Coenzyme Q10 Supplementation Rescues Renal Disease in Pdss2kd:Kd Mice With Mutations in Prenyl Diphosphate Synthase Subunit 2

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Coenzyme Q10 supplementation rescues renal disease in Pdss2kd/kd mice

with mutations in prenyl diphosphate synthase subunit 2

Ryoichi Saiki,1 Adam L. Lunceford,1 Yuchen Shi,1 Beth Marbois,1 Rhonda King,2 Justin Pachuski,2

Makoto Kawamukai,3 David L. Gasser,2 and Catherine F. Clarke1

1

Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles,California; 2 Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and 3 Department of Life Science and Biotechnology, Shimane University, Matsue, Japan

Submitted 30 July 2008; accepted in final form 7 September 2008

Saiki R, Lunceford AL, Shi Y, Marbois B, King R, Pachuski J,Kawamukai M, Gasser DL, Clarke CF. Coenzyme Q10 supplemen-tation rescues renal disease in Pdss2kd/kd mice with mutations inprenyl diphosphate synthase subunit 2. Am J Physiol Renal Physiol295: F1535–F1544, 2008. First published September 10, 2008;doi:10.1152/ajprenal.90445.2008.—Homozygous mice carrying kd (kidney disease) mutations in the gene encoding prenyl diphosphatesynthase subunit 2 (Pdss2kd/kd ) develop interstitial nephritis and even-tually die from end-stage renal disease. The PDSS2 polypeptide inconcert with PDSS1 synthesizes the polyisoprenyl tail of coenzyme Q(Q or ubiquinone), a lipid quinone required for mitochondrial respi-ratory electron transport. We have shown that a deficiency in Qcontent is evident in Pdss2kd/kd mouse kidney lipid extracts by 40 daysof age and thus precedes the onset of proteinuria and kidney diseaseby several weeks. The presence of the kd (V117M) mutation inPDSS2 does not prevent its association with PDSS1. However,heterologous expression of the kd mutant form of PDSS2 togetherwith PDSS1 in Escherichia coli recapitulates the Q deficiency ob-served in the Pdss2kd/kd mouse. Dietary supplementation with Q10

provides a dramatic rescue of both proteinuria and interstitial nephritisin the Pdss2kd/kd mutant mice. The results presented suggest that Qmay be acting as a potent lipid-soluble antioxidant, rather than byboosting kidney mitochondrial respiration. Such Q10 supplementationmay have profound and beneficial effects in treatment of certain formsof focal segmental glomerulosclerosis that mirror the renal disease of the Pdss2kd/kd mouse.

ubiquinone; mitochondrial lipid metabolism; genetic renal disease;focal segmental glomerulosclerosis; antioxidant

COENZYME Q (Q or ubiquinone) is an essential factor of aerobicrespiration and oxidative phosphorylation. Q also functions asa lipid-soluble antioxidant in cellular biomembranes and scav-enges reactive oxygen species (4, 7). Yeast strains not able toproduce Q show sensitivity to oxidative stresses such as hy-drogen peroxide and autooxidized polyunsaturated fatty acids(10, 44). Q is a dietary supplement and is also used in

therapeutic treatments for neurodegenerative and cardiovascu-lar diseases and for statin-induced symptoms of Q deficiency(11, 35).

Q is composed of a benzoquinone ring with a polyisoprenoidside chain of varying length. Living organisms possess distinctisoforms of Q depending on the length of the isoprenoid sidechain (17). For example, humans produce Q10, mice producepredominantly Q9 and small amounts of Q10, Saccharomycescerevisiae produce Q6, and Escherichia coli produce Q8. The

difference in the tail length of Q is determined by the long-chain polyprenyl diphosphate synthase in each organism (30).Long-chain trans-prenyl diphosphate synthases catalyze thecondensation step for the isoprenoid tails from farnesyl diphos-phate (C15) or geranylgeranyl diphosphate (C20) and isopen-tenyl diphosphate (C5). Structural analyses and site-directedmutagenesis have shown that short- and long-chain polyprenyldiphosphate synthases contain seven conserved regions desig-

nated as domains I through VII (Fig. 1 A) and two aspartate-richmotifs (DDXXD) that are substrate binding sites in associationwith Mg2 (12, 18, 28, 29, 45). These analyses suggest thatamino acid residues at the fourth and fifth positions before theaspartate-rich motif in domain II and two amino acid residuesin domain II have an important role in determining the lengthof the isoprenoid tails.

Polyprenyl diphosphate synthases are classified into twotypes of homo- or heterocomplexes. Most of the short poly-prenyl diphosphate synthases (C10–C25) in both prokaryotesand eukaryotes are homodimers (27). In contrast, long-chain(C30–C50) polyprenyl diphosphate synthases are more diver-sified. In E. coli, octaprenyl diphosphate synthase forms ahomodimer necessary for synthesis of the octaprenyl diphos-phate tail for both Q and menaquinone (16), whereas theenzymes that supply hexa- and heptaprenyl diphosphate formenaquinone in the genus Micrococcus and Bacillus are het-erodimers (47). The Coq1 polypeptide in S. cerevisiae func-tions on its own to produce hexaprenyl diphosphate; however,when expressed in fission yeast, Schizosaccharomyces pombe,it functions as a partner protein to produce decaprenyl diphos-phate (49). In S. pombe, mice, and humans, long-chain prenyldiphosphate synthases are composed of two subunits (PDSS1/ Dps1 and PDSS2/Dlp1) and form heterotetramers (39, 40).PDSS2/Dlp1, which is subunit 2 of nonaprenyl or decaprenyldiphosphate synthase in fission yeast and mammals, possessesthe conserved domains of a prenyl diphosphate synthase.

However, the subunit 2 lacks aspartate-rich motifs in domainsII and VI (39, 40). PDSS2 orthologs also are found in Dro-sophila, Xenopus, and mammals. Therefore, the heterotetramerconformation of long-chain prenyl diphosphate synthasesmight be standard in higher eukaryotes.

