Am J Physiol Renal Physiol 295: F1535F1544, 2008.

First published September 10, 2008; doi:10.1152/ajprenal.90445.2008.

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; 2Department 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 supplementation rescues renal disease in Pdss2kd/kd mice with mutations in
prenyl diphosphate synthase subunit 2. Am J Physiol Renal Physiol
295: F1535F1544, 2008. First published September 10, 2008;
doi:10.1152/ajprenal.90445.2008.Homozygous mice carrying kd
(kidney disease) mutations in the gene encoding prenyl diphosphate
synthase subunit 2 (Pdss2kd/kd) develop interstitial nephritis and eventually die from end-stage renal disease. The PDSS2 polypeptide in
concert with PDSS1 synthesizes the polyisoprenyl tail of coenzyme Q
(Q or ubiquinone), a lipid quinone required for mitochondrial respiratory electron transport. We have shown that a deficiency in Q
content is evident in Pdss2kd/kd mouse kidney lipid extracts by 40 days
of age and thus precedes the onset of proteinuria and kidney disease
by several weeks. The presence of the kd (V117M) mutation in
PDSS2 does not prevent its association with PDSS1. However,
heterologous expression of the kd mutant form of PDSS2 together
with PDSS1 in Escherichia coli recapitulates the Q deficiency observed in the Pdss2kd/kd mouse. Dietary supplementation with Q10
provides a dramatic rescue of both proteinuria and interstitial nephritis
in the Pdss2kd/kd mutant mice. The results presented suggest that Q
may be acting as a potent lipid-soluble antioxidant, rather than by
boosting kidney mitochondrial respiration. Such Q10 supplementation
may have profound and beneficial effects in treatment of certain forms
of focal segmental glomerulosclerosis that mirror the renal disease of
the Pdss2kd/kd mouse.

COENZYME Q (Q or ubiquinone) is an essential factor of aerobic

respiration and oxidative phosphorylation. Q also functions as
a lipid-soluble antioxidant in cellular biomembranes and scavenges reactive oxygen species (4, 7). Yeast strains not able to
produce Q show sensitivity to oxidative stresses such as hydrogen peroxide and autooxidized polyunsaturated fatty acids
(10, 44). Q is a dietary supplement and is also used in
therapeutic treatments for neurodegenerative and cardiovascular diseases and for statin-induced symptoms of Q deficiency
(11, 35).
Q is composed of a benzoquinone ring with a polyisoprenoid
side chain of varying length. Living organisms possess distinct
isoforms of Q depending on the length of the isoprenoid side
chain (17). For example, humans produce Q10, mice produce
predominantly Q9 and small amounts of Q10, Saccharomyces
cerevisiae produce Q6, and Escherichia coli produce Q8. The

difference in the tail length of Q is determined by the longchain polyprenyl diphosphate synthase in each organism (30).
Long-chain trans-prenyl diphosphate synthases catalyze the
condensation step for the isoprenoid tails from farnesyl diphosphate (C15) or geranylgeranyl diphosphate (C20) and isopentenyl diphosphate (C5). Structural analyses and site-directed
mutagenesis have shown that short- and long-chain polyprenyl
diphosphate synthases contain seven conserved regions designated as domains I through VII (Fig. 1A) and two aspartate-rich
motifs (DDXXD) that are substrate binding sites in association
with Mg2 (12, 18, 28, 29, 45). These analyses suggest that
amino acid residues at the fourth and fifth positions before the
aspartate-rich motif in domain II and two amino acid residues
in domain II have an important role in determining the length
of the isoprenoid tails.
Polyprenyl diphosphate synthases are classified into two
types of homo- or heterocomplexes. Most of the short polyprenyl diphosphate synthases (C10 C25) in both prokaryotes
and eukaryotes are homodimers (27). In contrast, long-chain
(C30 C50) polyprenyl diphosphate synthases are more diversified. In E. coli, octaprenyl diphosphate synthase forms a
homodimer necessary for synthesis of the octaprenyl diphosphate tail for both Q and menaquinone (16), whereas the
enzymes that supply hexa- and heptaprenyl diphosphate for
menaquinone in the genus Micrococcus and Bacillus are heterodimers (47). The Coq1 polypeptide in S. cerevisiae functions on its own to produce hexaprenyl diphosphate; however,
when expressed in fission yeast, Schizosaccharomyces pombe,
it functions as a partner protein to produce decaprenyl diphosphate (49). In S. pombe, mice, and humans, long-chain prenyl
diphosphate 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 decaprenyl
diphosphate synthase in fission yeast and mammals, possesses
the conserved domains of a prenyl diphosphate synthase.
However, the subunit 2 lacks aspartate-rich motifs in domains
II and VI (39, 40). PDSS2 orthologs also are found in Drosophila, Xenopus, and mammals. Therefore, the heterotetramer
conformation of long-chain prenyl diphosphate synthases
might be standard in higher eukaryotes.
Recently, several diagnostic examples of inherited Q deficiency, 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, UCLA

Dept. of Chemistry and Biochemistry, 607 Charles E. Young Dr. E., Los
Angeles, CA 90095-1569 (e-mail: cathy@chem.ucla.edu).

