ABSTRACT
Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease despite higher mitochondrial content. We sought to illuminate noncanonical, cell-specific roles for CoQ, independently of the electron transport chain (ETC). Here, we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway rather than ETC dysfunction. Single-nucleus RNA-Seq from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound, ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.
Subject(s)
Ataxia/metabolism , Indenes/pharmacology , Kidney Diseases/metabolism , Lipid Peroxidation/drug effects , MAP Kinase Signaling System/drug effects , Mitochondrial Diseases/metabolism , Muscle Weakness/metabolism , Podocytes/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Pyrazoles/pharmacology , Ubiquinone/deficiency , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolism , Animals , Ataxia/drug therapy , Ataxia/genetics , Ataxia/pathology , Drug Delivery Systems , HEK293 Cells , Humans , Kidney Diseases/drug therapy , Kidney Diseases/genetics , Kidney Diseases/pathology , Lipid Peroxidation/genetics , MAP Kinase Signaling System/genetics , Mice , Mitochondrial Diseases/drug therapy , Mitochondrial Diseases/genetics , Mitochondrial Diseases/pathology , Muscle Weakness/drug therapy , Muscle Weakness/genetics , Muscle Weakness/pathology , Podocytes/pathology , Proto-Oncogene Proteins B-raf/genetics , RNA-Seq , Ubiquinone/genetics , Ubiquinone/metabolismABSTRACT
Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.
