Nephrol Dial Transplant (2004) 19: 337-341
© ERA–EDTA 2004; all rights reserved
Original Article
Impact of the supplementation of kidney mass on blood pressure and progression of kidney disease
Mai Ots,
Julia L. Troy,
Helmut G. Rennke,
Harald S. Mackenzie and
Barry M. Brenner
Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, USA
Correspondence and offprint requests to: Mai Ots, Renal Division, Department of Internal Medicine, University of Tartu, 6 Puusepa Strasse, 51014, Tartu, Estonia. Email: mai.ots{at}kliinikum.ee
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Abstract
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Background. To test the hypothesis that nephron mass is an independent
determinant of arterial pressure, the effects of augmenting
renal mass by isograft transplantation were studied in the model
of secondary hypertension.
Methods. The effects of isograft transplantation or sham operation on blood pressure, proteinuria, remnant kidney mass, glomerular filtration rate and glomerulosclerosis were assessed in 5/6 nephrectomized (5/6 NPX) rats.
Results. Systolic blood pressure was lowered on average by 
35 mmHg and glomerular hyperfiltration was attenuated in the remnant kidneys of transplant recipients. Markedly lower urinary protein excretion rates and glomerulosclerosis scores in the remnant kidney accompanied these supplemental transplants to values roughly one-third of those from sham-operated rats.
Conclusions. The data show that reduced renal mass per se is the major factor in the development and maintenance of arterial hypertension and glomerular injury in 5/6 NPX rats and these changes can be reversed by supplementing renal mass. The data provide strong support for the notion that renal mass is a significant, independent determinant of arterial pressure.
Keywords: blood pressure; glomerulosclerosis; nephron number; renal mass; transplantation
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Introduction
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A close relationship between the kidney and arterial hypertension
has long been appreciated: severe hypertension inevitably injures
the kidney; equally, hypertension almost invariably accompanies
and aggravates chronic renal disease [
1]. Aside from such extremes,
however, the role of the kidney in blood pressure regulation
and the pathogenesis of all but a few ‘secondary’
forms of hypertension have remained ill-defined. Guyton and
others regarded the kidney as the ultimate determinant of blood
pressure through its regulation of extracellular fluid volume
[
2]. This implied that a renal defect must exist for sustained
hypertension to become established. The nature of the renal
‘defect’ in hypertension has usually been envisaged
as a qualitative difference, most often of tubule sodium reabsorption
[
3]. In 1988, in a departure from the conventional view, Brenner
et al. put forward an alternative hypothesis implicating deficient
quantity of kidney (i.e. nephron number) as the renal defect
[
4]. Although a substantial body of evidence may be cited in
support of this hypothesis [
5], rigorous experimental confirmation
has been difficult to obtain. To investigate directly the hypothesis
that kidney mass is an independent determinant of blood pressure,
we used transplantation techniques to assess the effects of
augmenting renal mass on blood pressure in the rat 5/6 nephrectomy
model of secondary hypertension [
6,
7]. Nephron mass was augmented
by transplanting an isogeneic kidney, thus conferring a substantial
increment in nephron number on the 5/6 nephrectomy recipient.
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Subjects and methods
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Experimental animals were kept in a climate-controlled vivarium
where animals housed under standard conditions on a 12-h light/dark
cycle, fed with rodent chow (Purina 5001, Laboratory Rodent
Diet is a Constant Nutrition
TM formulation recommended for rats,
PMI Feeds. St Louis, MO, sodium content 0.4%) and allowed free
access to water. Munich Wistar rats (
n = 27) underwent left
nephrectomy and selective ligation of branches of the right
renal artery such that only one-sixth of the total renal mass
remained viable. All surgical procedures were performed under
methohexital anaesthesia (50 mg/kg, i.p.) using aseptic precautions.
