Fig 2: 2HA-PC1-CTT (CTT) colocalizes and interacts with mitochondrial enzyme NNT.A Workflow for identification of protein interactors of mitochondrial-associated pools of PC1 and its fragments. Mitochondria from Pkd1F/H-BAC and WT mice were solubilized, crosslinked (3 mM DTSSP), and immunoprecipitated with anti-HA antibodies. Recovered proteins were identified by mass spectrometry. B Anti-HA immunoprecipitates from mitochondrial fractions were blotted with HRP-conjugated anti-HA, revealing recovery of the CTF of full-length 3FLAG-PC1-3HA as well as PC1-CTT-3HA fragments from Pkd1F/H-BAC but not WT control mice. C Volcano plot of PC1 and PC1-CTT interactors from Pkd1F/H-BAC vs WT kidneys (Colored dots: P < 0.05 determined by two-tailed Fisher’s exact test; n = 3 per group). The amount of NNT co-immunoprecipitating with CTT from Pkd1F/H-BAC kidneys was >16-fold greater than that in WT control immunoprecipitates (P < 10-10). D Anti-NNT immunoprecipitates from kidney lysates were immunoblotted with HRP-conjugated anti-HA, revealing coimmunoprecipitation of the 3FLAG-PC1-3HA CTF as well as PC1-CTT-3HA fragments from Pkd1F/H-BAC but not WT control mice. E Representative immunofluorescence (100×) image showing localization of the 2HA-PC1-CTT construct, identified with an anti-PC1-C-terminus antibody, expressed by transient transfection in HEK293 cells. CTT colocalizes in mitochondria with endogenous NNT (yellow arrows). As previously reported17, CTT was also observed in nuclei of a subset of transfected cells (red arrow). Scale bar:10 µm. F The mitochondria-associated fraction of CTT in (E) was assessed through Mander’s colocalization analysis (20 individual cells from 14 independent images, 3 biological replicates), revealing a 0.8535 overlap coefficient, indicating extensive overlap between CTT and NNT distributions. As expected, the TOMM20/NNT colocalization coefficient (measured as an experimental positive control) is slightly but significantly greater. Data are shown as mean ± SEM. Pairwise comparison was performed using two-tailed Student’s t-test. Colocalization of TOMM20 and NNT is shown in Supplementary Fig. 3. G Immunoblotting of total kidney lysates from WT “N” and “J” mice, confirming the presence and absence of NNT, respectively. H Immunoblotting of anti-HA immunoprecipitates from mouse kidney lysates, revealing immunoprecipitation of CTT in both “N” and “J” Pkd1-KO + CTT mice. NNT coimmunoprecipitation is detected exclusively in immunoprecipitates from N-Pkd1-KO + CTT mice. See Supplementary Data 1 for the complete comparative proteomic analysis depicted in (C). Source data are provided as a Source Data file.

Fig 3: CTT expression in pre-cystic mice reveals pronounced CTT-dependent redox modulation and distinct changes in metabolic profile.A Schematic representation of the experimental timeline for assessment of untargeted comparative metabolomics in pre-cystic mice at 10 weeks of age. B Comparative analysis between pre-cystic N-Pkd1-KO ± CTT revealed no statistically significant change in KW/BW ratio. Cystic mouse cohorts are composed of 50–60% female and 40–50% male mice. C, D Kidney function is preserved in 10-week-old N-Pkd1-KO and N-Pkd1-KO + CTT mice, as revealed by normal and comparable values of serum creatinine (C) and BUN (D) in both groups. E PCA plot of LC–MS-based metabolomic data from 10-week-old N-Pkd1-KO + CTT vs N-Pkd1-KO. Each individual mark corresponds to a different sample (n = 5 or 6 mice per group as shown in figure) and its location in the plot is determined by the relative contributions of subsets of metabolites to the variance among samples. F Volcano plot showing differences in metabolic profiling of the kidney extracts from 10-week-old N-Pkd1-KO ± CTT mice. The vertical lines in each panel mark twofold changes; horizontal lines mark P < 0.05 determined by Student’s t-test; n = 5 mice for N-Pkd1-KO + CTT and n = 6 mice for N-Pkd1-KO mice. The labeled colored dots indicate specific metabolites with significant fold changes. Complete untargeted comparative metabolomic analysis is provided in Supplementary Data 3. G, H LC–MS detection of NAD(P)(H) cofactors showing