Chromosomal Mapping of a Major Quantitative Trait Locus Regulating Compensatory Renal Growth in the Rat
MICHAL PRAVENEC*,,
VACLAV ZIDEK*,
ALENA MUSILOVA*,
VLADIMIR KEN*,,
VLASTA BILA and
ROBERT DI NICOLANTONIO
*Institute of Physiology, Czech Academy of Sciences, Czech Republic,
Victoria, Australia Institute of Biology and Medical Genetics, 1st Medical Faculty, Charles
University, Czech Republic, Victoria, Australia Department of Physiology, University of Melbourne, Parkville, Victoria,
Australia
Correspondence to Dr. Michal Pravenec, Institute of Physiology, Academy of
Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech
Republic. Phone/Fax : +420 2 475 2297 ; E-mail :
pravenec{at}biomed.cas.cz
Abstract. Despite extensive research conducted over the past
century,the mechanisms of compensatory renal growth (CRG) remain a mystery.
Insightinto the mechanisms that regulate CRG might be gained by identifying
geneticfactors that influence this complex phenotype. In a large setof
recombinant inbred strains derived from the spontaneouslyhypertensive rat and
the Brown Norway rat, a genome scan forquantitative trait loci (QTL) that
regulate CRG was performed.The CRG score was expressed as a ratio of the
weight of theremnant right kidney at 8 wk of age to the weight of the left
kidneyat 5 wk of age, both adjusted for body weight. QTL mapping was
performedusing Map Manager QT and the strain distribution patterns ofmore
than 600 genetic markers. It was found that CRG after unilateralnephrectomy
is a multifactorially determined trait with a substantialgenetic component.
The heritability of CRG approached 40%. Genomewide scan analysis revealed
significant evidence of linkageto a region of rat chromosome 4 designated
Crg 1 that accountedfor more than 50% of the additive genetic
variance of CRG inthe recombinant inbred strains. The detection of a major
QTLinfluencing CRG in the rat should provide new opportunitiesfor
identifying mechanisms that regulate this historically enigmaticphenomenon
and may also have implications for research on thepathogenesis of end-stage
kidney disease.
The ability of a solitary kidney to undergo compensatory growthhas been
recognized since the time of Aristotle
(1,2).
However,despite extensive research conducted over the past century,the
mechanisms of compensatory renal growth (CRG) remain a mystery.Given mounting
evidence that nephron mass may be a criticalfactor in the progression of
multiple forms of chronic renalfailure
(3), the search for factors
that regulate CRG has takenon renewed importance. Although kidney weight has
been reportedto be a highly heritable trait, the ability of genetic factors
tomodulate the phenomenon of CRG is unknown
(4). Insight intothe
mechanisms that regulate CRG might be gained by identifyingquantitative trait
loci (QTL) that influence this complex phenotype.It has recently been
proposed that hypertrophy may be a prerequisitefor progression of renal
injury and that attenuation of compensatorygrowth can limit the otherwise
relentless progression of kidneydamage that occurs in various forms of
chronic renal insufficiency(5,
6). Thus, identification of
factors that modulate CRG couldultimately have implications for the treatment
of chronic renalfailure and the prevention of end-stage kidney disease.
Accordingly,in a large set of recombinant inbred strains, we determinedthe
influence of genetic factors on the phenomenon of CRG inthe rat and sought to
map QTL that regulate this complex phenotype.
Animals
Genetic studies were performed in 29 recombinant inbred (RI)strains
derived from spontaneously hypertensive rats (SHR/Ola,referred to as SHR) and
normotensive Brown Norway rats (BN-Lx/Cub,referred to as BN)
(7,
8). The SHR progenitor strain
descendsfrom inbred SHR originally obtained from the National Institutesof
Health. The BN progenitor is a BN congenic strain that carriesa segment of
chromosome 8 from the polydactylous PD/Cub strain
(7).All strains have been
maintained in Prague by inbreeding formore than 15 yr. The RI strains were
derived from (SHR X BN)F2population : The F2 rats were paired off at random,
and eachof these F2 pairs was used to generate a new inbred strain by
repeatedbrother X sister mating of the offspring for at least 20 generations.