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

ubiquinone; mitochondrial lipid metabolism; genetic renal disease;

focal segmental glomerulosclerosis; antioxidant

http://www.ajprenal.org

0363-6127/08 $8.00 Copyright 2008 the American Physiological Society

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COENZYME Q10 SUPPLEMENTATION RESCUES RENAL DISEASE

Fig. 1. Amino acid sequence alignment of

mouse nonaprenyl diphosphate synthase subunit 1 (PDSS1/mSPS1) and subunit 2 (PDSS2/
mDLP1) isoforms. A: typical long-chain polyisoprenyl diphosphate synthases, such as
PDSS1 and PDSS2-L, have conserved domains
I through VII. An arrow shows the location of
the kd/kd mutation V117M within conserved
domain I. B: alternative splicing produces
PDSS2-Short (PDSS2-S) containing exons
13b and PDSS2-Long (PDSS2-L) containing
exons 13a and 4 8. PDSS2-S contains only
domains I through III. The predicted polypeptide produced from the Pdss2 BAC clone
256E1 is also shown. One transgenic line of
B6.kd/kd mice harboring an insertion of this
BAC clone 256E1 exhibits sporadic rescue
from kidney disease.

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, or
CABC1/COQ8 are significantly lower than in samples from
healthy individuals. Addition of either decylubiquinone or Q2, two
soluble Q analogs, restored oxidative phosphorylation activity in
cultured skin fibroblasts (19, 26) and muscle extracts (23) of the
affected patients. Therefore, the respiratory deficiency and the
symptoms in the patients are attributed to primary Q deficiency. In
some cases, prolonged Q supplementation relieves the symptoms
caused by Q deficiency (37). The reasons for the different responses to Q supplementation are still unclear.
In this study we have examined the nature of the Q deficiency in the Pdss2kd/kd mouse, which harbors a V117M
mutation in PDSS2 and develops kidney disease in early
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adulthood (33, 34). The V117 residue is invariant in PDSS2

polypeptides from fission yeast to humans (39). Based on
immunohistochemical profiling and electron microscopy, the
phenotype 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 in
kidney lipid extracts by 40 days of age and that dietary
supplementation with Q provides a dramatic rescue of the
proteinuria and interstitial nephritis. The results suggest that if
human renal patients are identified who have PDSS2 mutations
similar to that of the Pdss2kd/kd mouse, Q supplementation may
have profound and beneficial effects.
MATERIALS AND METHODS

Mice. All animal studies have been approved by the University

of California, Los Angeles (UCLA) and University of Pennsylvania institutional review boards. Mice harboring the Pdss2kd/kd