Recently, several diagnostic examples of inherited Q defi-ciency, caused by mutations in Pdss1, Pdss2, human COQ2(hCOQ2), or CABC1/COQ8 have been reported (8, 19, 23, 25,26, 36). These patients have severe Q10 deficiency and severe

Address for reprint requests and other correspondence: C. F. Clarke, UCLADept. of Chemistry and Biochemistry, 607 Charles E. Young Dr. E., LosAngeles, CA 90095-1569 (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement ”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Renal Physiol 295: F1535–F1544, 2008.First published September 10, 2008; doi:10.1152/ajprenal.90445.2008.

0363-6127/08 $8.00 Copyright © 2008 the American Physiological Societyhttp://www.ajprenal.org F1535



mitochondrial disease phenotypes such as encephalomyopathy,ataxia, renal diseases, cerebellar atrophy, and hyperlactatemia.Oxidative phosphorylation activities in fibroblasts (19, 23, 25, 26),muscle extracts (9, 23), or muscle mitochondria (25) from these

patients harboring a point mutation on Pdss1, Pdss2, hCOQ2, orCABC1/COQ8 are significantly lower than in samples fromhealthy individuals. Addition of either decylubiquinone or Q2, twosoluble Q analogs, restored oxidative phosphorylation activity incultured skin fibroblasts (19, 26) and muscle extracts (23) of theaffected patients. Therefore, the respiratory deficiency and thesymptoms in the patients are attributed to primary Q deficiency. Insome cases, prolonged Q supplementation relieves the symptomscaused by Q deficiency (37). The reasons for the different re-sponses to Q supplementation are still unclear.

In this study we have examined the nature of the Q defi-ciency in the Pdss2kd/kd mouse, which harbors a V117Mmutation in PDSS2 and develops kidney disease in early

adulthood (33, 34). The V117 residue is invariant in PDSS2polypeptides from fission yeast to humans (39). Based onimmunohistochemical profiling and electron microscopy, thephenotype of the Pdss2kd/kd mouse resembles the collapsing

glomerulopathy variant of focal segmental glomerulosclerosis(3). We have shown that a deficiency in Q content is evident inkidney lipid extracts by 40 days of age and that dietarysupplementation with Q provides a dramatic rescue of theproteinuria and interstitial nephritis. The results suggest that if human renal patients are identified who have PDSS2 mutationssimilar to that of the Pdss2kd/kd mouse, Q supplementation mayhave profound and beneficial effects.

MATERIALS AND METHODS

Mice. All animal studies have been approved by the Universityof California, Los Angeles (UCLA) and University of Pennsylva-nia institutional review boards. Mice harboring the Pdss2kd/kd

Fig. 1. Amino acid sequence alignment of mouse nonaprenyl diphosphate synthase sub-unit 1 (PDSS1/mSPS1) and subunit 2 (PDSS2/ mDLP1) isoforms. A: typical long-chain poly-isoprenyl diphosphate synthases, such asPDSS1 and PDSS2-L, have conserved domainsI through VII. An arrow shows the location of the kd/kd mutation V117M within conserveddomain I. B: alternative splicing producesPDSS2-Short (PDSS2-S) containing exons1–3b and PDSS2-Long (PDSS2-L) containingexons 1–3a and 4–8. PDSS2-S contains onlydomains I through III. The predicted polypep-tide produced from the Pdss2 BAC clone256E1 is also shown. One transgenic line of B6.kd/kd mice harboring an insertion of thisBAC clone 256E1 exhibits sporadic rescuefrom kidney disease.

F1536 COENZYME Q10 SUPPLEMENTATION RESCUES RENAL DISEASE

AJP-Renal Physiol • VOL 295 • NOVEMBER 2008 • www.ajprenal.org



missense mutation (V117M) on the B6 genetic background, line Gtransgenic mice expressing a truncated form of Pdss2, and B6. Alb/ cre,Pdss2loxP/loxP mice, in which the Pdss2 gene is knocked out inhepatocytes, have been described previously (14, 33, 34). Assaysof urine albumin were performed with ELISA kits from BethylLaboratories as described previously (33). When the mice wereeuthanized, hematoxylin and eosin (H&E)-stained sections of thekidneys were prepared. The sections were examined blindly and

scored as follows: 0, no tubular dilatation and no mononuclear cellinfiltrates; 1, small focal areas of cellular infiltration and tubulardilatation involving 10% of the cortex; 2, involvement of up to25% of the cortex; 3, involvement of up to 50% of the cortex; 4,extensive damage involving 75% of the cortex.

Preparation of mouse kidney and liver homogenates. Whole kid-neys and lobes of livers were dissected from euthanized mice andstored at 80°C until homogenization. Samples were homogenized in5 ml of 1 PBS, pH 7.4 (137 mM NaCl, 8 mM Na2HPO4, 2.7 mMKCl, and 1.5 mM KH2PO4) at 4°C, with a total of 10 strokes with atight-fitting Teflon pestle rotating at maximal speed with a FisherScientific Lab Stirrer LR400A. The homogenate was centrifuged at1,000 g for 5 min, supernatants were removed to fresh vials, andprotein concentrations were measured using the bicinchoninic acidassay (Pierce, Rockford, IL). Aliquots of each homogenate were

transferred to 50-ml glass tubes and stored at 80°C until extracted.Q10 supplementation. Mice were provided Q10 supplements (Tish-

con LiQsorb, 100 mg/ml) in their drinking water. In a pilot experi-ment, three males and two females were given 100 mg Q10 /400 mlwater (0.25 mg/ml), beginning at 47 days of age. While on this dose,both females had litters. After weaning, the progeny received 0.50 mgQ10 /ml water. Mice consumed 12–16 ml of water per day; thus atthis dose, the mice received 200 mg Q10 kg body wt1

day1.Some of their offspring were given a dose of 400 kg bodywt1

day1, beginning at the time they were weaned. Expression of murine PDSS1 and PDSS2 prenyl diphosphate syn-

thase subunits in E. coli. E. coli strain DH5 was used for generalplasmid construction and protein expression (42). E. coli cells weregrown in Luria Bertani (LB) medium with appropriate antibiotics suchas 100 g/ml carbenicillin, 100 g/ml ampicillin, and 50 g/ml

kanamycin. The pGEX-KG (GE Healthcare Bio-Sciences, Piscat-away, NJ) and pET-28c (Novagen, Madison, WI) plasmids served asvectors.

