Essential role of cleavage of Polycystin-1 at G protein-coupled receptor proteolytic site for kidney tubular structure

Abstract

Polycystin-1 (PC1) has an essential function in renal tubular morphogenesis and disruption of its function causes cystogenesis in human autosomal dominant polycystic kidney disease. We have previously shown that recombinant human PC1 is cis-autoproteolytically cleaved at the G protein-coupled receptor proteolytic site domain. To investigate the role of cleavage in vivo, we generated by gene targeting a Pkd1 knockin mouse (Pkd1(V/V)) that expresses noncleavable PC1. The Pkd1(V/V) mice show a hypomorphic phenotype, characterized by a delayed onset and distal nephron segment involvement of cystogenesis at postnatal maturation stage. We show that PC1 is ubiquitously and incompletely cleaved in wild-type mice, so that uncleaved and cleaved PC1 molecules coexist. Our study establishes a critical but restricted role of cleavage for PC1 function and suggests a differential function of the two types of PC1 molecules in vivo.

Fig. 1.

Generation of the Pkd1^V/V mouse by gene targeting. (A) Structure of various Pkd1 alleles. (i) Targeting vector. Neo, FRT-flanked (red triangle) PGK-neomycin selection cassette; TK, thymidine kinase gene; S, SpeI. Exons are depicted by solid boxes. The T3041V mutation is indicated. (ii) Pkd1 WT allele, with exon number shown. (iii) Pkd1^NeoV allele. The expected size of the SspI or SpeI restriction fragments for WT and Pkd1^NeoV alleles by using the 5′ probe (within exon 15 outside of the vector) is shown. The expected size of the 3′ long-range PCR product is also shown. (iv) Pkd1^V allele. A single FRT site of 47 bp is retained at the site of Neo insertion. (B) Genomic Southern blot showing correct targeting of the ES clone G8 by detection of 9.9-kb (SspI) and 10.4-kb (SpeI) Pkd1^NeoV-specific bands by the 5′ probe. The 3′ long-range genomic PCR confirmed correct targeting (data not shown). (C) Removal of Neo in the offspring of the Pkd1^NeoV/+:FLP mating as shown by the three-primer PCR-based strategy. (i) Primer positions and the expected size of the PCR product of the each allele. (ii) PCR result showing successful removal of Neo by amplification of the Pkd1^V-specific 329-bp product (Upper) only in the FLP-positive offspring (Lower). −, negative control for PCR. (D) RT-PCR from WT, Pkd1^V/+ (V/+), and Pkd1^V/V (V/V) kidneys at P9. The expected size of the PCR product using exon 23 and 27 specific primers is shown (Upper). A single product of expected size was amplified from each genotype (Lower). DNA size marker is shown on right. (E) DNA sequence of the RT-PCR products in D at the cleavage size.

Fig. 2.

Postnatal development of cystic kidney in Pkd1^V/V mice. General appearance of the mice and their kidneys at P9 (A) and P16 (B). Note that Pkd1^V/V kidneys are enlarged, pale, and cystic compared with kidneys from WT and heterozygous (V/+) littermates. (C) Survival curve of WT (n = 33), V/+ (n = 62), and V/V (n = 29) mice. The median age of survival of Pkd1^V/V mice is 23 days. (D) Elevated BUN level in the Pkd1^V/V mouse as compared with that in Pkd1^V/+ and WT littermates at P14 and P23.

Fig. 3.