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COENZYME Q10 SUPPLEMENTATION RESCUES RENAL DISEASE

missense mutation (V117M) on the B6 genetic background, line G

transgenic mice expressing a truncated form of Pdss2, and B6.Alb/
cre,Pdss2loxP/loxP mice, in which the Pdss2 gene is knocked out in
hepatocytes, have been described previously (14, 33, 34). Assays
of urine albumin were performed with ELISA kits from Bethyl
Laboratories as described previously (33). When the mice were
euthanized, hematoxylin and eosin (H&E)-stained sections of the
kidneys were prepared. The sections were examined blindly and
scored as follows: 0, no tubular dilatation and no mononuclear cell
infiltrates; 1, small focal areas of cellular infiltration and tubular
dilatation involving 10% of the cortex; 2, involvement of up to
25% 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 kidneys and lobes of livers were dissected from euthanized mice and
stored at 80C until homogenization. Samples were homogenized in
5 ml of 1 PBS, pH 7.4 (137 mM NaCl, 8 mM Na2HPO4, 2.7 mM
KCl, and 1.5 mM KH2PO4) at 4C, with a total of 10 strokes with a
tight-fitting Teflon pestle rotating at maximal speed with a Fisher
Scientific Lab Stirrer LR400A. The homogenate was centrifuged at
1,000 g for 5 min, supernatants were removed to fresh vials, and
protein concentrations were measured using the bicinchoninic acid
assay (Pierce, Rockford, IL). Aliquots of each homogenate were
transferred to 50-ml glass tubes and stored at 80C until extracted.
Q10 supplementation. Mice were provided Q10 supplements (Tishcon LiQsorb, 100 mg/ml) in their drinking water. In a pilot experiment, three males and two females were given 100 mg Q10/400 ml
water (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 mg
Q10/ml water. Mice consumed 1216 ml of water per day; thus at
this dose, the mice received 200 mg Q10 kg body wt1 day1.
Some of their offspring were given a dose of 400 kg body
wt1 day1, beginning at the time they were weaned.
Expression of murine PDSS1 and PDSS2 prenyl diphosphate synthase subunits in E. coli. E. coli strain DH5 was used for general
plasmid construction and protein expression (42). E. coli cells were
grown in Luria Bertani (LB) medium with appropriate antibiotics such
as 100 g/ml carbenicillin, 100 g/ml ampicillin, and 50 g/ml
kanamycin. The pGEX-KG (GE Healthcare Bio-Sciences, Piscataway, NJ) and pET-28c (Novagen, Madison, WI) plasmids served as
vectors.
cDNAs prepared from either B6 or B6.Pdss2kd/kd liver (34) were
used as template DNA to PCR amplify the short and long isoforms of
Pdss2. The 0.8-kb amplicon containing the Pdss2-Short isoform
cDNA was generated with B6 liver cDNA as template and two
oligonucleotide primers, 5-EcoRI-mDLP1 5-CCGAATTCGAATGAGCTCCGGCAG-3 (creates an EcoRI site) and 3-SalI-mDLP1Short, 5-ACGCGTCGACTTACTTCATGTTGTCACTGCC-3 (creates a SalI site). The amplified 0.8-kb fragment was digested with
EcoRI and SalI enzymes and inserted into the same sites of pET28c to
yield pET28c-PDSS2-S. The pET28c-PDSS2-S plasmid was digested
with XbaI and SalI, and the resulting fragment was cloned into the
same sites of pGEX-mSPS1 (39) to construct pM12S. Glutathione
S-transferase (GST)-tagged-PDSS1 and His6-tagged-PDSS2-Short are
coexpressed from the Tac promoter of pM12S.
A similar scheme was used to generate pM12L and pM12Lkd. The
1.2-kb amplicon containing the Pdss2-Long isoform cDNA was
generated with B6 liver cDNA as template and the oligonucleotide
primers, 3-SalI-mDLP1-L, 5-ACGCGTCGACTCAAGAAAATCTGGTCACAGC-3 (creates a SalI site) and 5-EcoRI-mDLP1. The
amplified 1.2-kb fragment was digested with EcoRI and SalI enzymes
and inserted into the same sites of pET28c to yield pET28c-PDSS2-L.
The pET28c-PDSS2-L plasmid was digested with XbaI and SalI, and
the resulting fragment was cloned into the same sites of pGEXmSPS1 to construct pM12L. The same sequence of steps was used to
construct pM12Lkd, except that cDNA prepared from B6.Pdss2kd/kd
mouse liver was used as template DNA. pM12L and pM12Lkd drive
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expression of GST-tagged-PDSS1 and either His6-tagged-PDSS2Long form or His6-tagged-PDSS2-Long form harboring the kd missense mutation (V117M).
DH5 harboring pM12S, pM12LB6, pM12Lkd, or the empty
vector pGEX-KG were inoculated in 5 ml of LB medium with
ampicillin and cultured at 30C, 250 rpm, for 18 h. Aliquots of each
bacterial culture (500 l) were used to inoculate 50 ml of fresh LB
medium with carbenicillin and incubated at 30C, 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 concentration of 0.5 mM, and cells were incubated at 30C, 250 rpm, for
4 h. Cells were collected and stored at 20C for lipid extraction.
Q measurements: electrochemical and UV detection. A known
amount of the internal standard Q6 was added to all samples and
calibration curve standards before lipid extraction. The same amount
of Q6 was also added to the Q9 and Q10 standards prepared at different
concentrations to generate an extracted standard curve. Lipid extractions were then performed on Q9 and Q10 standards, mouse tissue
homogenates, 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 vortexed
for 1 min and centrifuged at 910 g, and the top layer of petroleum
ether was removed and transferred to a separate vial. Fresh petroleum
ether (6 ml) was added to the remaining aqueous phase and vortexed
for 1 min. The vials were subjected to centrifugation as before, and the
second petroleum ether layer was removed. The process was repeated
once more, and the combined upper organic phase was dried under N2
and resuspended in 200 l of methanol. The quinones were then
separated and quantified by reverse-phase (RP)-HPLC connected to
an electrochemical detector as described previously (15), with the
following exceptions: the precolumn electrode was set at 650 mV to
oxidize all hydroquinones, and a Gilson 118 UV/Vis detector was
utilized to detect quinones (275 nm) as they eluted from the column.
The amounts of Q9 and Q10 in the standards and samples were
calculated via the calibration curve.
Q measurements: RP-HPLC-multiple reaction monitoring. Samples and standards were extracted as described above, except with
0.05 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) isolated from Rhodospirillum rubrum were used as internal standards.
RP-HPLC-multiple reaction monitoring (MS/MS) was performed on a
4000 Q Trap-Hybrid triple-quad linear ion trap analyzer (Applied
Biosystems/MDS Sciex) with an autosampler (CTC Analytics/LEAP
Technologies, HTC PAL; 10-l injections, 20-l loop) and a Turbo-V
source with the ESI probe inserted. Reverse-phase sample separation
was performed via a pentafluorophenyl propyl column (5 m, 4.6
100 mm; Phenomenex, Torrance, CA). The mobile phase was 95:5
acetonitrile-water containing 0.1% formic acid (2 ml/min). The mass
spectrometer was set to the positive ion mode with the following
parameters: declustering potential, 61 V for RQ9, 91 V for Q9 and
RQ10, 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 reaction
monitoring (MRM) mode: 210-ms dwell; MRM transitions: m/z
781.5/182.1 for RQ9, m/z 795.6/197.1 for Q9, m/z 848.7/182.1 for
RQ10, and m/z 863.7/197.0 for Q10. Quantification was via embedded
Analyst software version 1.4.2 (Applied Biosystems/MDS Sciex).
Coprecipitation of PDSS1 and PDSS2 polypeptides. To evaluate
whether PDSS2-Long-kd interacts with PDSS1, we adapted an affinity purification to isolate GST-PDSS1, GST, His-PDSS2-L, and HisPDSS2-L kd. GST-PDSS1 and GST were purified using glutathione
Sepharose 4B (GE Healthcare BioSciences), and His-PDSS2-L and
His-PDSS2-L kd were purified using Ni-NTA Superflow (Qiagen)
according to the manufacturers instructions. Briefly, BL21 (DE3) E.
coli cells harboring pET28c-Pdss2-L, pET28c-Pdss2-L kd, pGEX-