cDNAs prepared from either B6 or B6.Pdss2kd/kd liver (34) wereused as template DNA to PCR amplify the short and long isoforms of Pdss2. The 0.8-kb amplicon containing the Pdss2-Short isoformcDNA was generated with B6 liver cDNA as template and twooligonucleotide primers, 5- EcoRI-mDLP1 5-CCGAATTCGAAT-GAGCTCCGGCAG-3 (creates an EcoRI site) and 3-SalI-mDLP1-Short, 5-ACGCGTCGACTTACTTCATGTTGTCACTGCC-3 (cre-ates a SalI site). The amplified 0.8-kb fragment was digested with EcoRI and SalI enzymes and inserted into the same sites of pET28c toyield pET28c-PDSS2-S. The pET28c-PDSS2-S plasmid was digestedwith XbaI and SalI, and the resulting fragment was cloned into thesame sites of pGEX-mSPS1 (39) to construct pM12S. Glutathione

S -transferase (GST)-tagged-PDSS1 and His6-tagged-PDSS2-Short arecoexpressed from the Tac promoter of pM12S.

A similar scheme was used to generate pM12L and pM12Lkd. The1.2-kb amplicon containing the Pdss2-Long isoform cDNA wasgenerated with B6 liver cDNA as template and the oligonucleotideprimers, 3-SalI-mDLP1-L, 5-ACGCGTCGACTCAAGAAAATCT-GGTCACAGC-3 (creates a SalI site) and 5- EcoRI-mDLP1. Theamplified 1.2-kb fragment was digested with EcoRI and SalI enzymesand inserted into the same sites of pET28c to yield pET28c-PDSS2-L.The pET28c-PDSS2-L plasmid was digested with XbaI and SalI, andthe resulting fragment was cloned into the same sites of pGEX-mSPS1 to construct pM12L. The same sequence of steps was used toconstruct pM12Lkd, except that cDNA prepared from B6.Pdss2kd/kd

mouse liver was used as template DNA. pM12L and pM12Lkd drive

expression of GST-tagged-PDSS1 and either His6-tagged-PDSS2-Long form or His6-tagged-PDSS2-Long form harboring the kd mis-sense mutation (V117M).

DH5 harboring pM12S, pM12LB6, pM12Lkd, or the emptyvector pGEX-KG were inoculated in 5 ml of LB medium withampicillin and cultured at 30°C, 250 rpm, for 18 h. Aliquots of eachbacterial culture (500 l) were used to inoculate 50 ml of fresh LBmedium with carbenicillin and incubated at 30°C, 250 rpm, until the

cell density reached an optical density (OD600) of 0.4. Isopropyl--D-thiogalactoside (IPTG; Fisher Biotech) was added to a final con-centration of 0.5 mM, and cells were incubated at 30°C, 250 rpm, for4 h. Cells were collected and stored at 20°C for lipid extraction.

Q measurements: electrochemical and UV detection. A knownamount of the internal standard Q6 was added to all samples andcalibration curve standards before lipid extraction. The same amountof Q6 was also added to the Q9 and Q10 standards prepared at differentconcentrations to generate an extracted standard curve. Lipid extrac-tions were then performed on Q9 and Q10 standards, mouse tissuehomogenates, and E. coli cell pellets by using a standard method.Each sample or standard was mixed with 0.5 ml of water, 9 ml of methanol, and 6 ml of petroleum ether. The mixtures were vortexedfor 1 min and centrifuged at 910 g, and the top layer of petroleumether was removed and transferred to a separate vial. Fresh petroleum

ether (6 ml) was added to the remaining aqueous phase and vortexedfor 1 min. The vials were subjected to centrifugation as before, and thesecond petroleum ether layer was removed. The process was repeatedonce more, and the combined upper organic phase was dried under N 2

and resuspended in 200 l of methanol. The quinones were thenseparated and quantified by reverse-phase (RP)-HPLC connected toan electrochemical detector as described previously (15), with thefollowing exceptions: the precolumn electrode was set at 650 mV tooxidize all hydroquinones, and a Gilson 118 UV/Vis detector wasutilized to detect quinones (275 nm) as they eluted from the column.The amounts of Q9 and Q10 in the standards and samples werecalculated via the calibration curve.

Q measurements: RP-HPLC-multiple reaction monitoring. Sam-ples and standards were extracted as described above, except with0.05 ml of water, 0.9 ml of methanol, and 0.6 ml of petroleum ether.