a ~5-fold change in NADH/NAD+ (G) and a ~6-fold change in NADPH/NADP+ (H) ratios in 10-week N-Pkd1-KO ± CTT mice. Data are normalized to mean values observed in N- Pkd1-KO mice. Data are depicted with the mean ± SEM. Pairwise comparisons were performed using two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig 4: Expression of CTT partially suppresses cystic disease in a rapidly progressive orthologous mouse model of ADPKD when generated on an NNT-competent background.A Development of the 2HA-PC1-CTT;Pkd1fl/fl;Pkhd1-Cre mouse model. We crossed the 2HA-PC1-CTT;Pkd1fl/fl;Pax8rtTA;TetO-Cre mouse model on the “N” background (N-Pkd1-KO + CTT) with the previously characterized developmental Pkd1fl/fl;Pkhd1-Cre (Pkhd1-Cre;Pkd1-KO)22–24,41 ADPKD mouse model, generated on the “J” background. Breeders with a Pkd1fl/+;Pkhd1-Cre genotype were used, due to the constitutive activity of the Pkhd1-Cre that initiates collecting duct-specific Cre-mediated recombination during embryonic stages and prevents animals with the complete Pkd1fl/fl;Pkhd1-Cre from reaching sexual maturity. B Comparative analysis of NJ F1 (purple box) generation mice showing differences in KW/BW ratio. Of note, no phenotype differences were observed in NJ WT mice that do or do not express CTT (NJ;Pkhd1-Cre + CTT and NJ;WT, respectively). Red dots represent the animals depicted in (E). C Comparative analysis of NN F2 (blue box) generation mice showing differences in KW/BW ratio. Of note, no phenotype differences were observed in NN WT mice that do or do not express CTT (NN;Pkhd1-Cre + CTT and NN;WT, respectively). Red dots represent the animals depicted in (F). D Comparative analysis of JJ F2 (red box) generation mice showing differences in KW/BW ratio. Of note, no phenotype differences were observed in JJ WT mice that do or do not express CTT (JJ;Pkhd1-Cre + CTT and JJ;WT, respectively). Red dots represent the animals depicted in (G). E–G H&E-stained kidney sections (4×) from p14 NJ F1 (purple box), NN F2 (blue box), and JJ F2 (red box) mice. Each group of sections is representative of the average KW/BW ratio of the entire cohort. Scale bar: 2 mm. Data are depicted with the mean ± SEM. Multiple group comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. H&E-stained kidney sections from all of the cystic mice included in these 3 cohorts are provided in Supplementary Fig. 7. Source data are provided as a Source Data file.

Fig 5: Expression of polycystin-1 C-terminal tail (CTT) suppresses cystic disease in an orthologous mouse model of ADPKD.A Design of the 2HA-PC1-CTT;Pkd1fl/fl;Pax8rtTA;TetO-Cre (Pkd1-KO + CTT) mouse model. We generated transgenic mice that carry a BAC-2HA-PC1-CTT transgene inserted in the Rosa26 locus and preceded by a neomycin resistance (NeoR) STOP cassette flanked by loxP sequences. These mice were crossed with the previously characterized Pkd1fl/fl;Pax8rtTA;TetO-Cre mouse model of ADPKD in which exons 2–4 of the Pkd1 gene are flanked by loxP sequences22,74. Cre-mediated recombination of these second-generation 2HA-PC1-CTT;Pkd1fl/fl;Pax8rtTA;TetO-Cre mice via doxycycline induction promotes 2HA-PC1-CTT protein expression and loss of full-length PC1 protein expression in tubular epithelial cells. B–D Comparative analysis of N-Pkd1-KO, N-Pkd1-KO + CTT, and N-WT mice showing differences in KW/BW ratio (B), BUN (C), and serum creatinine (D). Cystic mouse cohorts are composed of 53–58% female and 42–47% male mice. Red dots represent the animals depicted in (E). E Representative H&E-stained kidney sections (4×) from 16-week N-Pkd1-KO, N-Pkd1-KO + CTT, and N-WT mice. Each group of sections is representative of the average KW/BW ratio of the entire cohort and is illustrative of the inherent variability associated with this mouse model22,32. Scale bar: 2 mm. Non-parametric data are depicted with the median and interquartile range (blue bars). Multiple group comparisons were performed using Kruskal–Wallis test followed by Dunn’s multiple-comparisons test. H&E-stained kidney sections from all of the cystic mice included in this cohort are provided in Supplementary Fig. 1. Source data are provided as a Source Data file.