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COENZYME Q10 SUPPLEMENTATION RESCUES RENAL DISEASE

mSPS1 (39), and pGEX-KG empty vector grown in 50 ml of LB

medium plus appropriate antibiotics were induced by 0.5 mM IPTG
for 3 h at 30C. After centrifugation to collect cells, the pellets were
sonicated for 12 cycles of 10-s sonication and 10-s intervals on ice in
buffer 1, composed of 50 mM sodium phosphate, 0.5 M sodium
chloride, 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 4C. The samples were centrifuged at 18,000 g for 20 min at 4C,
and supernatants were subsequently transferred to new tubes.
To purify the GST-PDSS1 and GST polypeptides, the supernatants
containing the solubilized proteins were incubated with glutathione
Sepharose 4B resin for 1 h at room temperature and subjected to three
washes with buffer 1. The bound proteins were subsequently eluted by
10 mM reduced glutathione in buffer 1. To purify the His-PDSS2-L
and His-PDSS2-L kd, the supernatant with 10 mM imidazole was
incubated with Ni-NTA Superflow resin for 1 h at room temperature,
washed three times in buffer 1 containing 20 mM imidazole, and
eluted in buffer 1 with 200 mM imidazole.
The purified proteins with each combination of PDSS1 and B6- and
kd-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 kd
protein 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 control
at the same time. After the precipitation step, the resins were washed
three times with 1 PBS with 0.1% Tween 20, 5 mM MgCl2, and 20
mM imidazole, and then precipitated proteins were analyzed by
Western blotting.
Generation of polyclonal antisera to the PDSS2 polypeptides.
Polyclonal antisera were obtained from rabbits by using a standard
immunization protocol (Cocalico Biologicals, Reamstown, PA). To
generate antibody against PDSS2, the pET28c-PDSS2-L plasmid
expressing the full-length of PDSS2 with a 6-His tag at the NH2
terminus was used to transform E. coli strain BL21 (DE3) (Novagen).
The fusion protein was induced by 0.5 mM IPTG (Fisher Biotech) at
30C for 3 h and purified from the bacterial lysate under denaturing
conditions (8 M urea in 1 PBS, adjusted to pH 7.4) by Ni-NTA
Superflow resin (Qiagen). Elution fractions containing partially purified 6 histidine-fused PDSS2 were concentrated by Amicon Ultra10,000 MWCO (Millipore). These procedures were repeated until a
total of 2 mg of the PDSS2 protein was recovered. The concentrated
sample was subjected to SDS-PAGE on an 11% gel, 1.5 mm thick.
After staining of the gel with Coomassie blue, the band corresponding
to PDSS2 was excised and used as antigen. The specificity of the
antisera was determined by immunoblotting with E. coli-produced
PDSS2 polypeptide without any epitope tags. The antisera were
affinity 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- and
151-day-old B6.Pdss2kd/kd mice were only 1520% of that
present in similarly aged B6 wild-type littermates (33). Young
B6.Pdss2kd/kd mice appear to grow and mature normally and
onset of kidney disease is variable but usually is evident no
earlier than 8 wk of age (14, 34). Given this relatively late
onset of kidney disease, it is important to determine the age at
which 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 that
is dramatic in the B6 wild-type littermates. The content of Q9
and Q10 in kidney lipid extracts of B6 wild-type littermates 75
days of age or older was significantly higher compared with
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Fig. 2. Kidney Q9 and Q10 levels are significantly lower in B6.Pdss2kd/kd mice
than in B6 wild-type mice. Wild-type B6, heterozygous B6./kd, and mutant
B6.Pdss2kd/kd mice were killed at the ages shown, and Q levels were measured
in kidney homogenates prepared from each mouse. Error bars indicate the
standard deviation in 3 separate measurements of the same lipid extract. Levels
of Q9 and Q10 in 123- and 151-day-old B6.Pdss2kd/kd mice and in 115- and
149-day-old B6 mice were previously reported (33).

