Generation of heterozygous PKD1 mutant pigs exhibiting early-onset renal cyst formation

Masahito Watanabe,Kazuhiro Umeyama,Kazuaki Nakano,Hitomi Matsunari,Toru Fukuda,Kei Matsumoto,Susumu Tajiri,Shuichiro Yamanaka,Koki Hasegawa,Kazutoshi Okamoto,Ayuko Uchikura,Shuko Takayanagi,Masaki Nagaya,Takashi Yokoo,Hiromitsu Nakauchi,Hiroshi Nagashima

doi:10.1038/s41374-021-00717-z

Masahito Watanabe, Kazuhiro Umeyama + Show 14 more

Open Access

https://doi.org/10.1038/s41374-021-00717-z

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Abstract

AbstractAutosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, manifesting as the progressive development of fluid-filled renal cysts. In approximately half of all patients with ADPKD, end-stage renal disease results in decreased renal function. In this study, we used CRISPR-Cas9 and somatic cell cloning to produce pigs with the unique mutation c.152_153insG (PKD1insG/+). Pathological analysis of founder cloned animals and progeny revealed that PKD1insG/+ pigs developed many pathological conditions similar to those of patients with heterozygous mutations in PKD1. Pathological similarities included the formation of macroscopic renal cysts at the neonatal stage, number and cystogenic dynamics of the renal cysts formed, interstitial fibrosis of the renal tissue, and presence of a premature asymptomatic stage. Our findings demonstrate that PKD1insG/+ pigs recapitulate the characteristic symptoms of ADPKD.Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the formation of cysts within the kidneys. The authors generated PKD1 heterozygous knockout (PKD1insG/+) pigs by CRISPR-Cas9 and somatic cell cloning techniques. The founder cloned animals and progeny showed the renal cyst formation from the neonatal stage, interstitial fibrosis of the renal tissue, and the presence of a premature asymptomatic stage. Their findings demonstrate that PKD1insG/+ pigs recapitulate the characteristic symptoms of ADPKD.

Highlights

Autosomal dominant polycystic kidney disease (ADPKD) affects one in every 500–1000 people worldwide and is the most common inherited kidney disease[1,2,3]
The PKD1insG/+ pigs produced in this study (Supplementary Table S4) with a mutation introduced into exon 1 of PKD1 (c.152_153insG) by CRISPR-Cas[9] exhibited a similar phenotype as human patients with ADPKD, including high penetrance of renal cyst formation
The progeny pigs faithfully reproduced the renal cystic phenotype, which is the major symptom of ADPKD

Summary

INTRODUCTION

Autosomal dominant polycystic kidney disease (ADPKD) affects one in every 500–1000 people worldwide and is the most common inherited kidney disease[1,2,3]. The gene dosage (haploinsufficiency) model hypothesizes that a reduction in PKD1 expression levels below a critical threshold leads to renal cyst formation[14,15] These rodent models have provided insights into ADPKD, the specific features of the models limit the ability to extrapolate the research outcomes to human patients. Received: 13 July 2021 Revised: 16 November 2021 Accepted: 27 November 2021 patients, who begin developing cysts during the fetal period, Analysis of CRISPR-Cas9-induced mutations in nuclear donor monoallelic PKD1 KO pigs do not display renal cysts neonatally. The resulting PKD1insG/+ cloned pigs displayed characteristics of an ADPKD model, including (i) neonatal renal cyst formation, (ii) progressive cyst development during animal growth, and (iii) sustained fertility after sexual maturation. The top 10 potential off-target sites were selected, and regions overlapping the sites were amplified by PCR using appropriate sets of primers (Supplementary Table S1) and

MATERIALS AND METHODS

RESULTS

DISCUSSION

34. Autosomal dominant polycystic kidney disease mutation database

Findings

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