PKD1 and PKD2 mRNA cis-inhibition drives polycystic kidney disease progression

Abstract

Autosomal dominant polycystic kidney disease (ADPKD), among the most common human genetic conditions and a frequent etiology of kidney failure, is primarily caused by heterozygous PKD1 mutations. Kidney cyst formation occurs when PKD1 dosage falls below a critical threshold. However, no framework exists to harness the remaining allele or reverse PKD1 decline. Here, we show that mRNAs produced by the noninactivated PKD1 allele are repressed via their 3'-UTR miR-17 binding element. Eliminating this motif (Pkd1^∆17) improves mRNA stability, raises Polycystin-1 levels, and alleviates cyst growth in cellular, ex vivo, and mouse PKD models. Remarkably, Pkd2 is also inhibited via its 3'-UTR miR-17 motif, and Pkd2^∆17-induced Polycystin-2 derepression retards cyst growth in Pkd1-mutant models. Moreover, acutely blocking Pkd1/2 cis-inhibition, including after cyst onset, attenuates murine PKD. Finally, modeling PKD1^∆17 or PKD2^∆17 alleles in patient-derived primary ADPKD cultures leads to smaller cysts, reduced proliferation, lower pCreb1 expression, and improved mitochondrial membrane potential. Thus, evading 3'-UTR cis-interference and enhancing PKD1/2 mRNA translation is a potentially mutation-agnostic ADPKD-arresting approach.

Fig. 1. Pkd1 mRNA is cis-repressed via its 3′-UTR miR-17 binding motif.

a Graphic illustration of the CRISPR/Cas9 approach used to delete the miR-17 motif from Pkd1 3′-UTR (Pkd1^Δ17). b PCR products obtained after amplification of tail DNA from mice with indicated genotypes. The lower band represents the Δ17 deletion. n = 3 for all genotypes. c 3′-UTR nucleotide sequence of wildtype (WT) and Pkd1^Δ17 alleles. The miR-17 binding motif and sgRNA PAM sites are highlighted in bold green and pink, respectively. The pink dashed line indicates the deleted nucleotides in Pkd1^Δ17. Sanger sequencing chromatogram depicting the nucleotide sequence of the Pkd1^Δ17 allele is shown. d H&E staining, Lotus Tetragonolobus Lectin labeling (LTL, a proximal tubule marker), Tamm-Horsfall protein immunostaining (THP, a loop of Henle maker), and Dolichos Biflorus Agglutinin labeling (DBA, a collecting duct marker) showing normal kidney histology in 8-week-old Pkd1^+/+ and Pkd1^Δ17/Δ17 mice. e–f Normal kidney-weight-to-body-weight (KW/BW) and serum blood urea nitrogen (BUN) levels in 8-week-old Pkd1^+/+ and Pkd1^Δ17/Δ17 mice. g–h Images and cyst index quantification of E13.5 Pkd1^+/+, Pkd1^Δ17/+, and Pkd1^Δ17/Δ17 kidneys grown for four days in culture media containing vehicle, 100 uM cAMP, or 100 uM cAMP plus 250 uM SAM. i Immunoblots depicting PC1 expression in Pkd1^+/+, Pkd1^Δ17/+, and Pkd1^Δ17/Δ17 ex-vivo kidneys treated with vehicle, cAMP, or cAMP plus SAM. Actin is used as the loading control. j Allele-specific qRT-PCR showing the quantity of Pkd1 mRNAs produced by the wildtype (+) and Δ17 alleles in E15.5 Pkd1^Δ17/+ kidneys (n = 5). Error bars indicate SEM. Statistical analysis: Two-tailed Student’s t-test (e, f); One-way ANOVA, Tukey’s multiple-comparisons test (h); Two-tailed Paired t-test (j). Source data are provided as a Source Data file.

Fig. 2. Monoallelic Pkd1 derepression alleviates polycystic kidney disease.