that of the B6.Pdss2kd/kd mice. To delineate the timing of the

increase in quinone content, kidneys were obtained from B6
wild-type mice at 20, 30, 40, 50, and 60 days of age, and the
content of Q9 and Q10 were determined by RP-HPLC/MS-MS
as described. As shown in Table 2, the content of Q9 and Q10
was already elevated in kidney lipid extracts prepared from
40-day-old mice. Thus the B6.Pdss2kd/kd mice develop kidney
disease 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 the
B6.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 contains
exons 15 of the Pdss2 gene (see Fig. 1B) and was used to
establish a rescued line of B6.Pdss2kd/kd mice (line G) (34).
The B6.Pdss2kd/kd-line G mice were the only one of 13 transgenic lines generated that expressed the Pdss2 transgene at a
high level. Mouse kidney lipid extracts prepared from
B6.Pdss2kd/kd and B6.Pdss2kd/kd-line G transgenic mice were
analyzed for Q9 and Q10 content as described in Fig. 2.
Remarkably, rescue of Q content in the line G mice was
sporadic. Intriguingly, a low content of Q9 and Q10 in the
transgene-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 require
both PDSS1 and PDSS2 subunits (39). The plasmid construct
pM12L allowed for coexpression of mouse PDSS1 and
PDSS2-Long in E. coli (Fig. 5A). Transformation of an ispB
prenyl diphosphate synthase mutant (E. coli strain KO229)
with this construct rescued aerobic growth and converted the
quinone synthesized in E. coli cells from Q8 to Q9 (Fig. 5B and
Table 5) (39). Coexpression of PDSS1 and PDSS2-LongV117M (from plasmid pM12Lkd) failed to rescue the E. coli
ispB mutant (data not shown). However, transformation of

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COENZYME Q10 SUPPLEMENTATION RESCUES RENAL DISEASE

Table 1. Content of Q9 and Q10 in kidney extracts of

B6.Pdss2kd/kd and B6 mice
Lipid Extract

10-day B6.kd/kd
10-day B6.kd/kd
10-day B6.kd/kd
10-day B6.kd/kd
37-day B6.kd/kd
123-day B6.kd/kd
151-day B6.kd/kd
180-day B6.kd/kd
180-day B6.kd/kd
180-day B6.kd/kd
20-day B6
75-day B6
111-day B6
115-day B6
149-day B6
160-day
Heterozygote

Average Q9,
pmol/mg Protein

Average Q10,
Pmol/mg Protein

51515
56913
1665.8
57224
29215
34118
37323
38213
26126
4422.9
57224
2,010147
1,950138
2,190194
2,330134

766.5
1058.2
269.0
908.9
293.6
507.5
398.4
532.6
3111
549.1
7621
35126
25420
42946
30222

2,350184

31326

Values are means SD.

wild-type DH5 E. coli with pM12Lkd allowed for the expression of Q9 in addition to Q8. E. coli DH5 harboring either
empty vector (pGEX-KG) or pM12S (directing coexpression
of PDSS1 and PDSS2-Short) failed to generate appreciable
levels of Q9, indicating that the PDSS2-Short form fails to
support prenyl diphosphate synthase activity. The Q9 content
in DH5 E. coli cells harboring pM12Lkd (34.8 7.6 pmol
Q9/mg wet weight cell pellet) was significantly lower than in
cells harboring pM12L (93.7 6.0 pmol Q9/mg wet weight
cell pellet). These results indicate that heterologous expression
of the kd mutant form of PDSS2 in E. coli recapitulates the
deficiency observed in the B6.Pdss2kd/kd mice. Capture of
GST-tagged PDSS1 coprecipitated PDSS2-Long and PDSS2Long-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 PDSS2Short were examined in liver mitochondria isolated from B6
wild-type, B6.Pdss2kd/kd, and B6.Alb/cre,Pdss2loxP/loxP mice.
Both PDSS2-Long and PDSS2-Short polypeptides were
readily detected in liver mitochondria isolated from B6 wildtype mice (Fig. 7). Steady-state levels of both polypeptides
were lower in the liver mitochondria of B6.Pdss2kd/kd mice,
suggesting that the lower Q9 and Q10 content in liver of the
B6.Pdss2kd/kd mice may result from a combination of impaired
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
20-day B6
30-day B6
30-day B6
40-day B6
40-day B6
50-day B6
50-day B6
60-day B6
60-day B6

21374
23114
45050
64160
1,08859
1,09560
1,437368
1,29885
1,855154
1,492210

222.0
252.0
344.0
547.0
12010
11510
15038
10726
16113
13018

Values are means SD at indicated ages.

