Adverse effects of hyperphosphatemia on myocardial hypertrophy, renal function, and bone in rats with renal failure

Kidney International, Vol. 66 (2004), pp. 2237–2244

Adverse effects of hyperphosphatemia on myocardial hypertrophy, renal function, and bone in rats with renal failure KATIA R. NEVES, FABIANA G. GRACIOLLI, LUCIENE M. DOS REIS, CARLOS A. PASQUALUCCI, ´ , and VANDA JORGETTI ROSA M.A. MOYSES Nephrology and Pathology Division, University of S˜ao Paulo, S˜ao Paulo, SP, Brazil

Adverse effects of hyperphosphatemia on myocardial hypertrophy, renal function, and bone in rats with renal failure. Background. Hyperphosphatemia and disturbances in calcium or parathyroid hormone (PTH) metabolism contribute to the high incidence of cardiovascular disease and renal osteodystrophy in chronic renal failure (CRF). We evaluated the effect of hyperphosphatemia on the cardiovascular system, on renal function, and on bone in experimental uremia. Methods. Wistar rats were submitted to parathyroidectomy (PTx) and 5/6 nephrectomy (Nx) with minipump implantation, delivering 1-34 rat PTH (physiologic rate), or were sham-operated and received vehicle. Only phosphorus content (low-phosphorus (LP) 0.2%; high-phosphorus (HP) 1.2%) differentiated diets. We divided the groups as follows: PTx +Nx +LP; sham + LP; PTx + Nx + HP; and sham + HP. Tail-cuff pressure and weight were measured weekly. After 2 months, biochemical, arterial, and myocardial histology and bone histomorphometry were analyzed. Results. Heart weight normalized to body weight (heart weight/100 g body weight) was higher in PTx + Nx + HP rats (PTx + Nx + HP = 0.36 ± 0.01 vs. sham + HP = 0.29 ± 0.01, PTx + Nx + LP = 0.32 ± 0.01, sham + LP = 0.28 ± 0.01) (P < 0.05). Serum creatinine levels were higher in PTx + Nx + HP rats than in PTx + Nx + LP rats (1.09 ± 0.13 vs. 0.59 ± 0.03 mg/dL) (P < 0.05). Levels of PTH did not differ significantly between the groups. Myocardial and arterial histology detected no vascular calcification or fibrosis. Bone histomorphometry revealed an association, unrelated to uremia, between HP diets and decreased trabecular connectivity. Conclusion. Myocardial hypertrophy, impaired renal function, and adverse effects on bone remodeling were associated with hyperphosphatemia and were not corrected by PTH replacement. Although no vascular calcification was observed in this model, we cannot rule out an adverse effect of hyperphosphatemia on the vascular bed. Our finding underscores the importance of phosphorus control in reducing morbidity and mortality in CRF patients.

Key words: hyperphosphatemia, renal osteodystrophy, vascular calcification, chronic renal failure, heart, phosphorus. Received for publication February 6, 2004 and in revised form May 8, 2004, and June 18, 2004 Accepted for publication June 29, 2004
C

2004 by the International Society of Nephrology

Controlling phosphorus retention has long been recognized as a key component in the clinical management of patients with chronic renal failure (CRF) [1]. In advanced CRF, renal phosphorus excretion is insufficient to remove the amount of dietary phosphorus that is absorbed daily. In addition, dialysis treatment does not correct phosphorus serum levels. As a consequence, hyperphosphatemia is commonly found in CRF patients, especially in those on dialysis [2]. The importance of managing phosphorus retention has been emphasized due to its role in the pathogenesis of secondary hyperparathyroidism (SHPT) [3]. However, disturbances in phosphorus metabolism have other significant adverse consequences [4]. Hyperphosphatemia may hasten the progression of renal disease [5], and recent studies have implicated hyperphosphatemia, either alone or in combination with the measures currently used to control it, in the substantially higher incidence of cardiovascular mortality, as well as of visceral and peripheral vascular calcification, seen in advanced CRF patients [2]. Cardiovascular disease is extremely common and accounts for at least 50% of deaths among this population [6, 7]. The exact mechanism whereby hyperphosphatemia contributes to excess mortality is unknown, but vascular calcification may be involved. Phosphorus probably induces calcification not only through indirect mechanisms but also by direct action at specific tissue sites. An in vitro study by Jono et al [8] showed that inorganic phosphate was able to transform vascular smooth muscle cells into calcifying cells by a direct mechanism involving the phosphate cotransporter Pit-1. This leads to the local formation of calcium- and phosphate-containing apatite crystals by cells that do not normally favor the deposition of such crystals. Hyperphosphatemia may also be indirectly involved through excessive secretion of parathyroid hormone (PTH) [9]. However, in patient studies, an isolated phosphorus effect cannot be evaluated because hyperphosphatemia is typically accompanied by elevated PTH, and therapeutic interventions can facilitate vascular calcification, worsen renal function, and adversely affect bone remodeling.