a Immunoblot showing reduced PC1 expression in Pkd1^RC/- compared to Pkd1^RC/+ cells. PC1 level was restored in Pkd1^RCΔ17/- cells. #1 and #2 refer to the two independent Pkd1^RCΔ17/- clonal cell lines. Actin serves as the loading control. n = 3 biologically independent samples. b–c Representative images and quantification showing increased 3D cyst size of Pkd1^RC/- compared to Pkd1^RC/+ cells cultured in Matrigel. Cyst size was normalized in Pkd1^RCΔ17/- cells. n = 300 cysts pooled from three independent experiments. d Heatmap showing alamarBlue-assessed proliferation of Pkd1^RC/- and Pkd1^RCΔ17/- cells in the absence (−) or presence (+) of 100 uM cAMP, 17 mM glucose, or 100 uM SAM. n = 8, each circle represents a biological replicate. e Representative images showing Mito-tracker labeling and anti-pCreb1 immunostaining in Pkd1^RC/+, Pkd1^RC/-, and Pkd1^RCΔ17/- cells. n = 3 biologically dependent experiments. f Gross kidney and H&E-stained kidney sections from 18-day-old mice with the indicated genotypes. Data from the progeny of the three founders are shown separately. Founder #1: Pkd1^RC/+ (n = 7), Pkd1^RC∆17/+ (n = 7), Pkd1^RC/- (n = 8), and Pkd1^RC∆17/- (n = 7). Founder #2: Pkd1^RC/+ (n = 15), Pkd1^RC∆17/+ (n = 19), Pkd1^RC/- (n = 15), and Pkd1^RC∆17/- (n = 17). Founder #3: Pkd1^RC/+ (n = 6), Pkd1^RC∆17/+ (n = 6), Pkd1^RC/- (n = 8), and Pkd1^RC∆17/- (n = 8). g Immunoblot showing PC1 expression in kidneys of 18-day-old mice with the indicated genotypes derived from the three founders. Actin serves as the loading control. n = 3 independent kidney samples for each genotype and founder. h, i KW/BW ratio and BUN levels in mice with the indicated genotypes are shown. Data from all three founders are shown. Founder#1 (blue circles), Founder#2 (light pink circles), and Founder#3 (orange circles). (j) Paired-end RNA-seq data showing the RC allele usage in Pkd1^RC/- (grey circles, n = 5), Pkd1^RCΔ17/- founder#2 (pink circles, n = 5), and Pkd1^RCΔ17/- founder#3 (orange circles, n = 5). Error bars indicate SEM. Statistical analysis: One-way ANOVA, Tukey’s multiple-comparisons test (c, h–j). Source data are provided as a Source Data file.

Fig. 3. Pkd1 derepression attenuates cyst-pathogenic events and disease progression.

a Gross kidney images and H&E-stained kidney sections from 18-week-old mice with the indicated genotypes derived from founder#3. miR-17 motif deletion was associated with sustained benefit and suppressed long-term PKD progression. n = 3 (Pkd1^RC/+), n = 3 (Pkd1^RC∆17/+), n = 8 (Pkd1^RC/-), and n = 7 (Pkd1^RC∆17/-). b KW/BW, BUN, and serum creatinine (Scr) levels in the 18-week-old progeny of founder#3 are shown. c Heatmap showing global mRNA expression profiles of kidneys from 18-day-old mice with the indicated genotypes. mRNAs that were dysregulated in Pkd1^RC/- compared to Pkd1^RC/+ kidneys but exhibited improved expression in Pkd1^RCΔ17/- kidneys were chosen for visualization. #3 = founder#3; #2 = founder#2. n = 3 (Pkd1^RC/+), n = 3 (Pkd1^RC∆17/+ founder 2), n = 3 (Pkd1^RC∆17/+ founder 3), n = 5 (Pkd1^RC/-), n = 5 (Pkd1^RC∆17/- founder 2), and n = 5 (Pkd1^RC∆17/- founder 3). (d) Representative images showing phospho-Histone-H3 (pHH3), Mannose Receptor C-Type 1 (MRC1), or pCreb1 immunostaining in kidney sections of 18-day-old and 18-week-old Pkd1^RC/- (n = 5) and Pkd1^RCΔ17/- (n = 5) mice. The sections were co-labeled with DBA to mark collecting duct-derived cysts. Error bars indicate SEM. Statistical analysis: One-way ANOVA, Tukey’s multiple-comparisons test (b). Source data are provided as a Source Data file.

Fig. 4. Pkd2 derepression retards cyst growth in Pkd1-mutant models.

a Immunoblot showing Polycystin-2 (PC2) expression in cells with the indicated genotypes. miR-17 motif deletion from Pkd2 3′-UTR leads to higher PC2 expression in Pkd1^RC/- cells. Actin serves as the loading control. #1 and #2 refer to the two independent Pkd1^RC/-; Pkd2^Δ17/Δ17 clonal cell lines. n = 3 biologically independent samples for both clones. b, c Representative images and quantification showing 3D cyst size of cells with indicated genotypes grown in Matrigel cultures. n = 300 cysts pooled from three independent experiments. d Representative images showing mitotracker labeling and anti-pCreb1 immunostaining in cells with the indicated genotypes. n = 3 biologically independent experiments. e Heatmap showing alamarBlue-assessed proliferation of Pkd1^RC/- and Pkd1^RC/-; Pkd2^Δ17/Δ17 cells in the absence (−) or presence (+) of cAMP, glucose, or SAM. n = 8, each circle represents a biological replicate. f H&E-stained kidney sections from 18-day-old mice with the indicated genotypes are shown. n = 3 (Pkd1^RC/+; Pkd2^+/+), n = 3 (Pkd1^RC/+; Pkd2^∆17/∆17), n = 8 Pkd1^RC/-; Pkd2^+/+), and n = 11 (Pkd1^RC/-; Pkd2^∆17/∆17). g Immunoblots showing PC2, Yap1, and c-Myc expression in kidneys of 18-day-old mice with the indicated genotypes (n = 3 for each group). h, i KW/BW and serum creatinine levels in 18-day-old mice with the indicated genotypes. j Representative images showing pHH3 and MRC1 immunostaining in kidney sections from 18-day-old mice with the indicated genotypes (n = 3 for each group). k Heatmap showing differential mRNA expression in kidneys of 18-day-old mice with the indicated genotypes (n = 3). mRNAs that were dysregulated in Pkd1^RC/- compared to Pkd1^RC/+ kidneys but exhibited improved expression in Pkd1^RC/-; Pkd2^Δ17/Δ17 kidney were chosen for heatmap visualization. Error bars indicate SEM. Statistical analysis: One-way ANOVA, Tukey’s multiple-comparisons test (b, h, and i). Source data are provided as a Source Data file.