Fig. 3. Differences in liver Q9 content in B6.Pdss2kd/kd mice and B6 wild-type

mice 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 liver
homogenates prepared from each mouse. Error bars indicate the standard
deviation in 3 separate measurements of the same lipid extract.

polyprenyl diphosphate activity and lower steady-state levels

of the PDSS2 polypeptide. Only small amounts of the PDSS2Long polypeptide were detectable in liver mitochondria isolated from B6.Alb/cre,Pdss2loxP/loxP mice, where the Pdss2
gene is knocked out in hepatocytes (33). A drastic decrease in
liver Q9 content was evident in these mice; the content of Q9
measured in two different animals (95 days old) using the
RP-HPLC-MS/MS assay (see MATERIALS AND METHODS) was
8.9 2.4 and 33.5 6.7 pmol Q9/mg liver protein, whereas
normal Q9 content in age matched controls was 10 times
higher.
Rescue of renal disease by Q10 supplementation. In a pilot
experiment, three male and two female B6.Pdss2kd/kd mice
were supplemented with 0.25 mg Q10/ml drinking water and
compared with their untreated littermates. When the mice were
tested at 132 days of age, the mutants had less urine albumin
than their untreated littermates (7.2 5.8 vs. 13.5 8.3 mg/24
h, respectively). However, this difference was not significant as
indicated by Students t-test (P 0.288). Therefore, beginning
at weaning, the offspring produced by the B6.Pdss2kd/kd-supTable 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
37-day B6.kd/kd
123-day B6.kd/kd
151-day B6.kd/kd
180-day B6.kd/kd
180-day B6.kd/kd
180-day B6.kd/kd
20-day B6
75-day B6
111-day B6
115-day B6
149-day B6
201-day B6
201-day B6
201-day B6

47644
8861
3325.5
33714
24417
25922
42829
42217
44416
29325
446162
80682
1,18054
1,02049
1,17072

Values are means SD at indicated ages.

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Fig. 4. Expression of a truncated form of Pdss2 in B6.Pdss2kd/kd mice restores

content 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 by
asterisks) were killed at the ages shown, and Q9 and Q10 levels were measured
in kidney homogenates taken from each mouse. Error bars indicate the
standard deviation in 3 separate measurements of the same lipid extract.

plemented mice received double the dose (0.50 mg Q10/ml

drinking water), equivalent to 200 mg Q10 kg body
wt1 day1 (see MATERIALS AND METHODS). After mice reached
at least 130 days of age, urine albumin was determined.
Supplemented mice had on average a fivefold reduction in the
urine albumin content compared with unsupplemented, agematched B6.Pdss2kd/kd mice, and their nephritis scores were
significantly lower (P 0.004, Students t-test) than those of
untreated B6.Pdss2kd/kd controls (Table 6). However, the content 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/mg
kidney protein) was not significantly different from the content
in the untreated B6.Pdss2kd/kd controls (n 3; 353.0 40.5
pmol Q9/mg kidney protein and 45.2 6.0 pmol Q10/mg
kidney protein). Doubling the dose to 400 mg kg body
Table 4. Content of Q9 and Q10 in kidney extracts of
B6.Pdss2kd/kd, B6.Pdss2kd/kd-line G, and B6 mice
Lipid Extract

Average Q9,
pmol/mg Protein

Average Q10,
pmol/mg Protein

123-day B6.kd/kd
151-day B6.kd/kd
180-day B6.kd/kd
180-day B6.kd/kd
70-day B6.kd/kd Tg
94-day B6.kd/kd Tg
94-day B6.kd/kd Tg
94-day B6.kd/kd Tg
157-day B6.kd/kd Tg
157-day B6.kd/kd Tg
157-day B6.kd/kd Tg
157-day B6.kd/kd Tg
75-day B6
111-day B6
149-day B6
149-day B6
143-day B6 Tg

26741
23721
20428
29924
1,53048
2,870107
3,060271
37222
54449
1,740106
1,750108
2,71095
2,25024
2,13036
2,730147
1,960154
2,490139

ND
ND
ND
ND
13511
2768.5
3364.6
239.1
242.7
12728
13930
2618.6
2178.2
1599.1
18912
13118
21911

Urine
Albumin, mg

0.17
0.16
0.14
4.20
7.40
0.26
0.60
0.15

0.12

Values are means SD at indicated ages. Tg, Pdss2 line G transgenic mice
(partial Pdss2 transgene contains exons 15). Albumin content in urine was
measured in each of the mice harboring the partial Pdss2 transgene. ND, not
detected.
AJP-Renal Physiol VOL

Fig. 5. Heterologous expression of the mutant kd form of Pdss2 in Escherichia

coli 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 Pdss1
inserted in frame after the carboxyl terminus of GST (GST-PDSS1) and
Pdss2-S (Short form) inserted in frame after a six-Histidine tag (His6-PDSS2S). pM12L expresses GST-PDSS1 and His6-PDSS2-L (Long form). pM12Lkd
expresses GST-PDSS1 and His6-PDSS2-L-V117M (Long form harboring the
kd mutation). B, BamHI; E, EcoRI; S, SalI, and Xb, XbaI. An asterisk shows
the kd/kd mutation position V117M, and rbs indicates a ribosomal binding
site from pET-28c to allow polycistronic expression of the mouse polypeptides. B: Q8, normally produced in E. coli, and Q9, generated by the heterologous expression of PDSS1 and PDSS2, were measured in lipid extracts
prepared from E. coli DH5 harboring each of the constructs designated in A.
E. coli strain KO229 harboring a deletion of the ispB prenyl diphosphate
synthase gene was rescued by pM12L. Error bars indicate the standard
deviation in 3 separate measurements of the same lipid extract.

