Fatty Acid Oxidation is Impaired in An Orthologous Mouse Model of Autosomal Dominant Polycystic Kidney Disease

Abstract

Background: The major gene mutated in autosomal dominant polycystic kidney disease was first identified over 20 years ago, yet its function remains poorly understood. We have used a systems-based approach to examine the effects of acquired loss of Pkd1 in adult mouse kidney as it transitions from normal to cystic state.

Methods: We performed transcriptional profiling of a large set of male and female kidneys, along with metabolomics and lipidomics analyses of a subset of male kidneys. We also assessed the effects of a modest diet change on cyst progression in young cystic mice. Fatty acid oxidation and glycolytic rates were measured in five control and mutant pairs of epithelial cells.

Results: We find that females have a significantly less severe kidney phenotype and correlate this protection with differences in lipid metabolism. We show that sex is a major determinant of the transcriptional profile of mouse kidneys and that some of this difference is due to genes involved in lipid metabolism. Pkd1 mutant mice have transcriptional profiles consistent with changes in lipid metabolism and distinct metabolite and complex lipid profiles in kidneys. We also show that cells lacking Pkd1 have an intrinsic fatty acid oxidation defect and that manipulation of lipid content of mouse chow modifies cystic disease.

Interpretation: Our results suggest PKD could be a disease of altered cellular metabolism.

Keywords: ADPKD; Autosomal Dominant Polycystic Kidney Disease; Diet; Fatty acid oxidation; Fatty acid oxidation defect; Lipid metabolism; Metabolism; Mouse model; Pkd1; Polycystic kidney disease; Systems biology.

Fig. 1

Sex is a major determinant of disease progression and gene expression profiles in a mouse PKD model. a) Kidney to body weight ratios (KBW) plotted over time starting 50 days after Pkd1 inactivation and showing more rapid and severe kidney disease in males (curve fitted for each sex/genotype group using generalized linear model with gamma distribution; the line for control female overlaps and is obscured by the control male line). b) Multidimensional scaling (MDS) plot representing similarity in gene expression patterns between samples and showing that sex and cystic stage are responsible for most of the difference. Each dot corresponds to one kidney (size: correlates with KBW). c) Representative kidney histology of mutant and control male and female mice showing cystic mutant male and less affected littermate mutant female. Boxes delimit group comparisons using all animals in the dataset and corresponding number of differentially expressed genes. d) Heatmap plot showing patterns of gene expression between male and female control kidneys. Each column is a sample and each row is a gene. The colored vertical bar on the left groups genes with very similar (correlated) expression patterns into modules. These clusters can be further grouped into 2 sets of correlated genes that we labeled “Meta-module 1” and “Meta-module 2” (demarcated on the right margin of the plot). The dendogram on the top shows that the expression pattern of these genes can separate male and female kidneys. e and f) Differential expression in cystic females mirror those in males. e) Cumulative distribution of p values comparing mutant vs. control at different age intervals in males and females. The curves show that the number of differentially expressed genes in females is similar to those in younger males, consistent with a delayed, but similar, disease process in females. f) Bar plot showing that the number of differentially expressed genes between mutant and controls correlates with age (disease severity), and is smaller in females, likely reflecting the delayed phenotype. In the more severe stage, most differentially expressed genes (orange) were differentially expressed in both males and females. Each bar corresponds to genes differentially expressed only in females (red), only in males (blue), or in both (orange).

Fig. 2

Metabolic profile of mouse kidneys. a and b) Principal component plots showing metabolites and complex lipid profiles in male mouse kidneys (n = 14; 8 mutants and 6 controls). The first principal component separates genotype and KBW (purple circles: mutant; green circles: control; circle size: proportional to KBW). c) Volcano plot showing fold change and p-value of identified metabolites (blue) and lipids (orange) in mouse kidneys. Blue lines demarcate significance thresholds: p < 0.05 and 2 fold change. Significantly different metabolites are marked in red and labeled. d) Heatmap plot showing relative expression of each significantly different metabolite in kidney samples. The bar graph at the bottom shows the corresponding KBW. The dendrogram at the top depicts sample distances and is labeled according to genotype (mutant: purple; control: green).

Fig. 3

Fatty acid oxidation, but not glycolysis, is defective in kidney epithelial cell lines. a and b) Fatty acid oxidation assay measuring oxygen consumption rate (OCR) in five kidney epithelial cell lines using palmitate as a substrate. a) OCR profile (line colors: different cell line pairs, dashed line: mutant (i.e. lacking Pkd1), full line: control; vertical line: injection of A: 1.5 μM oligomycin, B: 0.8 μM FCCP, C: 0.8 μM FCCP, D: 2 μM rotennone/4 μM antimycin A). b) Violin plot of maximum respiration rate measurements of different cell line pairs. Data points are the rates measured between vertical lines C and D of the OCR profile (shape interior color: genotype). Paired t test: n = 5 pairs, p-value = 0.0001367. c and d) Plots showing extracellular acidification rate (ECAR) as a readout for glyocolysis. c) ECAR as a percentage of baseline in two epithelial cell lines. The vertical dashed lines represent the sequential addition of 10 mM glucose, 1.5 μM oligomycin, 1.5 μM oligomycin and 50 mM 2-deoxy-d-glucose (2-DG). d) Violin plots showing the glycolytic capacity of the experiment in the c panels, calculated as the increase over baseline after the addition of oligomycin (shape interior color: genotype). Paired t test: n = 2 pairs, p-value = 0.691.

Fig. 4

Diet lipid content is positively correlated with severity of cystic kidney disease. a) In both tamoxifen-inducible (ER-CRE) and Ksp-Cre induced mouse model, kidney/body weight (KBW) in increased in animals fed NIH37 diet, which has higher lipid content. b) Body weight is not statistically different between the two diets.