Fig. 5. Acute Pkd1 and Pkd2 derepression attenuate PKD.

a–b qRT-PCR and immunoblot analysis showing Pkd1/PC1 and Pkd2/PC2 expression in Pkd1^RC/- cells transfected with vehicle (PBS), 100 uM control oligonucleotide, or 100 uM RGLS4326. c Images and quantification of 3D cyst size of Pkd1^RC/- cells cultured in Matrigel before (day 4) or after (day 7) transfection with vehicle (PBS), 100 uM control oligonucleotide, or 100 uM RGLS4326. d H&E-stained kidney sections from 18-day-old Pkd1^RC/- mice injected on P10, P11, P12, and P16 either with vehicle (PBS), 20 mg/kg control oligonucleotide, or 20 mg/kg RGLS4326. H&E-stained kidney section from untreated 18-day-old wildtype mouse is shown for reference. e–g KW/BW, BUN, and serum creatinine levels in 18-day-old Pkd1^RC/- mice treated with vehicle (PBS), 20 mg/kg control oligonucleotide, or 20 mg/kg RGLS4326 are shown. Data from untreated 18-day-old wildtype mice are shown as a reference. h H&E-stained kidney sections from 26-day-old Pkd1^RC/- mice injected on P16 and P17 with 20 mg/kg control oligonucleotide or 20 mg/kg RGLS4326. H&E-stained kidney sections from genetically matched but untreated 16-day-old Pkd1^RC/- mice are shown to depict disease prior to starting treatment. i–k KW/BW, BUN, and serum creatinine levels in untreated 16-day-old or treated 26-day-old Pkd1^RC/- mice are shown. l–n Pkd1^RC/- mice were injected on P16 and P17 with vehicle, 20 mg/kg RGLS4326, or 20 mg/kg control oligonucleotide. These mice then received their respective treatment regimen every week until 18 weeks of age. The fourth cohort of Pkd1^RC/- mice received 20 mg/kg RGLS4326 treatment on P16, P17, and bimonthly thereafter. l H&E-stained kidney sections of 125-day-old Pkd1^RC/- mice on the vehicle or RGLS4326 treatment are shown. m Kaplan-Meir survival curves of Pkd1^RC/- mice in the four treatment groups. Survival of untreated wildtype is shown as a reference. n KW/BW ratio in mice that survived till 125 days. Wildtype mice: n = 3; Pkd1^RC/- mice: n = 2 (vehicle treatment), n = 5 (weekly RGLS4326 treatment), and n = 7 (bi-monthly RGLS4326 treatment). Error bars indicate SEM. Statistical analysis: One-way ANOVA, Tukey’s multiple-comparisons test (a, c, e–g, i–k, n); Mantel-Cox (m). Source data are provided as a Source Data file.

Fig. 6. PKD1^Δ17 or PKD2^Δ17 reduce 3D cyst growth in primary human ADPKD cultures.

CRISPR/Cas9-editing was used to delete the miR-17 motif from PKD1 3′-UTR (PKD1^Δ17) or PKD2 3′-UTR (PKD2^Δ17) in primary ADPKD cultures from four human donors (#1 through #4). a, b Immunoblots showing higher PC1 expression in PKD1^Δ17 and higher PC2 expression in PKD2^Δ17 ADPKD cultures compared to their respective unedited (UE) parental ADPKD cultures. Protein bands are 460 kDa (a) and 110–120 kDa size (b). Actin serves as the loading control. c–f Images and quantification showing reduced cyst size of PKD1^Δ17 and PKD2^Δ17 compared to their respective unedited (UE) parental ADPKD cultures. g, h Images showing higher mitotracker labeling (red) and reduced pCREB1 immunostaining (green) in PKD1^Δ17 and PKD2^Δ17 ADPKD cultures compared to their respective unedited parental ADPKD cultures. n = 3 biologically independent experiments for each cell line. Errors bars represent SEM, Statistical analysis: Two-tailed Students t-test (e, f). Source data are provided as a Source Data file.