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We designed an experimental study involving diets with high phosphorus (HP) content that would induce hyperphosphatemia, while taking measures to ensure that PTH levels remained within the physiologic range. We did so in order to investigate the isolated effect of phosphorus on the development of cardiovascular calcification, cardiac fibrosis, and other cardiovascular changes, as well as the isolated effect of hyperphosphatemia on renal function and on bone in chronic experimental uremia. METHODS Experimental protocol A total of 32 male Wistar rats, obtained from our local breeding colony, with initial weights of 280 to 330 g, were used in this study. They were housed in individual cages in a light-controlled environment (12 hours on/12 hours off) at a constant temperature (25◦ C) and humidity (25%). They were divided into four groups. Animals from group 1 (N = 8) and group 3 (N = 8) were anesthetized with pentobarbital (50 mg/kg intraperitoneally) and then submitted to parathyroidectomy (PTx) involving microsurgical techniques using electrocautery. The animals were allowed to recover from surgery for 1 week. After this period, they were anesthetized as before and, if their ionized calcium (iCa) was <7.2 mg/dL, they were submitted to 5/6 nephrectomy (Nx), which consisted of removal of the right kidney and infarction of approximately two thirds of the left kidney. Simultaneous to the Nx procedure, normal PTH activity was restored by subcutaneous implantation of an Alzet Model 2mL 4 osmotic minipump (Alza Corp., Palo Alto, CA, USA), providing continuous infusion of 1-34 rat PTH [10] (0.022 lg/100 g/hour) (Sigma-Aldrich, St. Louis, MO, USA). On postNx day 28, the same animals were given light ether anesthesia and the osmotic minipump was replaced (pumping lifetime of 28 days) with another minipump set to the same infusion rate. Groups 2 (N = 8) and 4 (N = 8) were submitted to sham PTx and sham Nx (sham controls), including subcutaneous implantation of identical osmotic minipumps, delivering only vehicle (2% cysteine) (SigmaAldrich). Immediately after Nx, animals were fed identical diets of rodent chow (Harlan-Teklad, Indianapolis, IN, USA), with the following exceptions in phosphorus content: groups 1 [PTx +Nx +low phosphorus (LP)] and 2 (sham + LP) received a LP diet (P = 0.2%); groups 3 (PTx + Nx + HP) and 4 (sham + HP) received a HP diet (P = 1.2%). All diets were equal in their content of vitamin D, calcium (0.7%), protein (24%), and calories. A pair-feeding protocol was used. Weight measurement and tail cuff plethysmography (TCP) were performed weekly. A fluorochrome bone marker (Terramycin
) in a dose of 25 mg/kg was ip injected on days 11 and 12, as well as on days 4 and 5 before sacrifice. After 8 weeks of treatment, the animals were anesthetized and sacrificed through aor-