After the surgical procedures, rats were housed under standard
conditions with weekly determinations of systolic blood pressure
(SBP, mmHg) by the tail cuff method and 24 h protein excretion
rates (UprotV, g) from urine collections obtained from rats
individually housed in metabolic cages. Urine protein concentration
was measured by colorimeter after precipitation with 3% sulfosalicylic
acid. At 18 days after 5/6 nephrectomy designated week 0, rats
were divided into two groups matched for SBP and UprotV. Rats
then underwent renal isograft supplementation (I-Supp,
n = 14)
by orthotopic transplantation of the left kidney of genetically
similar Munich-Wistar donor rats (
Figure 1). Renal vessels and
ureter were attached by end-to-end anastomoses to the recipient
renal vessels and ureter, respectively, using 10-0 prolene.
Sham operated 5/6 nephrectomized rats (Sham-Op,
n = 13) underwent
mobilization of the left renal pedicle only and served as controls.
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Fig. 1. Experimental protocol of the present study. (A) Week –2: 5/6 nephrectomy; (B) week 0: remnant kidney hypertrophy; renal isograft supplementation; (C) week 8: decrease of remnant kidney hypertrophy, isograft nephrectomy; (D) week 10: remnant kidney hypertrophy.
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At week 8, glomerular filtration rate (GFR) (ml/min) was assessed
and all I-Supp rats then underwent transplant nephrectomy (
Figure 1).
GFR determinations were performed by inulin clearance [
8].
Briefly, rats were placed on a heated table to maintain body
temperature at 37°C, and tracheotomized. The left femoral
artery was catheterized for direct blood pressure measurement
and intermittent blood sampling. The right femoral vein was
catheterized for constant infusion of inulin (7.5%) in isotonic
saline at 20 µl/min. The left ureter was catheterized
with PE-10 for urine collection from the isograft, where present;
bladder catheterization allowed collection of urine from the
remnant kidney. After 60 min, two consecutive 15-min clearance
periods were completed. Clearance studies were repeated at week
10 and the study concluded at week 10 in subsets of I-Supp (
n = 6) and Sham-Op (
n = 5); in the remaining rats, the study concluded
after clearance measurements at week 12 (I-Supp,
n = 6; Sham-Op,
n = 6). Albumin excretion was measured with a rat-specific albumin
antibody. GFR measurements at week 12 were incomplete for technical
reasons in two rats from the Sham-Op group and data were omitted
from calculations of GFR and albumin excretion rate means. Two
I-Supp rats were excluded from the final analysis because of
hydronephrosis (ureter proximal to anastomosis >3 mm). Remnant
kidney dimensions were measured at weeks 0, 8, 10 and 12 from
which volume (
Kvol) was estimated using the formula for a prolate
ellipsoid.
Morphological studies
At the conclusion of the study, remnant kidneys were fixed for histopathological evaluation by retrograde aortic perfusion with 1.25% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at the measured arterial pressure of each rat. After perfusion-fixation, coronal sections (3–4 mm thick) through the mid-portion of the remnant kidney were taken, post-fixed in 10% buffered formaldehyde and processed for light microscopy by paraffin embedding. Glomerulosclerosis was scored on periodic acid-Schiff stained sections [8]. The number of glomeruli with segmental lesions was expressed as a percentage of the total number examined (>100 per rat).
Statistical methods
Data are presented as mean ± SEM. Group data were analysed by ANOVA techniques, with post-hoc testing where appropriate; correlations were assessed by stepwise regression using commercially available software packages for Macintosh computers. The null hypothesis was rejected at P < 0.05.
