The role of PKD proteins in regulating tubular morphology

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

As previously noted, we had developed a mouse line with eGPF knocked into the c-terminus of the Pkd1 locus and used it to identify the PC1 interactome in brain. The full description of the mouse line, its characterization, and the interactome were published this summer. Key aspects of the report are the following: 1) we have shown that adding a 3-HA epitope tag and EGFP to the C-terminus of PC1 had little if any effect on the proteins function; 2) we also showed that the level of expression of endogenous PC1-tagged with EGFP is below the level of detection using various microscopic methods without antibody amplification; 3) the most important finding was our identification of the first endogenous PC1 interactome. (The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD035250 and 10.6019/PXD035250.) While we intentionally used a method that was likely to generate some false positives, it is the approach recommended by leading experts in the field and our analyses suggest that the majority of hits were likely true positives. Three interesting aspects to these findings: a) PC2 was by far the strongest and most reliable binding partner, with most of the interactome comprised of either weak or transient direct interactors or pathway components bound to direct interactors; b) we found an enrichment for mitochondrial and cytoplasmic targets; c) we did not find many of the targets previously reported by others. Whether this reflects differences in methods, tissue source, or using endogenous PC1 rather than over-expression of recombinant protein as bait is unknown, but the relatively low stringency of washing should have maximized the likelihood of capturing and retaining true targets. We also verified that our approach captured the full length of PC1, ruling out a C-terminal bias introduced by using antibodies that targeted the C-terminus of the protein as a possible explanation for why our results differ from what has been published. Studies of other organs may help resolve this issue. Over the past year, we have collected enough young mouse kidneys to allow us to repeat the mass-spectrometry proteomics-based IP study. We expect to determine the PC1 interactome in kidney over the coming months, allowing us to compare the endogenous interactome of three organs (we have previously done placenta). Interestingly, one of the top targets was nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial protein important in the regulation of NADP/NADPH levels and reactive oxygen species (ROS). Given that a commonly used mouse strain, the C57Bl6J line, has a mutation in this gene, we had compared the severity of cystic disease in Pkd1 mutant mice in the C57Bl6J (mutant Nnt) and C57Bl6N (normal Nnt) backgrounds and found no difference. This year, another group also reported NNT as a binding partner of PC1, but in contrast to our findings, they reported that NNT was an important modifier of disease severity which could be corrected by re-expression of just the C-terminal 200aa of PC1 (CTT). We had previously published that this proteolytic product of the 4302aa full-length protein traffics to mitochondria and could rescue some mitochondrial properties in Pkd1 mutant cells. Further study will be required to resolve our different findings with respect to the effects of loss of Nnt on cystic disease. In other progress, we have published the results for our Pkhd1 complete knock-out mouse. In new studies done over the past year and included in the report, we discovered that Pkhd1del3-67 male mice have reduced fertility. A review of our records confirmed that the problem had already been present years earlier, when we performed in vitro fertilization using several Pkhd1 mutant lines and controls and observed low rates only in the Pkhd1del3-67 line. These observations suggest that reduced male fertility is a unique feature of the Pkhd1del3-67 line. While full characterization of this phenotype is beyond the scope of our program, it is notable that we had previously found a highly expressed 1.0kb mRNA transcript in testis that was detected with an exon 41 probe and not with one from exon 5. In an RNA-seq dataset from mouse testis, we found reads predominantly mapped to exons of 4930486I03Rik, a long non-coding RNA of unknown function embedded within Pkhd1. The gene is in the opposite orientation to Pkhd1 and its third exon overlaps with Pkhd1s exon 38 and is highly expressed in testis. Pkhd1 and 4930486I03Rik have mostly overlapping patterns of tissue expression, though there is an inverse relationship between their levels of expression, particularly in testis. We speculate that loss of this lncRNA is responsible for the reduced fertility of our Pkhd1del3-67 male mice, presumably by affecting sperm development. We also added two other analyses completed this past year to the published work. We had previously observed that kidney/body weight ratios of mutant female mice were slightly larger than in controls, a finding consistent with a previous report showing mild kidney dilatation only in females of a truncating Pkhd1 model. Given a prior report of altered planar polarity in phenotypically normal Pkhd1 mutant kidneys, we compared tubule diameter and cell number on cross sections of male and female mutant and wild type kidneys and found no difference. We also had hypothesized that compensatory pathways might prevent cystogenesis in certain genetic backgrounds or environmental conditions, so we performed single nucleus transcriptomic studies of Pkhd1 mutant and control kidneys. We had identified differentially expressed genes but found no evidence for functional pathway abnormalities. Network analysis suggests that ubiquitin-conjugation may be altered, an interesting finding given our earlier studies linking FPC, the Pkhd1 gene product, to the NEDD4-family of E3 ubiquitin ligases. Single nuclear RNA sequencing related datasets generated for this study can be found in the NCBI Sequence Read Archive (SRA) archive with BioProject record PRJNA911229. Cellrange output count matrices were deposisted in figshare with digital object identifier (DOI): 10.6084/m9.figshare.22726550; Supplementary snRNAseq analysis at 10.6084/m9.figshare.22720789; all other data also publicly available through Figshare. We have continued our characterization of the Pkhd1del3-67 eye phenotype. In an unexpected development, we found evidence to suggest there may be a previously unrecognized gene embedded within Pkhd1 that could be contributing to the phenotype through interactions with a previously described gene linked to the phenotype. Studies are underway to test this hypothesis. In other updates, we found that loss of Lad1 also appeared to have no effect on the cystic presentation in the adult onset Pkd1-loss model. We continue to explore mechanisms responsible for the downregulation of Lad1 in cells/tissues lacking Pkd1. With respect to our organoid studies, early results suggest that the mouse NPC-based cysts are properly oriented with their primary cilia extending into the central lumen. This is in sharp contrast to what has been reported for cysts produced by human iPSC-based nephron organoids. Studies currently underway are examining the transcriptional profile of WT vs Pkd1 mutant, undifferentiated NPCs to determine baseline differences based on genotype and time in culture. These will serve as the foundation for subsequent scRNA studies focused on differentiation pathways.