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. While we have not been able to use the eGFP-PC1 line to visualize PC1 in living cells as anticipated because of the very low expression level of the native protein, we have successfully used GFP nanobodies to reliably isolate PC1 and its partners. Using large quantities of tissue (P2 mouse head) we have been able to isolate a PC1 interactome which is enriched for mitochondrial proteins and components of metabolic pathways. We also identify nicotinamide nucleotide transhydrogenase (NNT) as a probable mitochondrial partner, linking PC1 to the regulation of reactive oxygen species (ROS). Loss of Nnt function had no significant effect on renal cystic disease in Pkd1 mutants but treatment of mice with early onset cystic disease with n-acetyl-cysteine (NAC) provided modest benefit only in the Nnt+/+ genetic background. These studies suggest that new methods and brighter tags will be required to track endogenous PC1, but this mouse model and interactome data will be useful for characterizing the endogenous PC1 interactome. The data also support our prior findings that the PC1 C-terminus localizes to mitochondria and regulates their function. A manuscript reporting these findings has been posted on bioRxiv (bioRxiv 2022.04.08.487705; doi: https://doi.org/10.1101/2022.04.08.487705) and submitted for publication. We also have expanded the study to include a second organ, one that has an established Pkd1 mutant phenotype. Our rationale is that by comparing the PC1-related proteome for a variety of organs we might find a core set of proteins common to all, and then sets of others that are tissue-specific. Encouragingly, Nnt was also reliably identified in the second organ, as were multiple other proteins. The lab has long-standing interest in using transcriptomics to understand PKD biology. We have recently been pursuing three lines of inquiry. In the first, we investigated the relationship between Lad1, which was consistently down-regulated in cystic samples, and Pkd1. Lad1 encodes a filamin-binding regulator of actin dynamics but little is known about its function. We found its expression decreased even prior to the onset of cyst formation, suggesting a possible causal relationship. We used CRISPR to generate two null alleles that each remove most of the gene and then examined the phenotype of Lad1 mutants and Pkd1/Lad1 double mutants. We found that Lad1 mutants appear normal, and loss of Lad1 in an early onset model of Pkd1 cystic disease had no effect. We soon expect to finish assessing the effects of Lad1 in an adult onset model. We currently are exploring mechanisms responsible for the downregulation of Lad1. In a second set of studies, we had collaborated with investigators at TIGEM (Italy) who had postulated that there would be a transcriptional signature for TFEB, a master regulator of lysosomal biogenesis, in Pkd1-mutant samples. They found that TFEB is the main driver of the cystic disease and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dube syndrome. We performed RNA-seq on 12 mutant and 12 WT kidneys and 29 Pkd1-mutant and 29 control samples from 8 cell lines and found no evidence of such a signature. The third approach has been directed at studying the livers of our Pkhd1 mutant mice. We worked out methods to isolate pools of single cells enriched for cell populations other than hepatocytes (<3% of final pool) for scRNA studies. We are currently comparing the transcriptional profiles of WT vs Pkhd1 vs Pkd1 cystic livers and validating findings. We have continued our characterization of the Pkhd1 del 3-67 eye phenotype. We have confirmed our prediction that double heterozygotes (for Pkhd1 del3-67 and another gene) develop the same eye phenotype as Pkhd1 homozygotes. Studies are underway to look at the developmental process accounting for this effect. Lastly, we have made significant progress in developing a novel organoid model for autosomal dominant polycystic kidney disease based on mouse nephron progenitor cells (NPCs). Antibodies that detect kidney lineage differentiation markers were used to sort NPCs from mouse E13.5 embryonic kidneys. NPCs sorted with our protocol could be maintained long term with nephrogenic potential. NPCs derived from Pkd1, Pkd2 or DPKD mutants spontaneously make cystic structures when grown in suspension. This is the first mouse NPC-based organoid system that appears to make cystic structures spontaneously and will be a useful tool to reveal mechanisms of cystogenesis in ADPKD.