Necrotic renal epithelial cell inhibits renal interstitial fibroblast activation: role of protein tyrosine phosphatase 1B

Abstract

Our recent studies showed that contents of necrotic renal proximal tubular cells (RPTC) from 2 × 10(6) cells/ml directly induced death of cultured renal interstitial fibroblasts. However, it remains unknown whether nonlethal number of necrotic RPTC would also alter the fate of renal interstitial fibroblasts. To address this issue, renal interstitial fibroblasts (NRK-49F) were exposed to necrotic RPTC supernatant (RPTC-Sup) obtained from 2 × 10(4) to 5 × 10(5) cells/ml. These concentrations of RPTC did not induce cell death, but led to inactivation of renal fibroblasts as indicated by reduced expression of α-smooth muscle actin and fibronectin, two hallmarks of activated fibroblasts. Concurrently, the same doses of necrotic RPTC-Sup suppressed phosphorylation of epidermal growth factor receptor (EGFR) and signal transducers and activators of transcription-3 (STAT3) in a time- and dose-dependent manner, but did not affect phosphorylation of platelet-derived growth factor receptor-β, AKT, and extracellular signal-regulated kinase 1/2. The presence of sodium orthovanadate, a general protein tyrosine phosphatase (PTP) inhibitor or TCS-401 (a selective PTP1B inhibitor), abrogated those effects of RPTC-Sup, whereas coincubation with the EGFR inhibitor (Gefitinib) or silencing of EGFR with siRNA preserved the ability of RPTC-Sup in suppressing renal fibroblast activation and STAT3 phosphorylation. Moreover, RPTC-Sup treatment induced PTP1B phosphorylation and its interaction with EGFR. Collectively, these results indicate that nonlethal necrotic RPTC-Sup can induce inactivation of renal interstitial fibroblasts, which occurs through a mechanism involved in PTP1B-mediated inhibition of EGFR signaling.

Fig. 1.

Effect of necrotic renal proximal tubular cells (RPTC) on viability of cultured NRK-49F. Normally cultured NRK-49F cells with 5% FBS were treated with indicated concentrations of RPTC supernatant (RPTC-Sup) for 48 h (A). NRK-49F cells treated with 1 mM H₂O₂ for 3 h were used as positive control to induce cell death (A-C). After treatments, cell lysates were prepared and subjected to immunoblot analysis with antibodies for cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), or GAPDH. A: representative immunoblots from 3 or more experiments. NRK-49F cells were treated with RPTC-Sup from 5 × 10⁵ cells/ml for 48 h or RPTC-Sup prepared from 2 × 10⁶ cells/ml for 24 h. After treatments, cells were stained with DAPI and photographed with fluorescent microscope to observe nuclei morphology (×200) and shrunken and condensed nuclei were counted (B). Values are means ± SD of 3 independent experiments (C). Bars with different letters (a, b) are significantly different from one another (P < 0.01).

Fig. 2.

Necrotic RPTC reduce the expression of α-SMA and fibronectin expression in cultured NRK-49F. Normally cultured NRK-49F cells with 5% FBS were treated with indicated concentrations of RPTC-Sup for 36 h (A and B) or cells were treated with necrotic RPTC-Sup prepared from 5 × 10⁵ cells/ml for the indicated time (12, 24, and 36 h; C and D) or cells were starved overnight with DMEM-F12 medium containing 0.5% FBS and then treated with necrotic RPTC-Sup in the presence or absence of TGF-β1 (1 ng/ml) for 24 h (E and F). Cell lysates were prepared and subjected to immunoblot analysis with antibodies for α-SMA, fibronectin, α-tubulin, or GAPDH (A, C, E). Representative immunoblots from 3 experiments are shown. The levels of α-SMA and fibronectin were quantified by densitometry and normalized with α-tubulin or GAPDH (B, D, F). Values are means ± SD of 3 independent experiments. Bars with different letters (a–d) are significantly different from one another (P < 0.01).

Fig. 3.

Necrotic RPTC reduce epidermal growth factor receptor (EGFR) phosphorylaton in NRK-49F. NRK-49F cells were treated for 36 h with RPTC-Sup prepared from an indicated number of cells (A and B) or treated with RPTC-Sup from 5 × 10⁵ cells/ml for the indicated time (C and D). The cell lysate was prepared after treatment and subjected to immunoblot analysis for phospho-EGFR (Tyr1068), phospho-platelet-derived growth factor receptor-β (PDGFRβ; Tyr751), EGFR, or PDGFRβ (A and C). Representative immunoblots from 3 experiments are shown. The phosphorylated and total levels of EGFR and PDGFRβ were quantified by densitometry and phosphorylated protein levels were normalized to total protein levels (B and D). Values are means ± SD of 3 independent experiments. Bars with different letters (a–d) are significantly different from one another (P < 0.01).

Fig. 4.