wt1 day1 did not improve the effectiveness of the treatment (Table 6). Examples of H&E-stained sections from the
kidneys of Q10-treated and untreated Pdss2kd/kd mice are
shown in Fig. 8, A and B, respectively.
DISCUSSION

Mice harboring the homozygous kd/kd mutation in Pdss2 show

normal growth and fertility but develop kidney disease as young
adults. Severe tubulointerstitial nephritis and tubular dilation are
accompanied by morphological disorders of mitochondria in kidney and liver (34). The kd/kd mutation (V117M) occurs within the
conserved domain I of PDSS2 (Fig. 1A). A decreased Q content
was observed in adult B6.Pdss2kd/kd mice (33). In this study we

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COENZYME Q10 SUPPLEMENTATION RESCUES RENAL DISEASE

Table 5. Content of Q8 and Q9 in extracts of Eschericia coli

expressing Pdss1 and isoforms of Pdss2
Lipid Extract

Average Q8, pmol/mg

Wet Weight

Average Q9, pmol/mg

Wet Weight

Empty vector
Pdss1 Pdss2-Short
Pdss1 Pdss2 V117M
Pdss1 Pdss2-Long

22621
14361
11515
750.4

2.80.5
2.21.1
357.6
946.0

(kDa)

Values are means SD.

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 content
in kidney lipid extracts of B6 wild-type mice 40 days of age or
older are significantly higher compared with that in
B6.Pdss2kd/kd mice. The normal increase at 30 40 days of age
may reflect unidentified physiological changes that occur at
weaning, which normally occurs soon after day 20. The failure
to increase the content of Q in kidneys of the B6.Pdss2kd/kd
mutant mice at 40 days of age precedes by at least several
weeks the renal disease that develops at about 8 10 wk of age.
In contrast, Q content in liver from B6.Pdss2kd/kd mice remains
similar to wild-type mice until later in adulthood (150 days
of age). This profound deficiency in Q content in the kidney
early in life is likely responsible for the renal disease phenotype.

Fig. 6. Coprecipitation of PDSS1 and PDSS2 polypeptides. His-PDSS2-L or

His-PDSS2-L-V117M polypeptides were incubated with GST-PDSS1 for 30
min 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 a
negative control at the same time (lane 1). Antibodies to GST (A) and the 6-His
tag (B) were utilized to recognize GST-PDSS1 and His-PDSS2 polypeptides,
respectively. The asterisks indicate nonspecific bands recognized by the GST
antibody. GST*(truncated), observed in lanes 2, 3, 5, and 6 in A, bottom, is
likely 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 coprecipitation
assay and correspond to lanes 1, 2, and 3, respectively. Similar amounts of
GST-PDSS1 were coprecipitated with both His-PDSS2-L and His-PDSS2-Lkd
fusion proteins (A, lanes 2 and 3, top). In contrast, GST did not coprecipitate
with the His-PDSS1-L fusion protein (A, lane 1, bottom).
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(kDa)

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

Mice express two polypeptide isoforms of PDSS2 due to

alternative splicing, PDSS2-Long (exons 1 8) and PDSS2Short (exons 13b) (Fig. 1B) (34). Previous work has shown
that PDSS2 forms a heterotetramer with PDSS1 and that both
PDSS1 and PDSS2-Long are required for polyprenyl diphosphate synthesis (39). In this study we have shown that coexpression of the short isoform of PDSS2 with PDSS1 in E. coli
fails to reconstitute synthesis of Q9. We hypothesize that the
PDSS2-Short form may repress nonaprenyl diphosphate synthase activity by forming an unproductive complex with
PDSS1. E. coli harboring a construct coexpressing PDSS1 and
PDSS2-Long-V117M (the kd mutant form) had significantly
lower Q9 content than did cells expressing PDSS1 and PDSS2Long (wild type). These results recapitulate the low Q content
observed in the B6.Pdss2kd/kd mice and suggest that the
V117M mutation in Pdss2 occurs in a conserved domain of
polyprenyl diphosphate synthase and partially impairs synthesis of nonaprenyl and decaprenyl diphosphate. The mutation
may affect enzymatic activity and/or heterotetramer formation.
Table 6. Effect of adding Q10 to the drinking water of
mutant mice
Mice

Dose of Q10,
mg kg1 day

Age, Days

B6
Normal water 17 17044
B6.Pdss2kd/kd Normal water 23 1274.7
B6.Pdss2kd/kd
200
19 14420.2
B6.Pdss2kd/kd
400
8 138.518.5
Values are means SD.

