A tumor necrosis factor-alpha-mediated pathway promoting autosomal dominant polycystic kidney disease

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is caused by heterozygous mutations in either PKD1 or PKD2, genes that encode polycystin-1 and polycystin-2, respectively. We show here that tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine present in the cystic fluid of humans with ADPKD, disrupts the localization of polycystin-2 to the plasma membrane and primary cilia through a scaffold protein, FIP2, which is induced by TNF-alpha. Treatment of mouse embryonic kidney organ cultures with TNF-alpha resulted in formation of cysts, and this effect was exacerbated in the Pkd2(+/-) kidneys. TNF-alpha also stimulated cyst formation in vivo in Pkd2(+/-) mice. In contrast, treatment of Pkd2(+/-) mice with the TNF-alpha inhibitor etanercept prevented cyst formation. These data reveal a pathway connecting TNF-alpha signaling, polycystins and cystogenesis, the activation of which may reduce functional polycystin-2 below a critical threshold, precipitating the ADPKD cellular phenotype.

Figure 1

Effects of TNF-α on FIP2 and PC2 in IMCD cells. a, Western blot analysis shows increased FIP2 protein levels but not PC2 protein levels upon TNF-α stimulation for 4 hr or 16 hr. b, Co-immunoprecipitation of endogenous FIP2 and PC2 with anti-PC2 or FIP2 antibody. TNF-α induction for 4 hr or 16 hr resulted in increased amounts of FIP2 pulled down with PC2, and PC2 pulled down with FIP2. IgG: control antibody. c and d, Double immunofluorescence staining of endogenous PC2 (green) and acetylated tubulin (tub, red) before (c) or after (d) treatment with TNF-α for 16 hr, observed with confocal microscopy. Scale bars, 10 μm. e and f, enlarged cilia images in the boxed regions in c and d, respectively. g and h, Transfection of siRNA#05 against FIP2 prevented TNF-α-induced mislocalization of PC2 in IMCD cells.

Figure 2

TNF-α triggers cyst formation in cultured embryonic kidneys and is present in human ADPKD cyst fluid. a, Treatment of wild-type cultured kidneys from E15.5 embryos with various concentrations of TNF-α for 5 days triggered cyst formation. Each vertical pair represents left and right kidneys from the same embryo. Scale bars, 500 μm. b, Treatment with different concentrations of TNF-α for 5 days triggered cyst formation in Pkd2^+/− cultured kidneys from E15.5 embryos. Scale bars, 500 μm. c, Immunoblot analysis of FIP2, TNFR-I and TACE protein levels in wild-type and Pkd2^+/− embryonic kidneys with or without TNF-α (6.25 ng/ml) stimulation for 5 days. The band intensity of FIP2 (top) and TNFR (bottom) are shown as histograms. d, e, Quantification of TNF-α levels in ADPKD human cysts by ELISA. The data represent 20 cysts from 10 ADPKD patients. The horizontal axis indicates cyst volumes; the vertical axis indicates TNF-α concentration (d); and the total TNF-α amount in each cyst (e). f, Immunoblot analysis of FIP2 and TNFR-I protein levels, comparing cultured normal human kidney (NHK) cells and ADPKD cyst lining (PKD). The band intensity of FIP2 (left) and TNFR (right) are shown as histograms.

Figure 3

TNF-α inhibitor, etanercept, prevents cyst formation in Pkd2^+/− mice. a, Kidney histology sections from Pkd2^+/− mice injected intraperitoneally, from week 8.5 to week 18.5, with etanercept at 125 μg/mouse/week (right column) or phosphate buffered saline (PBS) (middle and right columns). For control examples (-etanercept), the panel to the right of each kidney section displays an enlarged image of a cyst in that section. Scale bars, 4 mm. b, Quantitative result of etanercept treatment experiments. The number above each bar indicates the number of mice represented by the bar. p=0.0005 (Fisher’s exact test). c. A functional network connecting TNF-α signaling, polycystin complex and cystogenesis. Three double-negative feedback loops can be observed: between PC2 function and the physiological response that leads to TNF-α production; between PC2 level and FIP2 expression; between PC2 and TNFR expression. +: positive effect; −: negative effect. In red: genetic and non-genetic factors that could influence the pathway leading to cystogenesis.