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Results
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Both I-Supp and Sham-Op rats maintained similar growth rates
throughout the study, I-Supp rats averaging 270 ± 5 g
at transplantation, reaching 333 ± 7 g at week 8
vs 273
± 6 rising to 334 ± 4 g for Sham-Op rats. Body
weight was also examined separately in subsets of 10- and 12-week
rats (
Table 1) where no statistically significant difference
was found. Following transplantation, reductions in SBP of 25–50
mmHg
vs Sham-Op were observed in I-Supp rats, persisting after
the supplemented renal mass was removed by transplant nephrectomy
at week 8 (
Figure 2). Although SBP trended upwards at week 10,
at week 12, SBP remained significantly lower in I-Supp rats,
144 ± 4 mmHg
vs Sham-Op, 180 ± 6 mmHg (
P <
0.05). After supplemental transplantation a marked decrease
in UprotV was observed (
Figure 2). After transplant nephrec-tomy,
UprotV increased but remained significantly lower at week 10.
At week 12, UprotV averaged 45 ± 11 mg/day, a value not
significantly different from Sham-Op rats (66 ± 7 mg
/day). At week 8, GFR in the remnant kidney of the Sham-Op group
averaged 1.31 ± 0.1 ml/min, consistent with the presence
of single-nephron hyperfiltration in the remnant. In contrast,
in I-Supp rats, the contribution of the isograft (1.17 ±
0.1 ml/min) lessened the adaptive increase in the remnant kidney
in which GFR averaged 0.74 ± 0.07 ml/min (
P < 0.05
vs Sham-Op). Two weeks after transplant nephrectomy (week 10),
remnant kidney GFR in Sham-Op rats fell significantly to 0.64
± 0.11 ml/min whereas in I-Supp rats it remained stable
at 1.1 ± 0.13 ml/min (
Table 2). Similarly, albumin excretion
rate was significantly higher in Sham-Op rats at week 8 and
after the transplant nephrectomy at week 10 (
Table 3). Kidney
volume data are presented in
Table 3. After transplantation,
a significant decrease of previously hypertrophied remnant kidney
was observed, and after removing the isograft the remnant kidney
hypertrophied once again. The better-preserved remnant kidney
GFR at week 10 in I-Supp rats was associated with significantly
less histologic evidence of glomerular injury. On average, only
one-third as many glomeruli showed evidence of focal segmental
glomerulosclerosis (FSGS) in remnant kidneys of I-Supp rats
vs Sham-Op rats, both at 10 and 12 weeks (
Figure 3).
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Fig. 2. In I-Supp rats (black bars, n = 14) transplantation of a renal isograft at time 0 was followed by a reduction in SBP at week 1 and sustained until transplant nephrectomy at week 8. Subsequently, SBP rose towards the levels of the Sham-Op rats (hatched bars, n = 13), in whom SBP remained at hypertensive levels throughout. Blood pressure reduction after isograft supplementation was associated with a reduction in UprotV, whereas in Sham-Op rats, UprotV showed a tendency to rise progressively after week 1. All data: mean ± SEM. *P < 0.05 vs Sham-Op by ANOVA and Scheffé's test.
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Fig. 3. The percentages of glomeruli showing evidence of FSGS are shown for individual rats in circles. Squares represent group mean ± SEM. *P < 0.05 vs week 10 Sham-Op; *P<0.05 vs week 12 Sham-Op by ANOVA.
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Discussion
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Restoration of renal mass by supplemental transplantation in
rats who had moderate renal insufficiency after 5/6 NPX led
to significant lowering of blood pressure and slowed the progression
of glomerular injury in remnant kidneys. Mitchell and colleagues
[
9] have shown previously that adding an isograft to a 5/6 NPX
rat leads to reversal of both hypertrophy and hyperfiltration
of remnant nephrons. This latter study focused on kidney size
but did not measure effect to blood pressure, GFR or morphology
of remnant tissue. We therefore designed the present study to
answer the following questions: (i) does ‘isograft supplementation’
reverse systemic hypertension and impaired kidney function?
(ii) Can restoration of renal mass to rear-normal levels arrest
the progression of glomerulosclerosis in the remnant kidney?