Currently,most of the RI strains reached more than 45 generations of
brother-sisterinbreeding. Results of DNA fingerprint and PCR microsatellite
testshave confirmed that the progenitor and RI strains are highlyinbred
(7,
9).
Experimental Procedures
Five-week-old male rats were weighed and anesthetized with ether,and their
left kidneys were surgically removed and weighed.Three weeks later, body
weights were recorded and the rightkidneys were removed and weighed. The CRG
score was expressedas a ratio of the weight of the remnant right kidney at 8
wkof age (KW8) to the weight of the left kidney at 5 wk of age
(KW5).To adjust for increases in kidney weight associated with
normaldevelopment, kidney weights were corrected for body weightsusing an
allometric scaling factor of 0.75 in accordance withthe results of previous
allometric studies of kidney growthin the rat
(10,11,12,13,14).
The CRG score for each rat wascalculated as CRG =
[KW8/(BW8)0.75]/[KW5/(BW5)0.75].
No adjustmentswere made for any small systematic differences in weight that
mayoccur between right and left kidneys. The CRG score of a givenstrain was
determined by averaging the CRG results of multiplerats from that strain
(SHR, n = 9 ; BN, n = 10 ; RI strains,n = 4 to 5
per strain).
Genetic and Statistical Analyses
Heritability of CRG was estimated according to the method ofPlomin and
McClearn using the variances in CRG scores betweenand within the RI and
progenitor strains (15). The
calculationof heritability corrects for the doubling effects of inbreedingon
additive genetic variance and provides an estimate of thenarrow heritability
that would be expected in an F2 populationderived from the SHR and BN
progenitor strains (15). QTL
mappingwas performed using Map Manager QT (version b28)
(16) and thestrain
distribution patterns of more than 600 genetic markerspreviously mapped in
the RI strains (7). The marker
data setcovers approximately 1200 centiMorgans of the rat genome andhas
proved effective in genome scanning for QTL regulating avariety of complex
traits including BP, insulin resistance,and lipid levels
(8,
17,
18). Map Manager QT was used
to testfor single locus associations by regression analysis and the
significanceof each potential association measured using the likelihoodratio
statistics (LRS) of Haley and Knott
(19). The intervalregression
method of Map Manager QT was used to test for QTLwithin marker intervals. The
significance threshold for thegenome wide scan was empirically determined by
the Map ManagerQT permutation test, using the informative markers and 1000
permuteddata sets as recommended by Doerge and Churchill
(20). Significantlinkage was
defined in accordance with the guidelines of Landerand Kruglyak as
statistical evidence occurring by chance inthe genome scan with a probability
of 5% or less (21). Basedon
these criteria and the results of the permutation test, anLRS = 15.4
(corresponding to a lod score of 3.3) was establishedas the threshold for
significant linkage in the RI strain dataset. This empirically determined
threshold is nearly identicalto the significance threshold recently
recommended by Belknapand colleagues for genome wide scans in RI strains
(22). One-halfof the fraction
of variance attributable to each QTL in theRI strains was used to estimate
the QTL effect expected in acomparable F2 population derived from the SHR and
BN progenitors(to correct for the doubling effect of inbreeding on additive
geneticvariance) (15,
23). The fraction of genetic
variance contributedby each QTL was determined by dividing the estimated QTL
effectby the heritability.
Initial studies in the progenitor strains revealed a significantdifference
in the compensatory growth of SHR and BN kidneysfollowing unilateral
nephrectomy ; the mean CRG score of theSHR progenitor strain, 1.42 ±
0.05, was significantlygreater than that of the BN progenitor strain, 1.28
±.03(P = 0.025 by t test).
In the RI strains derived from the SHR and BN progenitors, thedistribution
of CRG scores was continuous, suggesting a polygenicmode of inheritance for
the trait (Figure 1). The BN
progenitorstrain exhibited the lowest CRG score, whereas the CRG scoreof the
SHR progenitor fell midway within the distribution ofCRG scores of the RI
strains (Figure 1). The
observation ofgreater CRG scores in certain RI strains than in either
progenitoris consistent with multifactorial inheritance and suggests the
possibilityof gene-gene interactions. The SHR progenitor was derived by
recurrentselective breeding for hypertension ; however, no correlationwas
observed between BP and CRG in the RI strains.