Rapid and progressive cystic dilation in Pkd1^V/V postnatal kidneys. (A) Overview of Pkd1^V/V kidneys of various postnatal stages in H&E-stained sections, with two to three animals analyzed for most of the stages. (Scale bars, 2 mm.) Note the intact papilla tip in P3–P14 kidneys. P22 and P28 kidneys are more enlarged (≈15 mm long) with further cystic expansion (data not shown). (B–D) H&E-stained sections of P5, P7, and P14 Pkd1^V/V kidneys, respectively. Note the intact glomeruli and proximal tubule (PT)-like structures. (E) Masson-trichrome (MT)-stained kidney section of P22 Pkd1^V/V mouse. Note the intact glomeruli and PT-like tubules surrounded by interstitial fibrosis. (F) H&E-stained section of P28 Pkd1^V/V kidney. Note the large size of the cysts and intact glomeruli and PT-like tubules (Inset). (G) MT-stained liver section of P14 Pkd1^V/V mouse. Note the fibrosis around the dilated biliary ducts.

Fig. 4.

Distal nephron segment origin of cysts in Pkd1^V/V kidneys. (A–H) Pkd1^V/V kidney sections of various postnatal stages were double-stained with segment-specific markers as indicated under each section. Lectin Lotus tetragonolobus (LTL), proximal tubule (PT); lectin Dolichos biflorus (DBA), collecting duct (CD) (26); AQP2, CD (27); Tamm–Horsfall protein (THP), both the thick ascending limb (TAL) and the distal convoluted tubule (DCT) (28); and Na-K-Cl Cotransporter 2 (NKCC2), TAL only (29). (I) Summary of cyst origin in Pkd1^V/V kidneys. Cysts are derived from DCT and CD (in red) (except the papilla tip), and PT and TAL are not dilated.

Fig. 5.

Cleavage patterns of endogenous PC1 in WT and Pkd1^V/V mice. Endogenous PC1 was immunoprecipitated by using anti-CC and detected on Western blot by anti-CC. The protein size marker and the position of uFL-PC1 and CTF are indicated. (A) Schematic diagram of the domain organization of mouse PC1. LRR, Leucine-rich repeat; R1 and R2–16, PKD repeats; CLD, C-type lectin domain; REJ, receptor for egg jelly domain. The cleavage site HL̂T³⁰⁴¹ within GPS and the resulting NTF (3,040 aa) and CTF (1,253 aa) are shown. The epitope recognized by anti-CC or anti-CT (the equivalent antibody against human PC1), and anti-LRR used in this study is indicated by a black bar. (B) Western blots demonstrating the specificity of anti-CC. It detects both C-terminally FLAG-tagged uFL and CTF from lysate of HEK cells with stable expression of mouse full-length Pkd1 cDNA (mPC1-F) after IP with either anti-FLAG or anti-CC (Right). These signals were not detected from untransfected HEK cells (data not shown). Note that this pattern is identical to that of exogenously expressed human recombinant (hPKD1-F) or endogenous PC1 of HEK cells (HEK). (C–I) Western blots showing PC1 cleavage pattern in MEFs of various genotypes as indicated (C) and whole E12 embryos of various genotypes as indicated (D). Bracket indicates nonspecific bands that are also present in E12.5 and E14.5 Pkd1^−/− embryos (Right). (E) WT embryos of various stages as indicated. (F) Postnatal kidneys of WT and Pkd1^V/Vmice at different stages. * indicates an unknown PC1-specific band that occurs only in Pkd1^V/V cystic kidneys. (G) Primary PT and CD cells isolated from P5 WT and Pkd1^V/Vkidneys (Left) and conditionally immortalized CD cells isolated from P14 WT and Pkd1^V/Vkidneys (Right). Same results were obtained for primary PT and CD cells isolated from P10 WT and Pkd1^V/Vkidneys, respectively (data not shown). The purity of the PT and CD cells is verified by using the segment-specific markers [AQP2 for CD; APN, aminopeptidase N, for PT (30)] on Western blot of total lysates, or DBA by immunofluorescence. (H) Various organs of P3 WT and Pkd1^V/Vmice. B, brain; L, liver; K, kidney; Lu, lung; H, heart. (I) Organs of WT mice at P19. S, spleen; P, pancreas. Note the low expression level of PC1 in kidney. Arrowhead in G and H indicates the high-molecular-weight band in Pkd1^V/V cells and organs that probably represents a modified uFL form.