The role of PKD proteins in regulating tubular morphology

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

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Abstract

Progress this past year has improved since last year but continues to be negatively impacted by Covid-19 in a variety of ways. Despite these hurdles, we have made significant progress on a number of our projects. As noted last year, we have investigated peroxisome biogenesis and activity in PKD cells and tissues. Using a variety of methods, we found that peroxisome number, biogenesis, and FA metabolism were not different in Pkd1 mutant cells. We also showed that PC1 minimally co-localized with peroxisomal markers, likely excluding this organelle as a significant target for PC1. While we did not exclude abnormalities in other peroxisomal functions in ADPKD (ie. ROS regulation), our results suggest that any connection between PC1 and peroxisome function is likely to be indirect. We have now completed our analyses and published a report summarizing our findings. As also noted last year, we had developed a mouse line with EGPF knocked into the c-terminus of the Pkd1 locus. As previously noted, we have searched for EGFP fluorescence in frozen specimens and we have immunostained fixed specimens with anti-HA and anti-GFP antibodies, all without success. We also have tested for detectable EGFP-PC1 expression in primary cells isolated from the mouse line. We initially selected MEFs since they have relatively high levels of PC1 expression and are readily available. This, too, failed to yield reproducible findings. We next tested LTL-positive renal epithelial cells (RECs), cultured to confluence and to a polarized state. Though the level of PC1 expression is considerably lower in RECs than MEFs, we reasoned that PC1 might localize differently and in a detectable way in cells that are polarized. While we were unable to detect GFP in living cells, we could detect EGFP-PC1 in the cilia of fixed cells. We briefly described these results at the FASEB PKD Catalyst Conference in March 2021, advising the PKD Centers to consider alternative strategies to make a mouse with trackable, endogenous PC1 expression. While we have not been able to use the EGFP-PC1 line to visual 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. The logistical challenges posed by PC1s low level of expression have been significant. Even though we can very efficiently clear lysates of PC1 using GFP-nanobodies, our pilot studies have shown that huge amounts of starting material (up to 20 young mice/genotype per IP) are required to reliably isolate PC1 and PC2 as the top products identified by mass spec. Once we had worked out the conditions, we performed three independent experiments and identified by mass spec multiple proteins that reproducibly co-isolate with PC1, including a number of mitochondrial targets. We have verified one of the targets, and using a combination of interventions and genetic studies we have shown that the interaction has functional consequences. We are currently completing the genetic studies and have begun preparing a manuscript. 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, the target identified in the first set of studies was also reliably identified in the second organ, as were multiple other proteins. We are currently evaluating the results. 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. Lad1 mutants generally appear normal; characterization of double mutants is ongoing. In a second set of studies, we are collaborating 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 have performed RNA-seq on 12 mutant and 12 WT kidneys and 29 Pkd1-mutant and 29 control samples from 8 cell lines, and the results are being analyzed. 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 have thus far sequenced a total of 132K cells (after filtering for low quality cells), and we are currently working through the results. Finally, we have continued our characterization of the Pkhd1 del 3-67 eye phenotype. We have generated additional data supporting our hypothesis, and we have initiated import of a mouse model that we can use to do the definitive and final test of our hypothesis. The prediction is that double heterozygotes (for Pkhd1 del3-67 and another gene) will develop the same phenotype as homozygotes for either gene.