tic puncture exsanguination. Serum samples were frozen at −20◦ C for later biochemical evaluation. Whole blood was also collected to determine hematocrit. The heart was excised, blotted, and weighed. We also determined the heart weight to body weight ratio (heart weight/100 g body weight). The thoracic aorta and the left femoral artery were carefully removed and, along with the heart, fixed in buffered formalin. All experimental procedures were conducted in accordance with the guidelines of the Standing Committee on Animal Research of the University of Sao ˜ Paulo. Analytic determination On the day of Nx surgery, whole blood was collected by retro-orbital puncture. A model AVL-9140 Autoanalyzer (AVL Scientific Corporation, Roswell, GA, USA) was used to measure iCa levels in this blood either immediately after collection or following sacrifice (in frozen samples). Serum creatinine was determined by using a colorimetric assay (modified Heinegard-Tiderstrom). ¨ Phosphorus was determined using a colorimetric assay (Labtest Lagoa Santa, MG, Brazil). A rat PTH IRMA kit (Immutopics, San Clemente, CA, USA) was used to measure PTH. Hematocrit was also determined. Morphologic techniques Bone histomorphometry. At the end of the experimental period, the left femur of each rat was removed, dissected free of soft tissue, immersed in 70% ethanol, and processed as previously described [11]. Using a K Jung microtome, distal femurs were cut into sections of 5 lm and 10 lm in thickness. The 5 lm sections were stained with 0.1% toluidine blue, pH 6.4, and at least two nonconsecutive sections were examined for each sample. Static, structural, and dynamic parameters of bone formation and resorption were measured at the distal metaphyses (magnification, ×250), 195 lm from the epiphyseal growth plate, in a total of 30 fields, using a semi-automatic image analyzer Osteomeasure (Osteometrics, Inc., Atlanta, GA, USA). Mineral apposition rate (MAR) was determined from the distance between the two terramycin labels, divided by the time interval between the two terramycin administrations and is expressed in micrometers per day. Mineralization lag time (MLT) is expressed in days. The percentage of double terramycin-labeled surface of total trabecular surface (MS/BS) and bone formation rate (BFR) completed the dynamic evaluation. Static histomorphometric indices included ratios of trabecular bone volume and osteoid volume to bone volume (BV/TV and OV/BV, respectively, expressed as percentages), as well as osteoid thickness (OTh), which is expressed in micrometers. Percentage of total trabecular surface is used to express areas of eroded surface (ES/BS), osteoid surface (OS/BS), osteoblast surface

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Table 1. Animal data

PTx + Nx + LP Sham + LP PTx + Nx + HP Sham + HP

Initial body weight g

Final body weight g

Food intake g/day

Heart weight g

Heart weight/100 g body weight

Tail cuff plethysmyography mm Hg

307.9 ± 11.8 310.3 ± 10.5 322.0 ± 10.0 311.1 ± 13.2

416.9 ± 12.3 432.3 ± 13.0 352.3b ± 16.8 397.9 ± 8.8

17.19 ± 0.54 17.59 ± 0.47 15.77c ± 0.38 17.53 ± 0.40

1.31 ± 0.05 1.21 ± 0.03 1.25 ± 0.04 1.14 ± 0.03

0.32 ± 0.01 0.28 ± 0.01 0.36b ± 0.01 0.29 ± 0.01

141.6a ± 2.11 114.9 ± 1.20 137.5c ± 1.31 109.6 ± 1.13

Abbreviations are: PTx, parathyroidectomy; Nx, 5/6 nephrectomy; HP, high phosphorus, LP, low phosphorus. P < 0.05, PTx + Nx +LP vs. sham + HP and sham + LP; b P < 0.05, PT x + N x + HP vs. all other groups; c P < 0.05, PTx +Nx + HP vs. sham + HP and sham + LP.

