Ciliary exclusion of Polycystin-2 promotes kidney cystogenesis in an autosomal dominant polycystic kidney disease model

Abstract

The human PKD2 locus encodes Polycystin-2 (PC2), a TRPP channel that localises to several distinct cellular compartments, including the cilium. PKD2 mutations cause Autosomal Dominant Polycystic Kidney Disease (ADPKD) and affect many cellular pathways. Data underlining the importance of ciliary PC2 localisation in preventing PKD are limited because PC2 function is ablated throughout the cell in existing model systems. Here, we dissect the ciliary role of PC2 by analysing mice carrying a non-ciliary localising, yet channel-functional, PC2 mutation. Mutants develop embryonic renal cysts that appear indistinguishable from mice completely lacking PC2. Despite not entering the cilium in mutant cells, mutant PC2 accumulates at the ciliary base, forming a ring pattern consistent with distal appendage localisation. This suggests a two-step model of ciliary entry; PC2 first traffics to the cilium base before TOP domain dependent entry. Our results suggest that PC2 localisation to the cilium is necessary to prevent PKD.

Fig. 1

Pkd2^lrm4 causes kidney cysts. Cysts develop in Pkd2^lrm4/lrm4 kidneys (a) at E15.5 but not in Pkd2^lrm4/+ (b) or in Pkd2^+/+ (c). Cysts are comparable to those seen in Pkd2^−/− kidneys at the same age (d). Scale bar: 100 µm. Boxed areas in a–d are represented in e–h. Glomerular cysts are visible in both Pkd2^lrm4/lrm4 (e) and Pkd2^−/− (f). n = 5 E15.5 kidneys from 5 independent litters were examined for each genotype. See Supplementary Fig. 2 for representative images of embryonic kidneys. Scale bar 25 µm

Fig. 2

Protein and RNA expression levels of PC2. a Western blots showing embryonic kidney lysates from E14.5 kidneys. The arrow indicates the PC2 band at ~110 KDa (the extra bands are due to non-specific binding of antibody, see Supplementary Fig. 6). The final lane represents Pkd2^lrm4/lrm4 kidney lysate. Tubulin loading control is shown at 50 KDa. b Graph represents protein levels normalised to tubulin loading control. Error bars represent SEM. n = 8 for each sample (pool of 3 kidney pairs). Pkd2^+/− (−/+) and Pkd2^lrm4/lrm4 (lrm4/lrm4) samples are reduced by 44.0% and 48.6%, respectively, compared to Wt (+/+). c Schematic of PC2 gene locus showing three transcripts predicted by ensembl. The positions of primers are indicated. The position of a loxP site that replaces exons 8–9 in the null allele is marked with a triangle and the position of the lrm4 point mutation is marked with an asterisk (*). d qPCR of RNA extracted from MEF cells reveals that PC2 transcription is not reduced in Pkd2^lrm4/lrm4 MEFs using primers covering three areas of Pkd2; Exons 2–3 (red), Exons 8–9 (blue), and Exons 12–13 (green) are the exons deleted in the null construct. Pkd2^+/+ (+/+), Pkd2^+/−(+/−), Pkd2^−/− (−/−) and Pkd2^lrm4/lrm4 (lrm4) MEF cells were assessed. Two sets of MEFs for each genotype were analysed and a technical repeat was performed for each sample analysed. See source data for densitometry values presented in b

Fig. 3

N-glycosylation pattern and cellular location of PC2. a Diagram depicting cleavage sites of PNGaseF and EndoH. PNGaseF cleaves between the innermost GlcNAc and asparagine of a high mannose glycoprotein. EndoH cleaves between two core GlcNAcs of an N-linked, high mannose glycoprotein. b Schematic of predicted glycosylation sites in PC2 (compiled from graphs in Supplementary Fig. 7). The arrows represent predicted sites of glycosylation. The black arrows represent sites shared by mouse and human. Unique sites are present in both human (green arrow) and mouse (blue arrow) sequences. The Pkd2^lrm4 mutation site is marked with a red star in the first extracellular loop. c MEF total cell lysates digested with PNGaseF or EndoH. To the right of each blot, a diagram represents the bands; a black asterisk (*) shows the undigested band and a red asterisk (*) indicates the enzyme-sensitive band. Both Wt (three left panels) and Pkd2^lrm4/lrm4 (right three panels) samples are PNGaseF and EndoH sensitive. Representative images of Pkd2^+/+ and Pkd2^lrm4/lrm4 cells stained with PC2(red) and d acetylated tubulin (green) or e PDI marking the ER. Cells show no overall changes in cellular PC2 localisation. PC2 is seen at the base of the cilium in mutant cells. White arrow denotes PC2 in the cilium in Wt cells. Scale bar 10 μm

Fig. 4

Ciliary localisation of PC2 protein. SIM images of MEF and kidney cell cilia showing PC2 localisation. a, b are representative of primary kidney cell cilia. In Wt MEF cells, CEP164, a component of the distal appendages of the mother centriole localises at the base of the cilium (c). In Wt kidney cilia (a) and Wt MEF cilia (d, f), PC2 localises along the length of the cilium. PC2 is seen in a ring structure at the base of the cilium in both Pkd2^lrm4/lrm4 kidney cilia (b) and MEF cilia (e, g) (white arrow). h Arl13b marks the ciliary membrane and PC2⁺ localises along the length of the cilium and is observed more densely at the cilium base. i PC2 is seen as a cluster at the base of the cilium. j Gamma tubulin localises at the base of the cilium and PC2⁺ can be seen to localise along the length of the cilium. k In mutant cells, a pool of PC2^lrm4 associates preferentially with the mother centriole (white arrow). In Pkd2^lrm4/lrm4 cells, PC2 clusters at the base of the cilium (white arrow). l CEP164 marks the distal appendages at the base of the cilium and PC2 localises along the length of the cilium as well as preferentially to the mother centriole. m The PC2^lrm4 cluster associates with the distal appendages. n The diagram indicates the position of each antibody with respect to the cilium with corresponding colours. Constituents of the ciliary axoneme or membrane: Ift88, acetylated tubulin and Arl13b are coloured magenta; PC2 is coloured green; Gamma tubulin, marking the basal body, is coloured blue; and cep164, marking the distal appendages, is coloured yellow

Fig. 5

PKD1 is not detected in the cilium in Pkd2^lrm4/lrm4 cells. In Wt MEFs, PKD1 localises along the length of the cilium. a–f enlarged from boxes in Merge on left. PC1 localises along the length of Wt cilia (a–c). In Pkd2^lrm4/lrm4 mutant cells, PC1 ciliary localisation is no longer observed. g Western blot analysis of MEF samples probed with an N-terminal-directed PC1 antibody and an antibody directed against PC2. β-Actin serves as a loading control. h Densitometry of PC1 and PC2 bands normalised to β-actin is represented as graphs. Error bars represent SEM. See source data for densitometry values presented in g