Fig 2: Ccl2 ablation reduces cyst formation in Pkd1 mutant mice Age from control, iPkd1 ?Tub, and iPkd1 ?Tub;iCcl2 ?Tub mice.Plasma blood urea nitrogen (BUN) from control, iPkd1 ?Tub, and iPkd1 ?Tub;iCcl2 ?Tub mice at 13.5 weeks.Representative periodic acid–Schiff (PAS)-stained kidney sections from control (n = 7), iPkd1 ?Tub (n = 6), and iPkd1 ?Tub;iCcl2 ?Tub (n = 7) mice at 13.5 weeks.Higher magnification of PAS-stained sections. Scale bar: 250 µm.Data information: In (A, B), each dot represents one individual mouse. Bars indicate mean. ANOVA followed by the Tukey–Kramer test. *P < 0.05, ns: not statistically different.

Fig 3: Tubule-derived CCL2 promotes macrophage recruitment and cyst formation in Pkd1-targeted kidneys Representative kidneys from control, iPkd1 ?Tub, and iPkd1 ?Tub;iCcl2 ?Tub mice at 13.5 weeks. See also Fig EV5.Kidney weight (KW)-to-body weight (BW) ratio in control, iPkd1 ?Tub, and iPkd1 ?Tub;iCcl2 ?Tub mice at 13.5 weeks. Ccl2 mRNA expression in kidneys from control, iPkd1 ?Tub, and iPkd1 ?Tub;iCcl2 ?Tub mice at 13.5 weeks.F4/80 immunostaining in kidneys from control, iPkd1 ?Tub, and iPkd1 ?Tub;iCcl2 ?Tub mice at 13.5 weeks. Representative images of n = 4 mice/group. Scale bars: 50 µm. Itgam mRNA expression in kidneys from control, iPkd1 ?Tub, and iPkd1 ?Tub;iCcl2 ?Tub mice at 13.5 weeks.Data information: In (B, C, E), each dot represents one individual mouse. Bars indicate mean. ANOVA followed by the Tukey–Kramer test, *P < 0.05, **P < 0.01.

Fig 4: Endothelial cell PC-1 stimulates vasodilation via eNOS, IK channel, and SK channel activation.(A) Representative traces illustrating reversible flow-mediated dilation in pressurized (80 mmHg) mesenteric arteries of Pkd1fl/fl and Pkd1 ecKO mice. (B) Mean dilation to flow (15 dyn/cm2) or ACh (10 µM). *p<0.05 versus Pkd1fl/fl. n=8 for each data set. (C) Representative diameter changes to stepwise increases in intravascular flow in pressurized (80 mmHg) mesenteric arteries from Pkd1fl/fl and Pkd1 ecKO mice. (D) Mean data. The Pkd1-sensitive component of flow-mediated vasodilation is shown in blue. n=4 each for Pkd1fl/fl and Pkd1 ecKO. *p<0.05 versus Pkd1fl/fl. (E–G) Regulation of flow (15 dyn/cm2)-mediated dilation by L-NNA (10 µM), apamin (300 nM), and Tram-34 (300 nM) in pressurized (80 mmHg) mesenteric arteries of Pkd1fl/fl and Pkd1 ecKO mice. (H) Mean data for inhibition of flow-mediated vasodilation (FMD) by L-NNA (Pkd1fl/fl n=8, Pkd1 ecKO n=10), Tram-34 (Pkd1fl/fl n=5, Pkd1 ecKO n=5), and apamin (Pkd1fl/fl n=5, Pkd1 ecKO n=5). Symbols illustrate #p<0.05 versus flow in the same genotype and *p<0.05 versus Pkd1fl/fl in the same condition. (I) Original Western blots illustrating effects of flow (15 dyn/cm2, 5 min, 37°C) on p-eNOS (S1176) and total eNOS proteins in Pkd1fl/fl and Pkd1 ecKO mesenteric arteries. (J) Mean data for flow-induced change (?) in proteins. Pkd1fl/fl n=4, Pkd1 ecKO n=6. * indicates p<0.05 versus static in the same genotype.

Fig 5: GPS cleavage is required for the ciliary trafficking of polycystin complex.(a) Confocal images of collecting duct cells isolated from Pkd1V/V kidneys (CDV/V) stained with antibodies against PC1(E4) or PC2. Arl13b or Ac-tubulin was used as a ciliary marker. The graph compares the percentage of cells with positive PC1 or PC2 in the wild-type CD (as shown in Fig. 1e or f) and CDV/V cells, from three independent experiments and presented as the mean±s.e.m.; ***P<0.001. The number of cells analysed (n) is indicated. (b) Confocal images of induced MDCKPC1V cells show the absence of ciliary PC1V and endogenous PC2. The graph compares the percentage of cells with positive PC1 or PC2 ciliary staining in MDCKPC1WT (as shown in Fig. 2f) and MDCKPC1V cells from three independent experiments, presented as the mean±s.e.m.; ***P<0.001. The number of cells analysed (n) is indicated. The white line in the XY scan in (a,b) indicates the path of the XZ scan. Scale bar, 10 µm. (c) Confocal images of cystic collecting ducts of the Pkd1V/V kidney stained with anti-PC2. Note that PC2 was not detectable in 17 of 17 cilia analysed. (d) N-glycosylation pattern of polycystin complex in Pkd1V/V MEFs versus wild-type MEFs. Polycystin complex was immunoprecipitated and analysed as in Fig. 3, with PC1 and PC2 bands indicated with colour code. Note that the co-precipitated PC2 from Pkd1V/V MEFs (lanes 4–6) was entirely EndoH sensitive, lacking EndoH-resistant PC2130 seen in wild-type MEFs (lanes 1–3). (e) N-glycosylation pattern of polycystin complex in Pkd1V/V kidney versus wild-type kidney. Polycystin complex was immunoprecipitated and analysed as in (d), expect that an anti-CC antibody was used to detect PC1. Note the presence of cleaved PC1CTF in wild-type kidney (lanes 1–3) but not in Pkd1V/V kidneys (lanes 4–6).