Primary coenzyme Q deficiency in Pdss2 mutant mice causes isolated renal disease - PubMed (original) (raw)

. 2008 Apr 25;4(4):e1000061.

doi: 10.1371/journal.pgen.1000061.

Marni J Falk, Volker H Haase, Rhonda King, Erzsebet Polyak, Mary Selak, Marc Yudkoff, Wayne W Hancock, Ray Meade, Ryoichi Saiki, Adam L Lunceford, Catherine F Clarke, David L Gasser

Affiliations

Primary coenzyme Q deficiency in Pdss2 mutant mice causes isolated renal disease

Min Peng et al. PLoS Genet. 2008.

Abstract

Coenzyme Q (CoQ) is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2(kd/kd) genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2(kd/kd) mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2(loxP/loxP) knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2(loxP/loxP) knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Generation of a Pdss2 Conditional Null Allele.

Generation of a Pdss2 conditional null allele, showing a map of the Pdss2 genomic locus and the targeting vector with exons represented by open boxes. The relative position of PCR primers (small arrowheads), loxP (large arrowheads), as well as cassettes encoding neomycin phosphotransferase (neo) are shown. Primers koF, koF2 and koR were used in PCR genotype analysis. Cre-mediated deletion results in either the Pdss2 null allele (deletion of exon 3 ) or the Pdss2 loxP/loxP allele (exon 2 flanked by loxP sites). Abbreviations: Ba, BamH I; Aa. Aat II; Xh, Xho I; Dr, Dra III.

Figure 2

Figure 2. Pdss2 Conditional Knockout Confirmation.

PCR products after laser capture microdissection, using primers for exon 2 (A) or exon 4 (B). Lanes: 1, Pdss2loxP/loxP glomerulus; 2, Pdss2loxP/loxP tubules; 3, B6.Podocin/cre,Pdss2loxP/loxP mouse 1, glomerulus; 4, B6.Podocin/cre,Pdss2loxP/loxP mouse 1 tubules; 5, B6.Podocin/cre,Pdss2loxP/loxP mouse 2, glomerulus; 6, B6.Podocin/cre,Pdss2loxP/loxP mouse 2 tubules; 7, B6.PEPCK/cre,Pdss2loxP/loxP mouse 1 glomerulus; 8, B6.PEPCK/cre,Pdss2loxP/loxP mouse 1, tubules; 9, B6.PEPCK/cre,Pdss2loxP/loxP mouse 2 glomerulus; 10, B6.PEPCK/cre,Pdss2loxP/loxP mouse 2, tubules.

Figure 3

Figure 3. Histologic Features of Renal Disease in Pdss2 Mutant and Control Mice.

Histologic features of renal disease in mutant and control mice (H & E-stained sections, original magnifications all 200x). A, B6.Podocin/cre,Pdss2loxP/loxP mouse (290 days old; 64 mg albumin; histologic score, 4+). B, B6.PEPCK/cre, Pdss2loxP/loxP (191 days old; 0.04 mg albumin; histologic score 0). C, B6.Pdss2kd/kd (146 days old; 15 mg albumin; histologic score, 3+). D, B6. Pdss2loxP/loxP mouse, (191 days old; 0.12 mg albumin; histologic score, 0). Note the prominent tubular dilatation and interstitial infiltrates in panels A and C, but absent in panels B and D.

Figure 4

Figure 4. Glomerular Electron Micrographs From Pdss2 Mutant and Control Mice.

Electron micrographs from mutant and control mouse kidney glomeruli; original magnifications all 10,000x; scale bar = 2 microns. A, B6, 362 days old; arrows show podocyte foot processes. B, B6.Pdss2kd/kd, 267 days old; arrows show regions of foot process effacement. C, B6.Pdss2loxP/loxP mouse, 248 days old; arrows show foot processes. D, B6.Podocin/cre,Pdss2loxP/loxP 290 days old; arrows show regions of foot process effacement.

Figure 5

Figure 5. CoQ Measurements in Pdss2 Missense, Conditional Knockout, and Strain-Matched Control Mice.

CoQ measurements in Pdss2kd/kd; Podocin/cre,Pdss2loxPloxP; PEPCK/cre,Pdss2loxPloxP; Alb/cre,Pdss2loxP/loxP; and strain-matched control mice (B6 and B6.Pdss2loxPloxP). A, Kidney levels of CoQ9 and CoQ10 are significantly lower in B6.Pdss2kd/kd (missense) mice than in B6 control mice. B, Kidney levels of CoQ9 and CoQ10 are similar in B6.Podocin/cre,Pdss2loxPloxP; B6.PEPCK/cre,Pdss2loxPloxP; B6.Alb/cre,Pdss2loxP/loxP; and control mice. The 92-day-old controls are B6.Pdss2loxP/+, and the 94-day-old control is B6.Pdss2loxP/loxP. C, Liver levels of CoQ9 in B6.Alb/cre,Pdss2loxP/loxP mice were below 30 picomoles/mg protein (*) while these same levels in B6.Podocin/cre,Pdss2loxPloxP and B6.PEPCK/cre,Pdss2loxPloxP mice were similar to those in control mice. The strains of the control mice in are the same as those in panel B.

