Role of P311 in interleukin-1α-induced epithelial to myofibroblast transition in kidney tubular epithelial cells

Figures & data

Table 1. Oligonucleotide primer sets for real-time PCR.

Gene	Primer sequences	Product length
P311	F: 5′-GCTCAAAATGGTTTACTAC-3 R: 5′-AAAAAGGGTGGAGGTA-3′	213 bp
α-SMA	F: 5′-ATCCGATAGAACACGGCATC-3′ R: 5′-AGTCACGCCATCTCCAGAGT-3′	275 bp
TGF-β1	F: 5′-TACAGGGCTTTCGCTTCAGT-3′ R: 5′-GTCCAGGCTCCAAATGTAGG-3	359 bp
GAPDH	F: 5′-GCAAGTTCAACGGCAGCA-3′ R: 5′-CGCCAGTAGACTCCACGAC-3′	140 bp

Figure 1. IL-1α-induced fibroblast-like morphological changes in cultured NRK52E tubular epithelial cells. (a) Confluent cells cultured in medium alone for 4 days to maintain the cobblestone epithelial morphological state and growth pattern. (b) Cells co-cultured with IL-1α showed marked hypertrophy, became elongated, and developed an invasive growth pattern. (c) Cells grown in medium alone displayed a normal aggregated epithelial growth pattern with apical-basal polarity and numerous microvillus on the surface. (d) Cells co-cultured with IL-1α showed fibroblast-like morphological transition.

Figure 2. Real time PCR analysis of P311, α-SMA and TGF-β1 mRNA expression in all groups.

Table 2. Expression of P311, α-SMA, and TGF-β1 in all groups.

Groups	Medium alone	IL-1α	IL-1α+ anti-P311
mRNA levels
P311	1.68E–04 ± 4.41E–05	1.29E–03 ± 2.99E–04*	3.12E–03 ± 5.31E–04*Δ
α-SMA	7.99E–03 ± 1.50E–03	0.56 ± 4.36E–02*	0.25 ± 1.00E–03*Δ
TGF-β1	1.83E–04 ± 5.57E–06	2.72E–04 ± 6.66E–06*	3.28E–04 ± 3.97E–05*Δ
Immunohistochemistry
P311 + cells (%)	2.04 ± 0.31	31.51 ± 3.55*	16.21 ± 2.38*Δ
α-SMA + cells (%)	4.12 ± 0.69	47.32 ± 1.80*	21.16 ± 2.01*Δ
Protein levels
P311/β-actin	0.17 ± 3.80E–3	0.33 ± 1.51E–3*	0.23 ± 5.42E–3*Δ
α-SMA/β-actin	0.56 ± 1.87E–2	0.97 ± 1.37E–2*	0.71 ± 2.94E–2*Δ
TGF-β1 (pg/mL)	19.30 ± 3.04	155.85 ± 1.64*	163.99 ± 0.91*Δ

Notes: Data are expressed as mean ± SD.

*, compared with medium alone group, p < 0.05; Δ, compared with IL-1α 10 ng/mL group, p < 0.05.

Figure 3. P311 protein and α-SMA protein in tubular epithelial-myofibroblast transition (TEMT). NRK52E cells were grown for four days on glass slides in the presence or absence of IL-1α or a neutralizing anti-P311 antibody. Cells expressing P311 protein or α-SMA protein were detected by immunohistochemistry. (a) Cells cultured in medium alone showed a normal epithelial morphology state. Only rare cells stained positive for P311. (b) Cells co-cultured with IL-1α caused marked TEMT. (c) The addition of neutralizing anti-P311 antibody produced much inhibition of P311 protein expression. (d) Cells cultured in medium alone showed a normal epithelial morphology and rare cells were stained positive for α-SMA. (e) Cells co-cultured with IL-1α caused marked TEMT. Strong α-SMA staining was demonstrated in many transformed cells. (f) The addition of neutralizing anti-P311 antibody obviously decreased the expression of α-SMA protein. This is one of three experiments that produced similar results.

Figure 4. Western blot analyses of P311 and α-SMA protein in NRK52E cells. Cells were cultured in the presence of medium alone, IL-1α, or IL-1α plus a neutralizing anti-P311 antibody.