction was set up in a 20 µL mixture containing 50 mM KCl, 10 mM Tris/HCl, 5 mM MgCl₂, 1 mM of each dNTPs, 2.5 µM random hexamers, 20 U Rnase inhibitor and 50 U of M-MLV reverse transcriptase. The reaction mixture was incubated for 60 min at 42℃, and then it was heated for 7 minutes at 90℃ in a thermocycler (Crocodile III, Oncor Co., Japan).

After cDNA synthesis by RT, the incubation mixture was split into two portions in order to amplify the TGF-β1 cDNA and the GAPDH cDNA using specific primers: for the TGF-β1 DNA, the sense primer was 5' GGA GGG GAA ATT GAG GGG TTT CGC 3', and the anti-sense primer was 5' TTA TGC TGG TTG TAC AGG GC 3'; for the GAPDH DNA, the sense primer was 5' CCC CAG GCA CCA GGG CGT GAT 3', and the anti-sense primer was 5' GGT CAT CTT GCG GTT GGC CTT GGG GT 3'. A semi-quantitative measurement of mRNA expression was performed by optimizing the amount of cDNA and number of PCR cycles as described previously7).

PCR was done in a 50 µL volume containing 50 mM KCl, 10 mM Tris/HCl (pH 8.3), 200 µM of each dNTPs, 50 pmol of each primer, 2 U Taq DNA polymerase and 1.5 mM MgCl₂. The mixture was first heated for 5 minutes at 95℃, and this was followed by 36 cycles: each cycle was 2.5 minutes at 95℃, 1.5 minutes at 55℃, 3 minutes at 72℃, and a final extension for 7 minutes at 72℃. For quantification of the PCR products and the house keeping gene, glyceraldehyde 3 phosphate dehydrogenase (GAPDH) PCR was performed in a parallel fashion in separate tubes.

To quantify the cDNAs for TGF-β1 in the glomeruli, competitive PCR was performed by adding decreasing amounts of mutant templates to the glomerular cDNA. For the TGF-β1 and GAPDH, deletion cDNA mutant templates for each of these were developed to create 51 and 39 bp deletions in the middle of the molecules, and this resulted in mutant cDNAs of 339 and 559bp, respectively.

Human TGF-β1 cDNA was amplified with the following primers: 5' GGA GGG GAA ATT GAG GGG TTT CGC 3' as the sense primer and 5' TTA TGC TGG TTG TAC AGG GC 3' as the antisense primer. Human GAPDH cDNA was amplified with the following primers: 5' CCA CCC ATG GCA AAT TCC ATG GCA 3' as the sense primer and 5' TCT AGA CGG CAG GTC AGG TCC ACC 3' as the antisense primer.

Competitive PCR was performed with the sample DNA by applying serial dilutions of the mutant DNA in the same tubes under the condition of 1 minute at 94℃, 1 minute at 60℃, and 3 minutes at 72℃ for 36 cycles for the TGF-β1 and 32 cycles for the GAPDH. The PCR products were electrophoresed in 2% agarose gel; they were eluted using a Qiagen DNA extraction kit and then sequenced to confirm the PCR products by using an automatic sequencing system (ABI Genetic Analyzer 310; PRISM, Branchburg Park, USA).

The ratio of the mutant DNA band density to the test DNA band density was calculated for each lane, and the data was plotted as a function of the amount of mutant template that was added to the reaction. The quantity of cDNA in the test sample was the amount at which the mutant/test band density was equal to one10).

Among the patients with biopsy-proven IgA nephropathy, eight patients (3 men and 5 women) having more than 10 glomeruli from their biopsy tissue were included. All the patients received angiotensin converting enzyme inhibitors during the follow up period. The renal biopsy procedure was approved by the Korea University Research and Education Institute Investigational Review Board, and informed consents were obtained from each patient. Blood urea nitrogen, serum creatinine, IgA and C3 concentrations were measured by Nephelometry (Behring, Germany). Creatinine clearance and daily proteinuria were also measured by employing 24 hours urine collections.

The morphological findings from the biopsy material such as the degree of glomerulosclerosis, tubulointerstitial changes, IgA staining pattern and Haas subclassification9) were determined "blindly" by two different renal pathologists. A semiquantitative score from 0 to 4+ was used to evaluate the degree of glomerulosclerosis: a 1+ lesion represented involvement of 25% of the glomerulus; a 2+ lesion represented involvement of 25~50% of the glomeruli; a 3+ lesion represented involvement of 5 0~75% of the glomeruli; and a 4+ lesion represented involvement of more than 75% of the glomeruli. Tubulointerstitial changes, including tubular dilatation, atrophy, proteinous casts, interstitial edema and mononuclear cell infiltrates were graded from 0 to 4+ (0=no change; 1+=change affecting <25% of the sample; 2+=change affecting 25~50% of the sample; 3+=change affecting 50~75% of the sample; 4+=change affecting >75% of the sample).

Data were summarized as means±SE. To compare the data in vitro and in vivo, the Mann-Whitney U test was used, and correlation was assessed by Pearson's correlation coefficient. p values <0.05 was considered statistically significant.

The IgA aggregates directly stimulated TGF-β1 mRNA expression in the cultured mesangial cell. The expression of TGF-β1 mRNA was significantly higher under the condition of IgA, whereas its expression was not changed under the condition of IgG aggregates (Figure 1). The expression of TGF-β1 mRNA in the IgA-treated mesangial cells during the incubation with PMA for 24 hours was not significantly different from the control. Similarly, in the case of a one hour pretreatment with calphostin C, there was no significant decrease in the mRNA expression of TGF-β1 (Figure 2).

The mean age of the eight patients (3 men and 5 women) was 27.4±8.5 yrs. The other basic characteristics are summarized in Table 1. Figure 3 shows the representative result of competitive PCR with glomeruli that were obtained from patients with IgA nephropathy. The intraglomerular TGF-β1/GAPDH mRNA ratio was significantly correlated with the levels of 24-hr proteinuria (r=0.781, p=0.022), serum creatinine (r=0.884, p=0.004), and tubulointerstitial changes (r=0.809, p=0.015). In addition, the intraglomerular TGF-β1 mRNA expression showed a significant negative correlation with the creatinine clearance (r=-0.0764, p=0.027), and it also showed positive correlation with glomerulosclerosis (r=0.646, p=0.084), but this was statistically insignificant. However, the TGF-β1 mRNA expression was not correlated with such other parameters as the peripheral blood IgA concentration, the intraglomerular immunoreactivity for IgA, and the Haas pathologic subclassification (Figure 4, 5).