Samples were resuspended in 200 l of ethanol. Rhodoquinone-9(RQ9) isolated from Ascaris suum and rhodoquinone-10 (RQ10) iso-lated from Rhodospirillum rubrum were used as internal standards.RP-HPLC-multiple reaction monitoring (MS/MS) was performed on a4000 Q Trap-Hybrid triple-quad linear ion trap analyzer (AppliedBiosystems/MDS Sciex) with an autosampler (CTC Analytics/LEAPTechnologies, HTC PAL; 10-l injections, 20-l loop) and a Turbo-Vsource with the ESI probe inserted. Reverse-phase sample separationwas performed via a pentafluorophenyl propyl column (5 m, 4.6 100 mm; Phenomenex, Torrance, CA). The mobile phase was 95:5acetonitrile-water containing 0.1% formic acid (2 ml/min). The massspectrometer was set to the positive ion mode with the followingparameters: declustering potential, 61 V for RQ9, 91 V for Q9 andRQ10, and 126 V for Q10; entrance potential, 10 V for all quinones;collision energy, 39 V for RQ9, 37 V for Q9, 45 V for RQ10, and 51

V for Q10; and collision exit potential, 12 V for RQ9 and 10 V for Q9,RQ10, and Q10. The samples were analyzed in the multiple reactionmonitoring (MRM) mode: 210-ms dwell; MRM transitions: m/z781.5/182.1 for RQ9, m/z 795.6/197.1 for Q9, m/z 848.7/182.1 forRQ10, and m/z 863.7/197.0 for Q10. Quantification was via embeddedAnalyst software version 1.4.2 (Applied Biosystems/MDS Sciex).

Coprecipitation of PDSS1 and PDSS2 polypeptides. To evaluatewhether PDSS2-Long-kd interacts with PDSS1, we adapted an affin-ity purification to isolate GST-PDSS1, GST, His-PDSS2-L, and His-PDSS2-L kd. GST-PDSS1 and GST were purified using glutathioneSepharose 4B (GE Healthcare BioSciences), and His-PDSS2-L andHis-PDSS2-L kd were purified using Ni-NTA Superflow (Qiagen)according to the manufacturer’s instructions. Briefly, BL21 (DE3) E.coli cells harboring pET28c-Pdss2-L, pET28c-Pdss2-L kd, pGEX-

F1537COENZYME Q10 SUPPLEMENTATION RESCUES RENAL DISEASE




mSPS1 (39), and pGEX-KG empty vector grown in 50 ml of LBmedium plus appropriate antibiotics were induced by 0.5 mM IPTGfor 3 h at 30°C. After centrifugation to collect cells, the pellets weresonicated for 12 cycles of 10-s sonication and 10-s intervals on ice inbuffer 1, composed of 50 mM sodium phosphate, 0.5 M sodiumchloride, 5% (wt/vol) sucrose, 1% (vol/vol) glycerol, and 1% (vol/vol)Tween 20 (pH 7.0). After addition of protease inhibitors (Complete,EDTA-free; Roche), the sonicated samples were rotated gently for 1 h

at 4°C. The samples were centrifuged at 18,000 g for 20 min at 4°C,and supernatants were subsequently transferred to new tubes.

To purify the GST-PDSS1 and GST polypeptides, the supernatantscontaining the solubilized proteins were incubated with glutathioneSepharose 4B resin for 1 h at room temperature and subjected to threewashes with buffer 1. The bound proteins were subsequently eluted by10 mM reduced glutathione in buffer 1. To purify the His-PDSS2-Land His-PDSS2-L kd, the supernatant with 10 mM imidazole wasincubated with Ni-NTA Superflow resin for 1 h at room temperature,washed three times in buffer 1 containing 20 mM imidazole, andeluted in buffer 1 with 200 mM imidazole.

The purified proteins with each combination of PDSS1 and B6- andkd -isoforms of PDSS2-L were suspended in 1 PBS with 0.1%Tween 20 and 5 mM MgCl2. His-PDSS2-L B6 or His-PDSS2-L kdprotein were incubated with GST-PDSS1 for 30 min and subsequently

precipitated by Ni-NTA Superflow resin. A coprecipitation assay of GST and His-PDSS2-L B6 also was performed as a negative controlat the same time. After the precipitation step, the resins were washedthree times with 1 PBS with 0.1% Tween 20, 5 mM MgCl2, and 20mM imidazole, and then precipitated proteins were analyzed byWestern blotting.

Generation of polyclonal antisera to the PDSS2 polypeptides.Polyclonal antisera were obtained from rabbits by using a standardimmunization protocol (Cocalico Biologicals, Reamstown, PA). Togenerate antibody against PDSS2, the pET28c-PDSS2-L plasmidexpressing the full-length of PDSS2 with a 6-His tag at the NH 2

terminus was used to transform E. coli strain BL21 (DE3) (Novagen).The fusion protein was induced by 0.5 mM IPTG (Fisher Biotech) at30°C for 3 h and purified from the bacterial lysate under denaturingconditions (8 M urea in 1 PBS, adjusted to pH 7.4) by Ni-NTA

Superflow resin (Qiagen). Elution fractions containing partially puri-fied 6 histidine-fused PDSS2 were concentrated by Amicon Ultra-10,000 MWCO (Millipore). These procedures were repeated until atotal of 2 mg of the PDSS2 protein was recovered. The concentratedsample was subjected to SDS-PAGE on an 11% gel, 1.5 mm thick.After staining of the gel with Coomassie blue, the band correspondingto PDSS2 was excised and used as antigen. The specificity of theantisera was determined by immunoblotting with E. coli-producedPDSS2 polypeptide without any epitope tags. The antisera wereaffinity purified as described previously (32).

RESULTS

Determination of Q content in kidney and liver of B6 wild-type and B6.Pdss2kd/kd mutant mice. Previous analyses

indicated that the content of Q9 and Q10 in kidneys of 123- and151-day-old B6.Pdss2kd/kd mice were only 15–20% of thatpresent in similarly aged B6 wild-type littermates (33). YoungB6.Pdss2kd/kd mice appear to grow and mature normally andonset of kidney disease is variable but usually is evident noearlier than 8 wk of age (14, 34). Given this relatively lateonset of kidney disease, it is important to determine the age atwhich low Q levels manifest. As shown in Fig. 2 and Table 1,the content of Q9 and Q10 in kidney lipid extracts of B6.Pdss2kd/kd mice failed to increase with age, an increase thatis dramatic in the B6 wild-type littermates. The content of Q9

and Q10 in kidney lipid extracts of B6 wild-type littermates 75days of age or older was significantly higher compared with