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Urine
Albumin, mg/24 h

Nephritis

0.240.56
18.2313.86
4.744.69
6.155.83

0.350.56
2.650.90
1.611.01
1.751.39

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COENZYME Q10 SUPPLEMENTATION RESCUES RENAL DISEASE

Fig. 8. Hematoxylin- and eosin-stained sections of kidneys from Q10-treated

and untreated B6.Pdss2kd/kd mice. A: no histological evidence of disease in a
162-day-old female treated with Q10. The amount of urine albumin measured
was 3.7 mg/24 h. B: severe interstitial nephritis in an untreated 128-day-old
female. The amount of urine albumin measured was 57.34 mg/24 h. Note the
severe interstitial inflammation, tubular dilatation, and glomerular crescents.
Magnification, 100.

However, capture of PDSS1 coprecipitates the PDSS2-LongV117M polypeptide, suggesting that the V117M mutation does
not prevent interaction of the two subunits.
Given that the short form of PDSS2 (exons 13b) failed to
restore Q biosynthesis, it is curious that the BAC clone 256E1,
which contains only a segment of the Pdss2 gene, established
a rescued line of B6.Pdss2kd/kd mice (line G) (34). However,
this clone includes exons 15 and in most cases rescued both
kidney disease and Q levels. Remarkably, rescue of kidney
disease and Q content in line G mice was sporadic. Intriguingly, a low Q content in transgene-positive mice was found to
correlate exactly with elevated levels of protein in urine.
The data suggest that the renal disease phenotypes in
Pdss2kd/kd homozygous mice are caused by a decreased content
of Q in the kidney. The renal disease could result from either
AJP-Renal Physiol VOL

inadequate respiratory function due to low levels of Q or

increased oxidative stress because QH2 is a potent antioxidant.
The partial rescue by dietary supplementation with Q10 is
consistent with both ideas. Several lines of evidence suggest
that Q/QH2 is serving a crucial antioxidant role. Conditional
knockout mice with deletions of Pdss2 in the glomerular
podocytes (B6.Podocin/cre,Pdss2loxP/loxP) fully recapitulate
the kd/kd phenotype (33). However, kidney disease does not
develop in the B6.PEPCK/cre,Pdss2loxP/loxP mice, in which
Pdss2 is deleted in renal tubular epithelial cells. Podocytes are
not thought to have an energetic requirement as high as that of
renal tubular epithelium, supporting the hypothesis that susceptibility to oxidative damage is the critical factor in the
Pdss2kd/kd model of kidney disease. The kidney content of Q10
was not enhanced by supplementation with Q10, a finding
consistent with other similar Q10 supplementation studies (21).
However, longer periods of supplementation (e.g., 17 mo) do
elevate both Q9 and Q10 kidney content (43). It is well established that short-term supplementation does impact levels of
Q10 in the liver and blood plasma (24). Hence, it seems likely
that the benefits of Q10 supplementation may be exerted on the
podocytes via their filtration of blood plasma components (46).
Although in this study we have not evaluated oxidative stress
or antioxidant status, other evidence that the Pdss2kd/kd mice
may be subject to oxidative stress is the finding that their
susceptibility to renal disease is markedly diminished by either
dietary restriction or a germ-free environment (13). Dietary
restriction is associated with decreased oxidative stress (48)
and has been shown to decrease lipid peroxidation products
and apoptosis in aged rat kidneys (22). Similarly, mice reared
in a germ-free environment recapitulate many aspects of dietrestricted animals and show enhanced AMP-activated protein
kinase activity (1), a hallmark of the response to dietary
restriction in yeast and worms (5).
It is possible that renal disease in Pdss2kd/kd mice results
from a defect in an isoprenylated product other than Q. The
PDSS2 polypeptide produces nona- and decaprenyl diphosphate, and studies of mevalonate labeling of mice and rats
suggest these long-chain prenyl groups may be utilized in
kidney protein prenylation (6, 31), similar to the better characterized protein posttranslational modification with farnesyl
or geranylgeranyl groups (20). However, the fact that disease
susceptibility is ameliorated by Q10 supplementation suggests
that Q itself is the most essential substrate with regard to this
phenotype. Patients with mutations in the COQ2 gene show
renal disease with collapsing glomerulopathy (2, 9), further
indicating the causative role is a deficiency in Q itself.
Several human Q10-deficient patients have responded dramatically to Q10 supplementation (36 38, 41). If their response
is primarily based on the antioxidant function of Q10 rather
than its role in mitochondrial respiration, then antioxidants that
are more readily taken up by the target tissues could be more
effective therapies than Q supplementation. Experiments are in
progress to determine whether other antioxidants may be more
effective 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

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COENZYME Q10 SUPPLEMENTATION RESCUES RENAL DISEASE

National Research Service Award issued from NIH Training Grant
GM007185. The LC-MS/MS determination of Q content was supported by
National Center for Research Resources Grant S10 RR024605. Histological
preparations were done in the Morphology Core for Molecular Studies in
Digestive and Liver Diseases at the University of Pennsylvania, which is
supported by NIH Grant P30 DK50306.

22.

23.

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