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



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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 NutritionTM 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.



View larger version (9K):
[in this window]
[in a new window]
 
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.

 
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.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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).


View this table:
[in this window]
[in a new window]
 
Table 1. Body weights (grams)

 


View larger version (51K):
[in this window]
[in a new window]
 
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.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Albumin excretion rate and glomerular filtration rate

 

View this table:
[in this window]
[in a new window]
 
Table 3. Remnant kidney volume and weight

 


View larger version (12K):
[in this window]
[in a new window]
 
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.

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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 [2124]. 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.



   Acknowledgments
 
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.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Klag MJ, Whelton PK, Randall BL. Blood pressure and end-stage renal disease in men. N Engl J Med 1996; 3341: 13–18[CrossRef]
  2. Guyton AC, Coleman TG, Cowley AV Jr, Scheel KW, Manning RD Jr, Norman RA Jr. Arterial pressure regulation. Overriding dominance of the kidneys in long-term regulation and in hypertension. Am J Med 1972; 525: 584–594
  3. Bianchi G, Niutta E, Ferrari P et al. A possible primary role for the kidney in essential hypertension. Am J Hypertens 1989; 22: 2S–6S
  4. Brenner BM, Garcia DL, Anderson S. Glomeruli and blood pressure. Less of one, more the other? Am J Hypertens 1988; 14: 335–347
  5. Keller G, Zimmer G, Mall G, Ritz E, Amann K. Nephron number in patients with primary hypertension. N Engl J Med 2003; 3482: 101–108[CrossRef]
  6. Koletsky S, Goodsit AM. Natural history and pathogenesis of renal ablation hypertension. Arch Pathol 1960; 69: 654–662[ISI][Medline]
  7. Novick AC, Gephardt G, Guz B, Steinmuller D, Tubbs RR. Long-term follow-up after partial removal of a solitary kidney. N Engl J Med 1991; 32515: 1058–1062
  8. Mackenzie HS, Tullius SG, Heemann UW et al. Nephron supply is a major determinant of long-term renal allograft outcome in rats. J Clin Invest 1994; 945: 2148–2152
  9. Mitchell TS, Lee S, Gittes RF. Hypotrophy of hypertrophied kidneys in response to renal transplantation in rats. Invest Urol 1974; 123: 213–217
  10. Mackenzie HS, Brenner BM. Antigen-independent determinants of late renal allograft outcome: the role of renal mass. Curr Opin Nephrol Hypertens 1996; 54: 289–296
  11. Ots M, Mackenzie HS, Troy JL, Rennke HG, Brenner BM. Effects of combination therapy with enalapril and losartan on the rate of progression of renal injury in rats with 5/6 renal mass ablation. J Am Soc Nephrol 1998; 92: 224–230
  12. Mackenzie HS, Brenner BM. Current strategies for retarding progression of renal disease. Am J Kidney Dis 1998; 311: 161–170
  13. Chertow GM, Brenner BM, Mackenzie HS, Milford EL. Non-immunologic predictors of chronic renal allograft failure: data from the United Network of Organ Sharing. Kidney Int Suppl 1995; 52: S48–S51[Medline]
  14. Sanchez-Fructuoso AI, Prats D, Marques M et al. Does renal mass exert an independent effect on the determinants of antigen-dependent injury? Transplantation 2001; 713: 381–386[CrossRef]
  15. Nyengaard JR, Bendtsen TF. Glomerular number and size in relation to age, kidney weight, and body surface in normal man. Anat Rec 1992; 2322: 194–201
  16. Hinchliffe SA, Sargent PH, Howard CV, Chan YF, van Velzen D. Human intrauterine renal growth expressed in absolute number of glomeruli assessed by the disector method and Cavalieri principle. Lab Invest 1991; 646: 777–784
  17. Merlet-Benichou C, Gilbert T, Muffat-Joly M, Lelievre-Pegorier M, Leroy B. Intrauterine growth retardation leads to a permanent nephron deficit in the rat. Pediatr Nephrol 1994; 82: 175–180
  18. Langley SC, Jackson AA. Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets. Clin Sci 1994; 862: 217–222
  19. Barker DJ, Osmond C, Golding J, Kuh D, Wadsworth ME. Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease. Br Med J 1989; 2986673: 564–567
  20. Hayman JM, Martin JM, Miller M. Renal function and the number of glomeruli in the human kidney. Arch Int Med; 1939; 64: 69–83
  21. Dahl LK, Heine M, Thompson K. Genetic influence of renal homografts on the blood pressure of rats from different strains. Proc Soc Exp Biol Med 1972; 1403: 852–856
  22. Bianchi G, Fox U, Di Francesco GF, Giovanetti AM, Pagetti D. Blood pressure changes produced by kidney cross-transplantation between spontaneously hypertensive rats and normotensive rats. Clin Sci Mol Med 1974; 475: 435–448
  23. Rettig R, Stauss H, Folberth C, Ganten D, Waldherr B, Unger T. Hypertension transmitted by kidneys from stroke-prone spontaneously hypertensive rats. Am J Physiol 1989; 2572: F197–F203
  24. Rettig R, Folberth C, Stauss H, Kopf D, Waldherr R, Unger T. Role of the kidney in primary hypertension: a renal transplantation study in rats. Am J Physiol 1990; 2583: F606–F611
  25. Curtis JJ, Luke RG, Dustan HP et al. Remission of essential hypertension after renal transplantation. N Engl J Med 1983; 30917: 1009–1015
Received for publication: 9. 1.03
Accepted in revised form: 17. 9.03





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Disclaimer
Request Permissions
Google Scholar
Articles by Ots, M.
Articles by Brenner, B. M.
PubMed
PubMed Citation
Articles by Ots, M.
Articles by Brenner, B. M.