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: Schematic representation and identification of PC1 cleavage sites and cleavage products.The N-terminal domain of PC1 undergoes autocatalytic cleavage at the G protein-coupled receptor Proteolytic Site (GPS), generating a large 3048-aa N-terminal fragment (NTF) that remains non-covalently attached to the 1254-aa C-terminal fragment (CTF)15. PC1 undergoes further cleavage at the C-terminal domain, giving rise to PC1 CTF fragments, comprised of transmembrane domains and the cytoplasmic PC1 C-terminal tail. An ~100-kDa transmembrane CTF localizes to the endoplasmic reticulum. Cleavage may occur within the C-terminal tail, generating PC1-CTT fragments ranging from 17 to 34-kDa that translocate to the nucleus and to mitochondria17–19. In the current study, we show that the 200 aa PC1-CTT can interact and increase the enzymatic activity of NNT in mitochondria, which produces a physiologically significant impact on mitochondrial redox, metabolism, and cystogenesis.

Fig 4: The interaction between NNT and CTT is critical to the CTT-mediated suppression of cystogenesis and tubular proliferation.A–C Comparative analysis of Pkd1-KO + CTT and Pkd1-KO in the “J” background revealed no significant change in KW/BW ratio (A), BUN (B), and serum creatinine levels (C). Cystic mouse cohorts are composed of 53–55% female and 45–47% male mice. Red dots represent the animals depicted in (E). D Quantification of tubular and cystic area in H&E-stained kidney sections from Pkd1-KO and Pkd1-KO + CTT mice on both “N” and “J” backgrounds, as determined by ImageJ using images of renal cross-sections shown in Supplementary Fig. 1. E Representative H&E-stained kidney sections (4×) from 16-week J-Pkd1-KO, J-Pkd1-KO + CTT, and J-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. F Representative immunofluorescence images (20×) showing tubular proliferation in Pkd1-KO and Pkd1-KO + CTT mice on “N” and “J” backgrounds, assessed by Ki67 staining (red). Tubular epithelial cells are identified by positive Na,K-ATPase a-subunit staining (green). Scale bar: 50 µm. G Quantification of tubular proliferation determined by the percentage of Ki67-positive nuclei in renal tubular epithelial cells (n = 9 images per mouse, 5 mice per group). Counting of Ki67 positive nuclei was performed by an individual blinded to the experimental conditions. Parametric data are depicted with the mean ± SEM (black bars). Pairwise comparisons were performed using two-tailed Student’s t-test (D, G). Multiple group comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test (A). 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 (B, C). 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.

Fig 5: CTT increases NNT activity in pre-cystic kidney tissue and cultured cells.A, B Kidney function is normal in all 10-week cohorts (N-Pkd1-KO, N-Pkd1-KO + CTT, and N-WT) used to assess NNT enzymatic activity, as revealed by BUN (A) and serum creatinine levels (B). Pre-cystic cohorts are composed of 45–50% female and 50–55% male mice. Data are depicted as means ± SEM. Multiple group comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. C, D Immunoblot of mitochondrial extracts from N-Pkd1-KO, N-Pkd1-KO + CTT, and N-WT kidneys (C). *St: rat heart mitochondrial extract; VDAC served as mitochondrial loading control. NNT expression (normalized to VDAC) was not significantly different across all groups of 10-week-old mice (D). Data are depicted as mean ± SEM. Multiple group comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. E NNT activity in mitochondria prepared from N-Pkd1-KO, N-Pkd1-KO + CTT, and N-WT kidneys, quantified by measuring rate of reduction of the NAD analog APAD. Samples were normalized to protein content. WT “J” mice served as negative controls, and confirmed the specificity of the assay by showing the expected absence of an upward slope. F Comparison of NNT activity among N-Pkd1-KO, N-Pkd1-KO + CTT, and N-WT mice, measured as ?OD/s/mg of protein. Data are depicted with the mean ± SEM. Multiple group comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. G Sequence of human PC1-CTT, highlighting the overlapping nuclear localization17 (aa 4134–4154, red letters) and mitochondrial-targeting19 sequences (aa 4129–4154, red letters and yellow highlight). H HRP-conjugated anti-HA immunoblotting of lysates from transfected Pkd1-/- cells revealing expression of both 2HA-PC1-CTT and 2HA-PC1-CTT?4134-4154. I NNT activity detected in mitochondrial extracts from Pkd1-/- cells transfected with 2HA-PC1-CTT (CTT), 2HA-PC1-CTT?4134–4154 (CTT?4134–4154) or empty pcDNA3.1 vector (EV). Absence of APAD reduction when NADPH is omitted from assay medium (-NADPH) confirms assay specificity. J Comparison of NNT activity among Pkd1-/- + EV, Pkd1-/- + CTT, and Pkd1-/- + CTT?4134–4154 mitochondrial extracts, measured as ?OD/s/µg of protein and normalized to Pkd1-/- + CTT mean values. Data are depicted with the mean ± SEM. Multiple group comparisons were performed using one-way ANOVA followed by Dunnet’s multiple-comparisons test, with Pkd1-/- + CTT serving as the reference group. Characterization of Pkd1 cells is shown in Supplementary Fig. 10a. Source data are provided as a Source Data file.