Figure 6

Figure 6. CoQ Deficiency Causes Mitochondrial Dysfunction in Pdss2 Mutant Mice.

Impact of coenzyme Q deficiency on mitochondrial function in B6.Pdss2kd/kd and B6.Alb/cre,Pdss2loxP/loxP mice compared to concurrent age- and strain-matched controls (B6 and B6.Pdss2loxP/loxP, respectively). Panels A, B, and C demonstrate polarographic results of freshly isolated liver mitochondria with substrates which specifically interrogate complex I-dependent (malate+glutamate), complex II-dependent (succinate), and complex IV-dependent (TMPD+ascorbate) OXPHOS capacity. B6.Pdss2kd/kd missense mutants have impaired complex I- and complex II-dependent OXPHOS capacity by 23% and 33%, respectively, with a 19% increase in complex IV-dependent OXPHOS capacity. B6.Alb/cre,Pdss2loxP/loxP mutants have impaired complex I- and complex II-dependent OXPHOS capacity by 20% and 40% respectively, with a 43% increase in complex IV-dependent OXPHOS capacity. Values represent state 3 (near maximal) oxygen consumption mean rate and standard error. Panel D summarizes electron transport chain activity assays for complexes I-III, II, III, and IV performed on remaining aliquots of liver mitochondria frozen following completion of polarography. Similar to polarographic results, I-III enzyme activity is significantly decreased by 19% in B6.Pdss2kd/kd missense mutants and by 41% in B6.Alb/cre,Pdss2loxP/loxP mutants when compared to pooled controls. Complex IV enzyme activity demonstrates a trend toward increase by 11% in B6.Pdss2kd/kd missense mutants and by 23% in B6.Alb/cre,Pdss2loxP/loxP mutants when compared to pooled controls, but does not reach the level of statistical significance, perhaps because of small sample size (n = 2 for remaining mitochondria from B6.Alb/cre,Pdss2loxP/loxP mutants). As expected, no change is seen in complex II or III activity for either mutant. Values represent mean enzyme activity and standard error. B6 and B6.Pdss2loxP/loxP ETC activity values were pooled to increase control sample size following demonstration of no significant differences between these strains. Statistical analyses comparing mutants and controls were performed by one-way ANOVA, where * indicates p<0.05 and ** indicates p<0.01.

Figure 7

Figure 7. Metabolic Pathway Alterations Are Seen by Expression Profiling in B6.Alb/cre,Pdss2loxP/loxP Mouse Liver.

Global genome expression profiling in B6.Alb/cre,Pdss2loxP/loxP mouse liver identifies concordant transcriptional alterations interpretable at the level of multiple metabolic pathways, which suggest significantly altered intermediary metabolism occurs despite an apparent absence of symptomatic disease. Extensive evolutionary concordance in upregulation of key biochemical pathways is seen in primary mitochondrial dysfunction, both in this mammalian Pdss2 liver-conditional knockout model of coenzyme Q deficiency and in a previously reported C. elegans gas-1(fc21) missense mutant model of primary complex I dysfunction . Biochemical pathways as curated from the KEGG online database (

http://genome.jp.kegg

) are indicated with the # of genes in each pathway (cluster size), normalized enrichment score (NES), statistical significance of altered pathway expression between mutant and wildtype controls (_p_-value), and false positive percentage in the form of a false discovery rate (FDR _q_-value) according to GSEA. Pathways are ranked by descending NES in the Pdss2 mutant (left data column). Comparison to previously reported complex I gas-1(fc21) missense C. elegans mutant dataset (middle data column) and a “validation” C. elegans dataset of 8 different complex I, II, and III missense and RNAi-interference generated mutants (right data column) is indicated by differential highlights . Font color denotes a pathway as relatively upregulated (red), downregulated (green), or unchanged (black).

Figure 8

Figure 8. Pdss2 Mutants Have Altered Amino Acid Profiles in Liver.

Pdss2 mutants have altered amino acid profiles in liver. Quantitative liver amino acid analysis detects significant differences in B6.Pdss2kd/kd missense mutants compared with B6 controls (Panels A and B), as well as in B6.Alb/cre,Pdss2loxP/loxP mutants compared with B6.Pdss2loxP/loxP controls (Panels C and D). To better demonstrate differences in all amino acids, results for three amino acids in highest abundance are shown separately (Panels B and D) with a greater scale compared to that used for the remainder of the amino acids present in relatively lower abundance (Panels A and C). Values represent mean +/− SEM. Statistical analyses comparing mutants and controls were performed by Student's t-test, where * indicates p<0.05 and ** indicates p<0.01.

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