To answer these questions supplemental isogeneic renal transplantation
was carried out in rats with pre-existing moderate glomerular
injury obtained 14–18 days after 5/6 NPX. The novelty
of this work results from the use of renal mass supplementation
as an experimental treatment modality for modulating the disease
progression. Our results are the first to show that supplementation
of isogeneic kidney mass indeed lowers arterial pressure in
experimentally acquired hypertension. Moreover, the results
of this study confirm that reduced renal mass is a major determinant
of elevated blood pressure and glomerular injury in the 5/6
nephrecomy model. This latter conclusion adds to our previous
findings that augmenting renal mass reduces proteinuria and
glomerulosclerosis in Fisher-Lewis rats [
8,
10], a normotensive
model of late renal allograft failure. Interestingly, similar
results were found by the author in the recent study where instead
of the above described method of the treatment of experimental
chronic renal failure antihypertensive drugs were used [
11].
Although pathogenetic mechanisms underlying the progression
of renal diseases appear to be multifactorial, disease progression
may be explained presently by relatively few mechanisms. Among
these hyperfiltration and glomerular capillary hypertension
have received the most attention. The studies of rats with partial
renal ablation have helped already to clarify these factors
involved in disease progression and much of the results obtained
in these studies appear to be applicable to human [
12]. Supplementation
of functioning extra renal mass greatly prevented the rate of
the disease progression in our study and this evidence support
therefore provides further for the conclusion that deficient
renal mass plays a key role in the pathogenesis of hypertension
and progressive renal injury [
13,
14].
We believe the implications of our observations are far reaching. In the human population nephron number follows a Gaussian distribution with an unusually large variance such that counts of 300 000 to 1 100 000 glomeruli per kidney span the normal range [15]. This extensive variability is attributable partly to genetic factors and partly to programming of nephron number during the later stages of gestation by local environmental factors. Intrauterine growth retardation, for example, may lead to formation of kidneys whose nephron endowment falls 15–40% below normal [16,17]. Moreover, low birth weight, a circumstance often associated with relative deficits in nephron number, presages later-life elevates in blood pressure in animals [18] and humans [19]. Our studies raise the possibility that deficiencies in nephron number, possibly with an attendant defect in sodium excretion, could explain this hypertensive risk. Yet, more than a half-century has elapsed since publication of the only human study demonstrating a close inverse correlation between nephron number and blood pressure [20].
Transplantation techniques have been used previously to explore the role of the kidney in rat models of genetically determined, spontaneous hypertension. Cross-strain transplant studies have shown that the susceptibility to develop hypertension follows the kidney and may be conferred on the normotensive control strain by transplanting a kidney from donor rats bearing the hypertensive trait; conversely, blood pressure may be lowered in hypertensive rats receiving a kidney from a normotensive control strain [21–24]. These findings may have counterparts in human renal transplantation [25]. In our experiment, adding renal mass by transplantation was followed by significant abatement of the severity of arterial hypertension, and, marked reduction of glomerular injury. The data provide strong support for the notion that reduced renal mass may be an important determinant of arterial pressure and, by inference, suggest that deficiencies in renal mass may contribute directly to the development and maintenance of hypertension. Furthermore, they reveal the extent to which nephron deficit is a determinant both of systemic blood pressure and the pace of renal disease progression. These findings represent a significant extension of our understanding of basic mechanisms of long-term blood pressure regulation and the pathogenesis and maintenance of arterial hypertension. In view of the profound implications for enhancing our understanding of human hypertension, nephron mass as an etiologic factor warrants further study.
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Acknowledgments
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These studies were conducted during Dr Ots’ tenure as
an International Society of Nephrology Fellow. Portions of this
study were published in abstract form. Ots M, Troy JL, Brenner
BM, Mackenzie HS. Isograft supplementation (I-Supp) slows the
progression of chronic experimental renal injury [Abstract].
J Am Soc Nephrol 1996; 9: 1861.
Conflict of interest statement. None declared.
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Received for publication: 9. 1.03
Accepted in revised form: 17. 9.03
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Abstract
Introduction