that of the B6.Pdss2kd/kd mice. To delineate the timing of theincrease in quinone content, kidneys were obtained from B6wild-type mice at 20, 30, 40, 50, and 60 days of age, and thecontent of Q9 and Q10 were determined by RP-HPLC/MS-MSas described. As shown in Table 2, the content of Q9 and Q10

was already elevated in kidney lipid extracts prepared from40-day-old mice. Thus the B6.Pdss2kd/kd mice develop kidneydisease several weeks after the normal increase in Q9 and Q10

content fails to occur.A similar trend was observed in liver, except that the

increase in B6 wild-type liver Q9 content occurred later (Fig. 3

and Table 3). Hence, a lower content of liver Q9 in theB6.Pdss2kd/kd mice was evident only after 149 days of age.Sporadic rescue of Q content B6.Pdss2kd/kd mutant mice by

a partial Pdss2 transgene. The BAC clone 256E1 containsexons 1–5 of the Pdss2 gene (see Fig. 1 B) and was used toestablish a rescued line of B6.Pdss2kd/kd mice (line G) (34).The B6.Pdss2kd/kd -line G mice were the only one of 13 trans-genic lines generated that expressed the Pdss2 transgene at ahigh level. Mouse kidney lipid extracts prepared fromB6.Pdss2kd/kd and B6.Pdss2kd/kd -line G transgenic mice wereanalyzed for Q9 and Q10 content as described in Fig. 2.Remarkably, rescue of Q content in the line G mice wassporadic. Intriguingly, a low content of Q9 and Q10 in thetransgene-positive mice was found to correlate with elevated

levels of albumin in the urine (Fig. 4 and Table 4).Functional analysis of PDSS2-Long, PDSS2-LongV177M,

and PDSS2-Short isoforms of prenyl diphosphate synthase.Prenyl diphosphate synthase activity has been shown to requireboth PDSS1 and PDSS2 subunits (39). The plasmid constructpM12L allowed for coexpression of mouse PDSS1 andPDSS2-Long in E. coli (Fig. 5 A). Transformation of an ispBprenyl diphosphate synthase mutant ( E. coli strain KO229)with this construct rescued aerobic growth and converted thequinone synthesized in E. coli cells from Q8 to Q9 (Fig. 5 B andTable 5) (39). Coexpression of PDSS1 and PDSS2-Long-V117M (from plasmid pM12Lkd) failed to rescue the E. coliispB mutant (data not shown). However, transformation of

Fig. 2. Kidney Q9 and Q10 levels are significantly lower in B6.Pdss2kd/kd micethan in B6 wild-type mice. Wild-type B6, heterozygous B6. /kd , and mutantB6.Pdss2kd/kd mice were killed at the ages shown, and Q levels were measuredin kidney homogenates prepared from each mouse. Error bars indicate thestandard deviation in 3 separate measurements of the same lipid extract. Levelsof Q9 and Q10 in 123- and 151-day-old B6.Pdss2kd/kd mice and in 115- and149-day-old B6 mice were previously reported (33).





wild-type DH5 E. coli with pM12Lkd allowed for the expres-sion of Q9 in addition to Q8. E. coli DH5 harboring eitherempty vector (pGEX-KG) or pM12S (directing coexpressionof PDSS1 and PDSS2-Short) failed to generate appreciablelevels of Q9, indicating that the PDSS2-Short form fails tosupport prenyl diphosphate synthase activity. The Q9 contentin DH5 E. coli cells harboring pM12Lkd (34.8 7.6 pmolQ9 /mg wet weight cell pellet) was significantly lower than incells harboring pM12L (93.7 6.0 pmol Q9 /mg wet weightcell pellet). These results indicate that heterologous expressionof the kd mutant form of PDSS2 in E. coli recapitulates thedeficiency observed in the B6.Pdss2kd/kd mice. Capture of GST-tagged PDSS1 coprecipitated PDSS2-Long and PDSS2-Long-kd (Fig. 6). Thus the V117M mutation in domain I of PDSS2-Long-kd does not prevent its association with PDSS1.

Steady-state polypeptide levels of PDSS2-Long and PDSS2-Short were examined in liver mitochondria isolated from B6wild-type, B6.Pdss2kd/kd , and B6. Alb/cre,Pdss2loxP/loxP mice.Both PDSS2-Long and PDSS2-Short polypeptides werereadily detected in liver mitochondria isolated from B6 wild-type mice (Fig. 7). Steady-state levels of both polypeptideswere lower in the liver mitochondria of B6.Pdss2kd/kd mice,suggesting that the lower Q9 and Q10 content in liver of theB6.Pdss2kd/kd mice may result from a combination of impaired

polyprenyl diphosphate activity and lower steady-state levelsof the PDSS2 polypeptide. Only small amounts of the PDSS2-Long polypeptide were detectable in liver mitochondria iso-lated from B6. Alb/cre,Pdss2loxP/loxP mice, where the Pdss2

gene is knocked out in hepatocytes (33). A drastic decrease inliver Q9 content was evident in these mice; the content of Q9

measured in two different animals (95 days old) using theRP-HPLC-MS/MS assay (see MATERIALS AND METHODS) was8.9 2.4 and 33.5 6.7 pmol Q9 /mg liver protein, whereasnormal Q9 content in age matched controls was 10 timeshigher.

Rescue of renal disease by Q10 supplementation. In a pilotexperiment, three male and two female B6.Pdss2kd/kd mice

were supplemented with 0.25 mg Q10 /ml drinking water andcompared with their untreated littermates. When the mice weretested at 132 days of age, the mutants had less urine albuminthan their untreated littermates (7.2 5.8 vs. 13.5 8.3 mg/24h, respectively). However, this difference was not significant asindicated by Student’s t -test (P 0.288). Therefore, beginningat weaning, the offspring produced by the B6.Pdss2kd/kd -sup-

Fig. 3. Differences in liver Q9 content in B6.Pdss2kd/kd mice and B6 wild-typemice are evident by 149 days of age. Wild-type B6 and mutant B6.Pdss2kd/kd

mice were killed at the ages shown, and Q levels were measured in liverhomogenates prepared from each mouse. Error bars indicate the standarddeviation in 3 separate measurements of the same lipid extract.

