Janus kinase 2/signal transducer and activator of transcription 3 inhibitors attenuate the effect of cardiotrophin-like cytokine factor 1 and human focal segmental glomerulosclerosis serum on glomerular filtration barrier

Abstract

Recurrence of idiopathic focal segmental glomerulosclerosis (FSGS) after renal transplantation is believed to be caused by a circulating factor(s). We detected cardiotrophin-like cytokine factor 1 (CLCF1), a member of the interleukin 6 family, in the plasma from patients with recurrent FSGS. We hypothesized that CLCF1 contributes to the effect of FSGS serum on the glomerular filtration barrier in vitro. Presently, we studied the effect of CLCF1 on isolated rat glomeruli using an in vitro assay of albumin permeability (P(alb)). CLCF1 (0.05-100 ng/mL) increased P(alb) and caused maximal effect at 5-10 ng/mL (P < 0.001). The increase in Palb was analogous to the effect of FSGS serum. Anti-CLCF1 monoclonal antibody blocked the CLCF1-induced increase in P(alb) and significantly attenuated the effect of FSGS serum (P < 0.001). The heterodimer composed of CLCF1 and cosecreted molecule cytokine receptor-like factor 1 (CRLF1) attenuated the increase in P(alb) caused by CLCF1 or FSGS serum. Western blot analysis showed that CLCF1 upregulated phosphorylation of signal transducer and activator of transcription 3 (STAT3) (Tyr705) in glomeruli. This effect was diminished by the heterodimer CLCF1-CRLF1. Janus kinase 2 (JAK2) inhibitor BMS-1119543 or STAT3 inhibitor Stattic significantly blocked the effect of CLCF1 or FSGS serum on P(alb) (P < 0.001). These novel findings suggest that when monomeric CLCF1 increases P(alb), the heterodimer CLCF1-CRLF1 may protect the glomerular filtration barrier. We speculate that albuminuria in FSGS is related to qualitative or quantitative changes in the CLCF1-CRLF1 complex, and that JAK2 or STAT3 inhibitors may be novel therapeutic agents to treat FSGS.

Figure 1

1A. CLCF1 caused an increase in glomerular albumin permeability (P_alb). Isolated rat glomeruli were incubated with recombinant CLCF1 (0.05–100ng) for 15 minutes at 37°C. CLCF1 at 0.05ng/mL concentration caused significant increase in P_alb (P<0.05 vs. control). Maximal increase was observed at 5–10 ng/mL (P<0.001). N= 15 glomeruli from 3 rats (5 glomeruli from each rat) in each group. 1B. Anti-CLCF1 antibody blocked the effect of CLCF1 on P_alb. Isolated rat glomeruli were incubated with recombinant CLCF1 (5ng/mL) or with a mixture of CLCF1 and anti-CLCF1 monoclonal antibody (0.05–50µg /mL) for 15 minutes at 37°C. Control group included 5 µg human IgG. CLCF1 caused significant increase in P_alb (P<0.001 vs. Control) that was maximally blocked by antibody at 50 µg /mL concentration (*, P<0.005 vs. CLCF1 alone). N= 15 glomeruli from 3 rats (5 glomeruli from each rat) in each group. 1C. Anti-CLCF1 antibody blocked the effect of sera from patients with recurrent FSGS. Isolated glomeruli were incubated with FSGS serum samples from five individuals (20 µL/mL each) or with a mixture of each serum sample and anti-CLCF1 antibody (50µg/mL) for 15 minutes at 37°C. Control group included 20µL/mL of pooled normal serum. Each pair of black and grey bars represents one sample. Black bars show that each FSGS serum sample caused significant increase in P_alb (P<0.001 vs. Control). Each grey bar shows the effect of anti-CLCF antibody on the corresponding FSGS sample. Numeric values for P_alb and percent change are shown under each pair of black and grey bars for each sample. Average of all five samples with SEM (mean±SEM) shows that anti-CLCF1 antibody significantly blocked the effect of FSGS serum (P<0.005, n=5 specimens). Five glomeruli from 1 rat were observed to calculate P_alb for each sample.

Figure 2

2A. Heterodimer CLCF1-CRLF1 complex did not increase P_alb. Isolated rat glomeruli were incubated with recombinant CLCF1-CRLF1 (0.1–10ng/mL) for 15 minutes at 37°C. CLCF1-CRLF1 did not affect P_alb at any of the concentrations used. N=15 glomeruli from 3 rats (5 glomeruli from each rat) in each group. 2B. Heterodimer CLCF1-CRLF1 heterodimer blocked the effect of CLCF1 on P_alb. Isolated rat glomeruli were pre-incubated with CLCF1-CRLF1 (5–20ng/mL) for 15 minutes followed by addition of CLCF1 (5ng/mL) for 15 minutes at 37°C. CLCF1 alone caused a significant increase in P_alb (#, P<0.001 vs. control or CLCF1-CRLF1 alone) and pre-treatment with CLCF1-CRLF1 blocked the effect of CLCF1 in a dose-dependent manner (*, P<0.02 vs. CLCF1 alone; **, P<0.005 vs. CLCF1 alone). N=15 glomeruli from 3 rats (5 glomeruli from each rat) in each group.