a

(ObS/BS) and osteoclast surface (OcS/BS). Trabecular thickness (TbTh) and trabecular separation (TbSp) are expressed in micrometers. Trabecular number (TbN) is expressed per millimeter. Histomorphometric indices are reported using nomenclature recommended by the American Society of Bone and Mineral Research (ASBMR) [12]. All animal data were obtained through blind measurement. Cardiovascular histology. Cross-sections of the heart, thoracic aorta, and left femoral artery were embedded in paraffin, sectioned at 4lm, and stained with hematoxylineosin, Masson trichrome, von Kossa, Verhoeff, and Picrosirius. A pathologist performed a blind qualitative analysis looking for calcification or fibrosis. Statistical analysis Comparisons among the groups were made using oneway analysis of variance (ANOVA) and Tukey post-hoc test. Multivariate linear regression analysis was used to determine which independent variables significantly influenced the dependent variable (heart weight/100 g body weight, creatinine, and BV/TV). Independent variables included in the multivariate analyses were those that had proved to be significant in the univariate analysis. Graph Pad Prism version 3.03 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS for Windows, version 8.0 (SPSS, Inc., Chicago, IL, USA) software programs were used. Results are presented as mean ± SE. Values of P < 0.05 were considered statistically significant. RESULTS Body weight, food intake, TCP, and heart weight/100 g body weight Table 1 summarizes the animal data. The duration of the uremia and diet treatment experiments was 52 days. Initial body weight did not differ among the groups. At the end of the study, despite the pair-feeding protocol, PTx + Nx + HP rats had lower body weight. Compared to those in all other groups, PTx + Nx + HP rats presented slightly lower food intake. Initial TCP readings were similar among all groups. However, after 8 weeks of uremia, rats that were submitted to Nx (PTx + Nx + LP and PTx + Nx + HP) developed hypertension, al-

though arterial pressure was comparable between the two groups. Nonetheless, arterial pressure was significantly lower in the two control groups. The heart weight was similar among all groups. Heart weight normalized to body weight was significantly higher in the PTx + Nx + HP group than in any other group (Fig. 1). Biochemical findings Table 2 and Figure 2 show serum chemistries and hematocrit results from uremic rats and from controls. The continuous infusion of 1-34 rat PTH normalized serum PTH levels in those animals previously submitted to PTx. Sham animals who were fed an LP diet presented significant lower serum PTH levels. Rats submitted to Nx (PTx + Nx + LP and PTx + Nx + HP) developed renal failure, but those who were fed an HP diet (PTx + Nx + HP) had higher serum creatinine levels than did PTx + Nx + LP rats. In PTx + Nx + HP rats, hematocrit levels were lower. Animals who were submitted to Nx and fed an HP diet (PTx + Nx + HP) presented very low iCa levels (hypocalcemia). In this group, considerable hyperphosphatemia developed—evidenced by a pronounced elevation in calcium × phosphorus (Ca × P) product—even in the presence of hypocalcemia. Morphologic results Bone histomorphometry parameters revealed that normal bone remodeling (bone turnover and bone architecture) was not achieved in rats who were fed an HP diet (PTx + Nx + HP and their controls, sham + HP) These rats had lower bone volume and this effect was unrelated to uremia. Higher trabecular separation and lower trabecular numbers were also detected in rats consuming the HP diet (PTx + Nx + HP and their controls, sham + HP), denoting an osteoporosis effect of the HP diet. Although not statistically significant, a higher MLT was also observed in PTx + Nx + HP rats. Higher MARs were seen in uremic rats fed an LP diet (PTx + Nx + LP) and higher BFRs were seen in Nx animals (Table 3). The efficacy of the HP diet in inducing cardiovascular calcification and fibrosis was analyzed. Qualitative analysis involving myocardial and arterial histology detected no vascular calcification or significant fibrosis.

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Fig. 1. Animal data for tail-cuff plethysmyography (TCP), heart weight/body weight ratio, serum creatinine (Cr), and bone trabecular volume (BV/TV). (A) Animals submitted to 5/6 nephrectomy (Nx) [high phosphorus (HP) or low phosphorus (LP)] had higher TCP than sham animals. There were no differences between parathyroidectomy (PTx) + Nx + HP and PTX + Nx + LP regarding TCP. (B) Only PTx + Nx + HP animals had a higher heart weight/body weight ratio (HW/100 g BW), despite PTx + Nx + LP also had higher blood pressure. (C) Animals that were submitted to Nx had increased serum creatinine levels, but those fed with a HP diet had even higher than those fed with a LP diet. (D) Animals that received a high content of phosphorus on their diet had lower bone trabecular volume (BV/TV), and this was unrelated to uremia. #P < 0.05, PTx + Nx groups vs. sham groups; ∗ P < 0.05, PTx + Nx + HP vs. all other groups; φP < 0.05, PTx +Nx + LP vs. sham groups;
P < 0.05, HP vs. LP groups.