Figure 1. Distribution of means (± SEM). Compensatory renal growth (CRG)
scores in the spontaneously hypertensive (SHR), Brown Norway (BN), and
recombinant inbred (RI) strains. , RI strains that inherited the BN
allele ; , RI strains that inherited the SHR allele of the
D4Cebr7s17 marker, which was significantly associated with CRG.
Based on the variances in CRG scores within and between theRI strains, the
heritability of CRG was estimated to be 38%.Because of the absence of
heterozygotes in the RI strains, thisestimate of heritability reflects
additive genetic effects onCRG phenotype. Given finding of a substantial
genetic componentto CRG in the SHR-BN model, we scanned for QTL influencing
CRGin the RI strains. Genome scanning of the RI strains revealedsignificant
linkage of the CRG phenotype to the D4Cebr7s17 markeron chromosome 4
(LRS = 15.9, lod score = 3.5, P = 6.7 x 10-5)
(Table 1); the
D4Cebr7s17 marker is closely linked to the Cacnalc(D4Arb4)
(24) gene coding for Ca
channel, voltage-dependent,L type, alpha 1c subunit. Interval mapping across
chromosome4 revealed a distinct peak in the LRS at D4Cebr7s17 and a
sharpdrop off in the LRS at the adjacent flanking markers
(Figure 2).The narrow QTL peak
reflects the enhanced mapping resolutionafforded by the fourfold increase in
recombination events providedby RI strains
(25). This QTL region,
designated Crgl, accountsfor 54% of the additive genetic variance of
the CRG phenotypeand suggests the existence of a major gene regulating
compensatoryrenal growth in the rat. The mean CRG score of the RI strains
thatinherited the BN allele for the D4Cebr7s17 marker was greater
thanthe mean CRG score of the RI strains that inherited the SHRallele
(Table 1). Because neither the
SHR nor BN strain wasselectively bred for the CRG phenotype, it is
conceivable thatvariants promoting compensatory renal growth are likely to
coexistwith those that limit compensatory growth in both progenitorstrains.
No other chromosome region was identified that showedsignificant evidence of
linkage to the CRG phenotype. However,given the known power limitations of RI
strains for detectingQTL with minor effect sizes, it is possible that QTL
exertingmore modest effects on CRG are located in other regions of thegenome
(25). In this regard, it
should be noted that suggestiveevidence for linkage was obtained for the
D6Mit9 marker on chromosome6 and the D7Mit8 marker on
chromosome 7 (Table 1).
Figure 2. Interval mapping of Crgl. Likelihood ratio statistics from the Map
Manager QT linkage analysis are plotted across chromosome 4. Estimated
distances between markers are in centiMorgans determined with the Haldane map
function. The horizontal lines indicate the threshold for significance of the
likelihood ratio statistic determined by the Map Manager QT permutation test.
To convert likelihood ratio statistics to lod scores, divide by 4.6.
CRG has been observed since ancient times and has received serious
scientificinvestigation for over a century, but little is known aboutits
molecular or cellular basis. Although unilateral nephrectomyin newborn rats
can promote renal cell hyperplasia, the phenomenonof compensatory renal
growth in older animals predominantlyinvolves glomerular and tubular
hypertrophy (1,
26). A hostof growth factors
and other biochemical mediators have beenimplicated in the phenomenon of
renal cell hypertrophy, buttheir relative roles in CRG are unclear
(1,
2,
26). This ispartly related to
the difficulty in distinguishing between keymechanistic steps in CRG and the
plethora of secondary changesthat result from the complex physiologic
adjustments occurringafter unilateral nephrectomy. The current findings
demonstratethat in the SHR-BN model, the heritability of CRG may be
sufficientto enable the use of genetic dissection techniques to searchfor
key steps in the process and avoid pursuing phenomena thatare simply
secondary to biochemical or physiologic perturbationsthat might accompany
CRG.
We found that CRG after unilateral nephrectomy is a multifactorialtrait
with a substantial genetic component. The heritabilityof CRG approached 40%.