Figure 3

3A. Pre-treatment of glomeruli with CLCF1-CRLF1 heterodimer blocked the effect of FSGS serum on P_alb. Isolated glomeruli were pre-incubated with CLCF1-CRLF1 complex (5–50ng/mL) for 15 minutes followed by addition of FSGS serum (20µL/mL) and incubation for 15 minutes at 37°C. FSGS serum alone caused a significant increase in P_alb (#, P<0.001 vs. Control) that was blocked by 10ng/mL (*, P<0.02 vs. FSGS alone), 20ng or 50ng/mL (**, P<0.005 vs. FSGS alone). N= 15 glomeruli from 3 rats (5 glomeruli from each rat) in each group. 3B. Pre-treatment with FSGS serum prevented the protective effect of CLCF1-CRLF1 heterodimer on P_alb. Isolated glomeruli were pre-incubated with FSGS serum (20µ/mL) for 15 minutes at 37°C followed by addition of CLCF1-CRLF1 (5–50 ng/mL) for 15 minutes at 37°C. FSGS serum with or without CLCF1-CRLF1 increased P_alb significantly (*, P<0.001 vs. control). N= 15 glomeruli from 3 rats (5 glomeruli from each rat) in each group.

Figure 4

4A. Pre-incubation of glomeruli with CLCF1-CRLF1 heterodimer did not block the effect of TNFα or IL6. Isolated rat glomeruli were pre-incubated with CLCF1-CRLF1 (5–20 ng/mL) for 15 minutes followed by addition of TNFa (10ng/mL) or IL6 (1 ng/mL) and further incubation for 15 minutes at 37°C. TNFα and IL6 each caused a significant increase in P_alb compared to control (*, P<0.001 vs. control). CLCF1-CRLF1 did not block the increase in P_alb caused by TNFα or IL6. N=15 glomeruli from 3 rats (5 glomeruli from each rat) in each group. 4B and 4C. CLCF1-CRLF1 heterodimer blocked the CLCF1- induced phosphorylation of STAT3. Figure 4B shows a representative Western blot image to demonstrate the upregulation of STAT3 (Tyr705) phosphorylation by CLCF1 and the blocking effect of CLCF1-CRLF1 heterodimer. Control group represents untreated glomeruli. Glomeruli were incubated with CLCF1 (10ng/mL) for 15 minutes in one group. In additional groups, glomeruli were pre-incubated with heterodimer CLCF1-CRLF1 (10–40ng/mL) for 15 minutes followed by addition of CLCF1 (10ng/mL) and incubation for 15 minutes at 37°C. Thus, the ratio of CLCF1 to the heterodimer CLCF1-CRLF1 ranged from 1:1 to 1:4 (ng:ng) or a molar ratio of approximately 1:0.3 to 1:1.25. Total protein lysates were resolved by SDS-PAGE followed by Western blotting using anti-pSTAT3 (Tyr705) as the primary antibody. Figure 4C. The bar graph shows results of quantitative analysis of protein band intensities. Changes in STAT3 phosphorylation (Tyr705) were determined by semi-quantitative image analysis. Background subtracted intensities were normalized by the loading control β-actin. Ratios of intensities (Experimental/control) are presented in the bar graph. CLCF1 caused significant increase in pSTAT3 (Tyr705) (*, P<0.001 control vs. CLCF1 alone). CLCF1-induced increase in pSTAT3 (Tyr705) was attenuated by pretreatment with 30 or 40 ng/mL CLCF1-CRLF1 (*, P<0.001 vs. CLCF1 alone). Mean±SEM of three separate experiments are shown.

Figure 5

JAK2 inhibitor blocked the effect of CLCF1 or FSGS serum on P_alb. Isolated rat glomeruli were pre-incubated with JAK2 inhibitor BMS-911543 (BMS, 1–10nM) for 15 minutes followed by addition of CLCF1 (10ng) or FSGS serum (20µL) for 15 minutes at 37°C. BMS blocked the effect of CLCF1 or FSGS serum at 5 and 10 nM concentrations and increased P_alb. (*, P<0.001 vs. CLCF1 alone; #, P<0.001 vs. FSGS alone; Mean±SEM, N= 15 glomeruli from 3 rats, 5 glomeruli from one rat in each group).

Figure 6

STAT3 inhibitor blocked the effect of CLCF1 and FSGS serum on P_alb. Isolated rat glomeruli were pre-incubated with STAT3 inhibitor Stattic (0.001–1µM) for 15 minutes followed by addition of CLCF1 (10ng/mL). In separate experiments, glomeruli were pre-incubated with Stattic (0.1 or 1µM) for 15 minutes followed by FSGS serum (20µL/mL) for 15 minutes at 37°C. Pre-treatment of glomeruli with Stattic (0.05–1µM) significantly blocked the effect of CLCF1 (P<0.001 vs.CLCF1 alone). Pre-incubation with Stattic (1µM) blocked the effect of FSGS serum on P_alb. (#, P<0.001 vs. FSGS alone). N=15 glomeruli from 3 rats (5 glomeruli from each rat) in each group.