Table 2. Biochemical data

PTx + Nx + LP Sham + LP PTx + Nx + HP Sham+HP

Hematocrit %

Ionized calcium mmol/L

Phosphorus mg/dL

Calcium × phosphorus mg2 /dL2

Creatinine mg/dL

Parathyoid hormone pg/mL

42.9 ± 1.1 42.9 ± 0.5 39.1c ± 1.3 43.5 ± 0.5

1.19 ± 0.05 1.22 ± 0.05 0.61c ± 0.05 1.23 ± 0.07

5.65 ± 0.48 4.43 ± 0.39 15.71c ± 2.56 4.95 ± 0.33

52.09 ± 3.46 41.97 ± 1.73 67.79d ± 5.19 45.86 ± 2.46

0.59a ± 0.03 0.32 ± 0.01 1.09c ± 0.15 0.46 ± 0.03

114.9 ± 30.1 10.5b ± 2.8 86.8 ± 20.3 135.9 ± 28.6

Abbreviations are: PTx, parathyroidectomy; Nx, 5/6 nephrectomy; LP, low phosphorus; HP, high phosphorus. P < 0.05, PTx + Nx + LP vs. sham + HP and sham + LP; b P < 0.05, sham + LP vs. all other groups; c P < 0.05, PTx + Nx + HP vs. all other groups; d P < 0.05, PTx + Nx + HP vs. sham + HP and sham + LP. a

Correlation analysis Table 4 shows the results of the univariate analysis, revealing that heart weight/100 g body weight correlated positively and definitively with phosphorus levels (r = 0.76, P < 0.0001), Ca × P (r = 0.76, P < 0.0001), and creatinine (r = 0.75 P < 0.0001). Strong correlations are also shown between creatinine and phosphorus (r = 0.94, P < 0.0001) and between creatinine and Ca × P (r = 0.81, P < 0.0001). Trabecular bone volume (BV/TV) was correlated with serum calcium and with final body weight and reached an almost significant degree of correlation with serum phosphorus and with serum creatinine. In Table 5, multivariate linear stepwise regression results show that heart weight was dependent on phosphorus and TCP. Moreover, the correlation between hematocrit and myocardial hypertrophy did not reach the level of statistical significance (P = 0.059). Another important result was that levels of creatinine were dependent upon levels of phosphorus. TCP was also associated with creatinine, but did not reach a significant result (P =

0.052). Trabecular bone volume (BV/TV) was dependent on serum calcium and on serum phosphorus. DISCUSSION This study showed that, in rats with moderate renal failure that received an HP diet and physiologic PTH reposition, there was no cardiovascular calcification, despite the development of pronounced hyperphosphatemia and elevated Ca × P. However, isolated hyperphosphatemia was associated with the development of myocardial hypertrophy. We also demonstrated that kidney dysfunction could be aggravated by hyperphosphatemia resulting from an HP diet. Another important finding is that an HP diet was associated with the development of an osteoporosis-like bone lesion that is aggravated by uremia. Until now, no experimental model of vascular calcification in uremic rats has been established. We designed this study in an attempt to demonstrate the development of phosphorus-induced vascular calcification. In our

Neves et al: Hyperphosphatemia in rats with renal failure

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experimental study, we were unable to demonstrate the relationship between hyperphosphatemia and vascular calcification previously described in dialysis patients [2, 13]. There have been very few experimental studies evaluating vascular calcification in a uremic milieu. In one of those studies (by Ejerblad, Erikson, and Johansson [14]), in which uremia was induced in rats (with and without PTx), arterial calcification was observed until spontaneous death or for 36 weeks. These authors showed that the development of arterial changes was dependent upon both the grade and the duration of uremia, since such changes were found in only a few animals, who died immediately upon becoming severely uremic. The authors also pointed out that PTx largely prevented the development of calcification, indicating that SHPT plays an important role in the development of calcification. In our study, due to the shorter observation period, we intentionally induced only moderate renal failure in an attempt to minimize the effect of anemia and acidosis. In other studies not involving renal failure, vascular calcification has been associated with hyperlipidemia and with administration of nicotine, warfarin and high doses of vitamin D, as well as with combinations of these factors [15]. The calcification observed could not be attributed to phosphorus alone. Other important factors are calcium content and the proportion of calcium to phosphorus in the diet. These previous studies demonstrated arterial calcification using diets with high calcium/ phosphorus proportions and high calcium contents, perhaps favoring increased calcium levels (sometimes even