Table 1. Content of Q9 and Q10 in kidney extracts of B6.Pdss2kd/kd and B6 mice

Lipid ExtractAverage Q9,

pmol/mg ProteinAverage Q10,

Pmol/mg Protein

10-day B6.kd/kd 51515 766.510-day B6.kd/kd 56913 1058.210-day B6.kd/kd 1665.8 269.010-day B6.kd/kd 57224 908.937-day B6.kd/kd 29215 293.6123-day B6.kd/kd 34118 507.5151-day B6.kd/kd 37323 398.4180-day B6.kd/kd 38213 532.6180-day B6.kd/kd 26126 3111180-day B6.kd/kd 4422.9 549.120-day B6 57224 762175-day B6 2,010147 35126111-day B6 1,950138 25420115-day B6 2,190194 42946149-day B6 2,330134 30222160-day

Heterozygote 2,350184 31326

Values are means SD.

Table 2. Content of Q9 and Q10 in kidney extracts of young

B6 mice

Lipid Extract Average Q9, pmol/mg Protein Average Q10, pmol/mg Protein

20-day B6 21374 222.020-day B6 23114 252.030-day B6 45050 344.030-day B6 64160 547.040-day B6 1,08859 1201040-day B6 1,09560 1151050-day B6 1,437368 1503850-day B6 1,29885 1072660-day B6 1,855154 1611360-day B6 1,492210 13018

Values are means SD at indicated ages.

Table 3. Content of Q9 in liver extracts of B6. Pdss2kd/kd

and B6 mice

Lipid Extract Average Q9, pmol/mg Protein

28-day B6.kd/kd 47644

37-day B6.kd/kd 8861123-day B6.kd/kd 3325.5151-day B6.kd/kd 33714180-day B6.kd/kd 24417180-day B6.kd/kd 25922180-day B6.kd/kd 4282920-day B6 4221775-day B6 44416111-day B6 29325115-day B6 446162149-day B6 80682201-day B6 1,18054201-day B6 1,02049201-day B6 1,17072

Values are means SD at indicated ages.





plemented mice received double the dose (0.50 mg Q10 /mldrinking water), equivalent to 200 mg Q10 kg bodywt1

day1 (see MATERIALS AND METHODS). After mice reachedat least 130 days of age, urine albumin was determined.Supplemented mice had on average a fivefold reduction in theurine albumin content compared with unsupplemented, age-matched B6.Pdss2kd/kd mice, and their nephritis scores weresignificantly lower (P 0.004, Student’s t -test) than those of untreated B6.Pdss2kd/kd controls (Table 6). However, the con-tent of Q9 and Q10 of supplemented animals (n 7; 267.2 86.7 pmol Q9 /mg kidney protein and 37.1 16.1 pmol Q10 /mgkidney protein) was not significantly different from the content

in the untreated B6.Pdss2

kd/kd

controls (n 3; 353.0 40.5pmol Q9 /mg kidney protein and 45.2 6.0 pmol Q10 /mgkidney protein). Doubling the dose to 400 mg kg body

wt1

day1

did not improve the effectiveness of the treat-ment (Table 6). Examples of H&E-stained sections from thekidneys of Q10-treated and untreated Pdss2kd/kd mice areshown in Fig. 8, A and B, respectively.

DISCUSSION

Mice harboring the homozygous kd/kd mutation in Pdss2 shownormal growth and fertility but develop kidney disease as youngadults. Severe tubulointerstitial nephritis and tubular dilation areaccompanied by morphological disorders of mitochondria in kid-ney and liver (34). The kd/kd mutation (V117M) occurs within theconserved domain I of PDSS2 (Fig. 1 A). A decreased Q contentwas observed in adult B6.Pdss2kd/kd mice (33). In this study we

Fig. 5. Heterologous expression of the mutant kd form of Pdss2 in Escherichiacoli recapitulates the deficiency in Q biosynthesis observed in B6.Pdss2kd/kd

mice. A: pGEX-KG (empty vector) produces just the glutathione S -transferase(GST) polypeptide. pM12S contains the open reading frame (ORF) of Pdss1inserted in frame after the carboxyl terminus of GST (GST-PDSS1) andPdss2-S (Short form) inserted in frame after a six-Histidine tag (His 6-PDSS2-S). pM12L expresses GST-PDSS1 and His6-PDSS2-L (Long form). pM12Lkdexpresses GST-PDSS1 and His6-PDSS2-L-V117M (Long form harboring thekd mutation). B, BamHI; E, EcoRI; S, SalI, and Xb, XbaI. An asterisk showsthe kd/kd mutation position V117M, and “rbs” indicates a ribosomal bindingsite from pET-28c to allow polycistronic expression of the mouse polypep-tides. B: Q8, normally produced in E. coli, and Q9, generated by the heterol-ogous expression of PDSS1 and PDSS2, were measured in lipid extractsprepared from E. coli DH5 harboring each of the constructs designated in A.

E. coli strain KO229 harboring a deletion of the ispB prenyl diphosphatesynthase gene was rescued by pM12L. Error bars indicate the standarddeviation in 3 separate measurements of the same lipid extract.