Inhibition of P2X7 receptor does not affect the necrotic RPTC-Sup-induced reduction of α-SMA and fibronectin expression. Normally cultured NRK-49F cells with 5% FBS were treated with RPTC-Sup from an indicated number of cells (2 × 10⁴ to 5 × 10⁵ cells/ml) for 36 h or RPTC-Sup from 2 × 10⁶ cells/ml for 24 h (A and B). After treatments, cell lysates were prepared and subjected to immunoblot analysis with antibodies for P2X7 or α-tubulin (A). NRK-49F cells were transfected with rat-specific small interfering RNA (siRNA) targeting P2X7 or scrambled siRNA. At 24 h after transfection, cells were treated with necrotic RPTC for 36 h (C and D). Cell lysates were prepared and subjected to immunoblot analysis with specific antibodies against P2X7, α-SMA, fibronectin, or α-tubulin (C). Representative immunoblots from 3 experiments are shown. The levels of P2X7, α-SMA, and fibronectin were quantified by densitometry and normalized with α-tubulin (B and D). Values are means ± SD of 3 independent experiments. Bars with different letters (a–d) are significantly different from one another (P < 0.01).

Fig. 5.

Necrotic RPTC decrease STAT3 phosphorylation in NRK-49F. NRK-49F cells were treated for 36 h with RPTC-Sup prepared from an indicated number of cells (A) or treated with RPTC-Sup from 5 × 10⁵ cells/ml for the indicated time (C). The cell lysate was prepared after treatments and then subjected to immunoblot analysis for phospho-STAT3 (Tyr705), STAT3, phospho- AKT, AKT, phospho-ERK 1/2, or ERK 1/2 (A and C). Representative immunoblots from 3 experiments are shown. The phosphorylated and total levels of STAT3, AKT, and ERK were quantified by densitometry and phosphorylated protein levels were normalized to total protein levels (B and D). Values are means ± SD of 3 independent experiments. Bars with different letters (a–d) are significantly different from one another (P < 0.01).

Fig. 6.

Effect of phosphatase inhibitors on necrotic RPTC-induced fibroblast inactivation. NRK-49F cells were pretreated with different concentrations of sodium orthovanadate (Na₃VO₄) for 1 h and then exposed to the necrotic RPTC-Sup prepared from 5 × 10⁵ cells/ml for an additional 36 h. Cell lysates were prepared and subjected to immunoblot analysis with specific antibodies against α-SMA, fibronectin, α-tubulin, phospho-EGFR, EGFR, phospho-STAT3, or STAT3 (A and C). Representative immunoblots from 3 experiments are shown. The levels of α-SMA and fibronectin were quantified by densitometry and normalized with α-tubulin (B). The phosphorylated and total levels of STAT3 and EGFR were quantified by densitometry and phosphorylated protein levels were normalized to total protein levels (D). Values are means ± SD of 3 independent experiments. Bars with different letters (a–d) are significantly different from one another (P < 0.01).

Fig. 7.

EGFR inhibition blocks the effect of Na₃VO₄ on necrotic RPTC-induced alterations in NRK-49F. NRK-49F cells were pretreated with gefitinib (2 μM) for 15 min and exposed to Na₃VO₄ (10 μM) for 1 h and then incubated with necrotic RPTC for an additional 36 h (A). NRK-49F cells were transfected with siRNA targeting EGFR or scrambled siRNA for 24 h. After transfection, cells were treated with necrotic RPTC for 36 h in the presence or absence of Na₃VO₄ (10 μM; B). Cell lysates were prepared and subjected to immunoblot analysis with specific antibodies against α-SMA, fibronectin, α-tubulin, phospho-EGFR, EGFR, phospho-STAT3, or STAT3 (A and B). Representative immunoblots from 3 experiments are shown.

Fig. 8.

Inhibition of protein tyrosine phosphatase (PTP)1B blocks necrotic RPTC-induced fibroblast inactivation. NRK-49F cells were pretreated with different concentrations of TCS-401 as indicated for 1 h and then incubated with necrotic RPTC-Sup for an additional 36 h. Cell lysates were prepared and subjected to immunoblot analysis with specific antibodies against α-SMA, fibronectin, α-tubulin, phospho-EGFR, EGFR, phospho-STAT3, or STAT3 (A and B). NRK-49F cells were pretreated with gefitinib (2 μM) for 15 min and then incubated with 2 μM TCS-401 for 1 h. After that, RPTC-Sup was added and incubated for an additional 36 h. Cell lysates were prepared and subjected to immunoblot analysis with specific antibodies against α-SMA, fibronectin, α-tubulin, phospho-EGFR, EGFR, phospho-STAT3, or STAT3 (C and D). Representative immunoblots from 3 experiments are shown.

Fig. 9.

Necrotic RPTC induce PTP1B tyrosine phosphorylaton and its interaction with EGFR. NRK-49F cells were treated with RPTC-Sup for 36 h and then cell lysates were subjected to immunoblotting for PTP1B and GAPDH (A). Normally cultured NRK-49F cells were treated with necrotic RPTC-Sup for 24 h and cells were harvested. Equal concentrations of protein from each sample were immunoprecipitated with PTP1B antibody and then subjected to Western blot analysis for PTP1B, PY20, or EGFR (B). Representative immunoblots from 3 experiments are shown.

Fig. 10.

Mechanisms of necrotic RPTC-Sup-induced inactivation of renal interstitial fibroblasts. Under normal culture condition, EGFR and STAT3 are activated, which is required for activation of renal interstitial fibroblasts. When cells are exposed to necrotic RPTC-Sup, PTP1B is activated, leading to EGFR dephosphorylation and subsequent inactivation of STAT3 and renal interstitial fibroblasts.