Genome wide scan analysis revealed significantevidence of linkage to a region
of rat chromosome 4 designatedCrgl that accounted for more than 50%
of the additive geneticvariance of CRG in the RI strains. Recently, we have
mappedthe Cacnalc (D4Arb4) gene coding for the Ca channel,
voltage-dependent,L type, alpha 1c subunit
(24) close to the peak of the
QTL linkage.The gene product is expressed in kidney proximal tubule
epithelialcells (27).
Furthermore, intracellular calcium has been shownto play an important role in
cellular growth and division
(28).In addition, it has been
reported that administration of calciumchannel blockers significantly
attenuates the degree of compensatoryrenal growth seen in mice
(29) and in rats with
experimentaldiabetes (30).
Taken together, these observations lead us tosuggest the Cacnalc as
a positional candidate gene in the CRGprocess. However, it should be
emphasized that this suggestionis largely speculative and that additional
functional studieswould be necessary to establish the role of
Cacnalc in CRG.
Based on conserved linkages between rat chromosome 4 and mousechromosome
6, other genes can also be regarded as positionalcandidates for the CRG
phenotype. For example, the gene forkidney androgen protein (Kap)
maps to this region of mouse chromosome6, and it is well known that androgens
are potent stimulatorsof renal growth in the intact rat
(31). The gene for growthand
differentiation factor 3 (Gdf3), a member of the transforminggrowth
factor-ß superfamily, also maps to the region ofmouse chromosome 6
homologous to Crgl in the rat. Gdf3 is ofparticular
interest because of recent studies showing that anothermember of the
transforming growth factor-ß superfamily,myostatin (formerly known as
Gdf8), functions as a negativeregulator of skeletal muscle growth
(32). Disruption of the
myostatingene causes skeletal muscle hypertrophy, suggesting that itnormally
inhibits the expansion of muscle mass
(32). It is conceivablethat
the kidney also produces a substance that restrains itsown growth and that
after unilateral nephrectomy, decreasedcirculating levels of such an
inhibitor allow for hypertrophyof the remaining kidney.
It has recently been proposed that hypertrophy may be a prerequisitefor
progression of renal injury and that attenuation of compensatorygrowth can
limit the otherwise relentless progression of kidneydamage that occurs in
various forms of chronic renal insufficiency
(6).Thus, identification of
mechanisms that limit the extent ofrenal growth could lead to new
opportunities for moderatingthe decline in renal function that typically
occurs in patientswith renal insufficiency. Accordingly, the genetic
dissectionof factors that regulate CRG might ultimately have implicationsfor
the treatment of chronic renal failure and the preventionof end-stage kidney
disease.
Acknowledgments
This work was supported in part by grants from the Ministryof Health of
the Czech Republic (No. 4812-3) and from the US-CZScience and Technology
Program (96005). Dr. Pravenec is an InternationalResearch Scholar of the
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Received for publication July 27, 1999.
Accepted for publication November 1, 1999.
EN*,
Top
Abstract
Introduction
en V,
Bílá V,
Szpirer C, Szpirer J, Wang J-M, Huang H, St. Lezin E, Spence MA, Floodman P,
Printz M, Lathrop M, Vergnaud G, Kurtz TW : Mapping of quantitative trait loci
for blood pressure and cardiac mass in the rat by genome scanning of
recombinant inbred strains. J Clin Invest9
6 : 1973-1978,1995
J, Scicli AG, Carretero OA,
Simonet L, Kurtz TW : Cosegregation of blood pressure with a kallikrein gene
family polymorphism. Hypertension17
: 242-246,1991
ek J,
Zídek V,
Musilová A,
Zdobinská M, Wang J-M, van Zutphen LFM, Kurtz
TW, Pravenec M : Quantitative trait loci influencing cholesterol and
phospholipid phenotypes map to chromosomes that contain genes regulating blood
pressure in the spontaneously hypertensive rat. J Clin
Invest 98 :856
-862, 1996[Medline]
, RI strains that inherited the BN
allele ; 
, RI strains that inherited the SHR allele of the
D4Cebr7s17 marker, which was significantly associated with CRG.