Table 4. Content of Q9 and Q10 in kidney extracts of B6.Pdss2kd/kd , B6.Pdss2kd/kd -line G, and B6 mice

Lipid ExtractAverage Q9,

pmol/mg ProteinAverage Q10,

pmol/mg ProteinUrine

Albumin, mg

123-day B6.kd/kd 26741 ND151-day B6.kd/kd 23721 ND180-day B6.kd/kd 20428 ND180-day B6.kd/kd 29924 ND70-day B6.kd/kd Tg 1,53048 13511 0.1794-day B6.kd/kd Tg 2,870107 2768.5 0.16

94-day B6.kd/kd Tg 3,060271 3364.6 0.1494-day B6.kd/kd Tg 37222 239.1 4.20157-day B6.kd/kd Tg 54449 242.7 7.40157-day B6.kd/kd Tg 1,740106 12728 0.26157-day B6.kd/kd Tg 1,750108 13930 0.60157-day B6.kd/kd Tg 2,71095 2618.6 0.1575-day B6 2,25024 2178.2111-day B6 2,13036 1599.1149-day B6 2,730147 18912149-day B6 1,960154 13118143-day B6 Tg 2,490139 21911 0.12

Values are means SD at indicated ages. Tg, Pdss2 line G transgenic mice(partial Pdss2 transgene contains exons 1–5). Albumin content in urine wasmeasured in each of the mice harboring the partial Pdss2 transgene. ND, notdetected.

Fig. 4. Expression of a truncated form of Pdss2 in B6.Pdss2kd/kd mice restorescontent of Q in kidney extracts to wild-type levels in most cases. Wild-type B6,mutant B6.Pdss2kd/kd , and line G B6.Pdss2kd/kd transgenic mice (indicated byasterisks) were killed at the ages shown, and Q9 and Q10 levels were measuredin kidney homogenates taken from each mouse. Error bars indicate thestandard deviation in 3 separate measurements of the same lipid extract.





have shown that Q9 and Q10 content in kidney lipid extracts of young B6.Pdss2kd/kd mice and their B6 wild-type littermates(20 days of age) are very similar. However, Q9 and Q10 contentin kidney lipid extracts of B6 wild-type mice 40 days of age orolder are significantly higher compared with that inB6.Pdss2kd/kd mice. The normal increase at 30–40 days of agemay reflect unidentified physiological changes that occur atweaning, which normally occurs soon after day 20. The failureto increase the content of Q in kidneys of the B6.Pdss2kd/kd

mutant mice at 40 days of age precedes by at least severalweeks the renal disease that develops at about 8 –10 wk of age.In contrast, Q content in liver from B6.Pdss2kd/kd mice remainssimilar to wild-type mice until later in adulthood (150 daysof age). This profound deficiency in Q content in the kidneyearly in life is likely responsible for the renal disease pheno-type.

Mice express two polypeptide isoforms of PDSS2 due toalternative splicing, PDSS2-Long (exons 1– 8) and PDSS2-Short (exons 1–3b) (Fig. 1 B) (34). Previous work has shown

that PDSS2 forms a heterotetramer with PDSS1 and that bothPDSS1 and PDSS2-Long are required for polyprenyl diphos-phate synthesis (39). In this study we have shown that coex-pression of the short isoform of PDSS2 with PDSS1 in E. colifails to reconstitute synthesis of Q9. We hypothesize that thePDSS2-Short form may repress nonaprenyl diphosphate syn-thase activity by forming an unproductive complex withPDSS1. E. coli harboring a construct coexpressing PDSS1 andPDSS2-Long-V117M (the kd mutant form) had significantlylower Q9 content than did cells expressing PDSS1 and PDSS2-Long (wild type). These results recapitulate the low Q contentobserved in the B6.Pdss2kd/kd mice and suggest that theV117M mutation in Pdss2 occurs in a conserved domain of polyprenyl diphosphate synthase and partially impairs synthe-

sis of nonaprenyl and decaprenyl diphosphate. The mutationmay affect enzymatic activity and/or heterotetramer formation.

Table 5. Content of Q8 and Q9 in extracts of Eschericia coliexpressing Pdss1 and isoforms of Pdss2

Lipid ExtractAverage Q8, pmol/mg

Wet WeightAverage Q9, pmol/mg

Wet Weight

Empty vector 22621 2.80.5Pdss1 Pdss2-Short 14361 2.21.1Pdss1 Pdss2 V117M 11515 357.6Pdss1 Pdss2-Long 750.4 946.0


Fig. 6. Coprecipitation of PDSS1 and PDSS2 polypeptides. His-PDSS2-L orHis-PDSS2-L-V117M polypeptides were incubated with GST-PDSS1 for 30min and subsequently precipitated by Ni-NTA Superflow resin (lanes 2 and 3).A coprecipitation assay of GST and His-PDSS2-L also was performed as anegative control at the same time (lane 1). Antibodies to GST ( A) and the 6-Histag ( B) were utilized to recognize GST-PDSS1 and His-PDSS2 polypeptides,respectively. The asterisks indicate nonspecific bands recognized by the GSTantibody. GST*(truncated), observed in lanes 2, 3 , 5 , and 6 in A , bottom, islikely to result from proteolysis of the GST-PDSS1 fusion protein. Lanes 4, 5,and 6 show the relative amounts of input protein used for each coprecipitationassay and correspond to lanes 1, 2, and 3, respectively. Similar amounts of GST-PDSS1 were coprecipitated with both His-PDSS2-L and His-PDSS2-Lkdfusion proteins ( A, lanes 2 and 3, top). In contrast, GST did not coprecipitatewith the His-PDSS1-L fusion protein ( A, lane 1, bottom).

(kDa)

(kDa)

Fig. 7. Western blot analysis of PDSS2 polypeptides in mice liver mitochon-dria. An anti-PDSS2 antibody was utilized to detect PDSS2 polypeptides in120 g of mouse mitochondria protein ( A). An existing amount of PDSS2-Land -S in B6 mouse mitochondria (lane 1) was significantly higher than inkd/kd and alb/cre mutant mice (lanes 2 and 3, respectively). PDSS2-S ispresent in relatively smaller amounts than PDSS2-L. An anti-yeast F1ATPsynthase subunit that cross-reacts with mouse F1ATP synthase subunitprovided a loading control ( B).

Table 6. Effect of adding Q10 to the drinking water of mutant mice

MiceDose of Q10,

mg kg1 day n Age, Days

UrineAlbumin, mg/24 h Nephritis

B6 Normal water 17 17044 0.240.56 0.350.56B6.Pdss2kd/kd Normal water 23 1274.7 18.2313.86 2.650.90B6.Pdss2kd/kd 200 19 14420.2 4.744.69 1.611.01B6.Pdss2kd/kd 400 8 138.518.5 6.155.83 1.751.39






However, capture of PDSS1 coprecipitates the PDSS2-Long-V117M polypeptide, suggesting that the V117M mutation does

not prevent interaction of the two subunits.Given that the short form of PDSS2 (exons 1–3b) failed to

restore Q biosynthesis, it is curious that the BAC clone 256E1,which contains only a segment of the Pdss2 gene, establisheda rescued line of B6.Pdss2kd/kd mice (line G) (34). However,this clone includes exons 1–5 and in most cases rescued bothkidney disease and Q levels. Remarkably, rescue of kidneydisease and Q content in line G mice was sporadic. Intrigu-ingly, a low Q content in transgene-positive mice was found tocorrelate exactly with elevated levels of protein in urine.

The data suggest that the renal disease phenotypes inPdss2kd/kd homozygous mice are caused by a decreased contentof Q in the kidney. The renal disease could result from either

inadequate respiratory function due to low levels of Q orincreased oxidative stress because QH2 is a potent antioxidant.The partial rescue by dietary supplementation with Q10 isconsistent with both ideas. Several lines of evidence suggestthat Q/QH2 is serving a crucial antioxidant role. Conditionalknockout mice with deletions of Pdss2 in the glomerularpodocytes (B6.Podocin/cre,Pdss2loxP/loxP) fully recapitulate

the kd/kd phenotype (33). However, kidney disease does notdevelop in the B6.PEPCK/cre,Pdss2loxP/loxP mice, in whichPdss2 is deleted in renal tubular epithelial cells. Podocytes arenot thought to have an energetic requirement as high as that of renal tubular epithelium, supporting the hypothesis that sus-ceptibility to oxidative damage is the critical factor in thePdss2kd/kd model of kidney disease. The kidney content of Q10

was not enhanced by supplementation with Q10, a findingconsistent with other similar Q10 supplementation studies (21).However, longer periods of supplementation (e.g., 17 mo) doelevate both Q9 and Q10 kidney content (43). It is well estab-lished that short-term supplementation does impact levels of Q10 in the liver and blood plasma (24). Hence, it seems likelythat the benefits of Q10 supplementation may be exerted on thepodocytes via their filtration of blood plasma components (46).Although in this study we have not evaluated oxidative stressor antioxidant status, other evidence that the Pdss2kd/kd micemay be subject to oxidative stress is the finding that theirsusceptibility to renal disease is markedly diminished by eitherdietary restriction or a germ-free environment (13). Dietaryrestriction is associated with decreased oxidative stress (48)and has been shown to decrease lipid peroxidation productsand apoptosis in aged rat kidneys (22). Similarly, mice rearedin a germ-free environment recapitulate many aspects of diet-restricted animals and show enhanced AMP-activated proteinkinase activity (1), a hallmark of the response to dietaryrestriction in yeast and worms (5).

It is possible that renal disease in Pdss2kd/kd

mice resultsfrom a defect in an isoprenylated product other than Q. ThePDSS2 polypeptide produces nona- and decaprenyl diphos-phate, and studies of mevalonate labeling of mice and ratssuggest these long-chain prenyl groups may be utilized inkidney protein prenylation (6, 31), similar to the better char-acterized protein posttranslational modification with farnesylor geranylgeranyl groups (20). However, the fact that diseasesusceptibility is ameliorated by Q10 supplementation suggeststhat Q itself is the most essential substrate with regard to thisphenotype. Patients with mutations in the COQ2 gene showrenal disease with collapsing glomerulopathy (2, 9), furtherindicating the causative role is a deficiency in Q itself.

Several human Q10-deficient patients have responded dra-

matically to Q10 supplementation (36–38, 41). If their responseis primarily based on the antioxidant function of Q10 ratherthan its role in mitochondrial respiration, then antioxidants thatare more readily taken up by the target tissues could be moreeffective therapies than Q supplementation. Experiments are inprogress to determine whether other antioxidants may be moreeffective than Q10 in preventing kidney disease in Pdss2kd/kd

mice.

GRANTS

This work was supported in part by National Institutes of Health (NIH)Grants GM45952 and AG19777 (to C. F. Clarke) and DK55852 (to D. L.Gasser). A. L. Lunceford received support from the Ruth L. Kirschstein

Fig. 8. Hematoxylin- and eosin-stained sections of kidneys from Q10-treatedand untreated B6.Pdss2kd/kd mice. A: no histological evidence of disease in a162-day-old female treated with Q10. The amount of urine albumin measuredwas 3.7 mg/24 h. B: severe interstitial nephritis in an untreated 128-day-oldfemale. The amount of urine albumin measured was 57.34 mg/24 h. Note thesevere interstitial inflammation, tubular dilatation, and glomerular crescents.Magnification, 100.





National Research Service Award issued from NIH Training GrantGM007185. The LC-MS/MS determination of Q content was supported byNational Center for Research Resources Grant S10 RR024605. Histologicalpreparations were done in the Morphology Core for Molecular Studies inDigestive and Liver Diseases at the University of Pennsylvania, which issupported by NIH Grant P30 DK50306.

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Coenzyme Q10 Supplementation Rescues Renal Disease in Pdss2kd:Kd Mice With Mutations in Prenyl Diphosphate Synthase Subunit 2