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Fig. 2. Animal data for serum parathormone, ionized calcium, and phosphorus and calcium × phosphorus product. (A) Sham animals that received a low phosphorus (LP) diet had lower serum parathyroid hormone (PTH) levels. Animals submitted to 5/6 nephrectomy (Nx) and parathyroidectomy (PTx) had similar PTH levels, and allowed an evaluation of isolated hyperphosphatemia. (B) PTx + Nx + high phosphorus (HP) animals presented lower serum ionized calcium (iCa) levels, higher serum phosphorus (P) levels (C) and a higher calcium × phosphorus (Ca × P) product (D). +P < 0.05, Ptx + Nx + LP vs. all other groups; ∗ P < 0.05, PTx + Nx + HP vs. all other groups.

with hypercalcemia). In addition, the physiologic PTH serum levels observed in our study could have been protective against calcification since it is suspected that high PTH levels are a necessary condition for the development of cardiovascular or even other soft tissue calcifications, perhaps due to elevated cytosolic calcium [16]. In our study, calcification was not detected, maybe because we used rats, which developed hypocalcemia and moderate renal failure, were treated only for 8 weeks and maintained normal levels of PTH. Perhaps the presence of high PTH levels, more severe uremia and normocalcemia or hypercalcemia is a necessary condition for arterial calcification development in rats. Otherwise, myocardial hypertrophy, as suggested by the higher heart weight to body weight ratio, was evident in the uremic hyperphosphatemic group (PTx + Nx + HP). This hypertrophy was also unrelated to PTH levels. A similar finding was described by Amann et al [17], who found significantly higher left ventricular weights in Nx rats fed an HP diet. However, the authors also found PTH levels to be higher in this group, making it difficult distinguish the role of phosphorus from that of PTH in hypertrophy. Therefore, to our knowledge, this is the first study to demonstrate that isolated hyperphosphatemia is associated to myocardial hypertrophy. This could happen through a disproportionate rise in collagen content, which would lead to myocardial fibrosis and structural remodeling of the hypertrophied myocardium. However, we were unable to demonstrate myocardial fibrosis, although a more sensitive analysis might. Nevertheless, this

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Abbreviations are: BV/TV, trabecular bone volume to bone volume; OV/BV, osteoid volume to bone volume; OTh, osteoid thickness; OS/BS, osteoid surface to bone surface; ES/BS, eroded surface to bone surface; Obs/BS, osteoblast surface to bone surface; OcS/BS, osteoclast surface to bone surface; TbSp, trabecular separation; TbN, trabecular number; TbTh, trabecular thickness; MS/BS, terramycin-labeled surface of total trabecular surface; MAR, mineral apposition rate; BFR/BS bone formation rate to bone surface; MLT, mineralization lag time; PTx, parathyroidectomy; Nx, 5/6 nephrectomy; LP, low phosphorus; HP, high phosphorus. a < P 0.05, PTx + Nx + LP vs. sham + LP; b P < 0.05, PTx + Nx + HP vs. LP groups; c P < 0.05, PTx + Nx + HP vs. sham groups; d P < 0.05, Sham + HP vs. LP groups; e P < 0.05, Sham + HP vs. all others.

4.47 ± 0.35 1.17a ± 0.10 0.05a ± 0.01 4.34 ± 0.59 3.10 ± 0.57 0.78 ± 0.05 0.02 ± 0.01 6.28 ± 1.83 5.88 ± 1.20 1.17 ± 0.19 0.08c ± 0.02 11.82 ± 5.13 3.32 ± 0.66 0.76 ± 0.09 0.03 ± 0.01 6.41 ± 1.17 4.33 ± 1.01 178.7 ± 17.88 4.39 ± 0.26 55.56 ± 2.88 3.16 ± 0.70 161.8 ± 9.35 4.53 ± 0.21 58.17 ± 2.59 3.72 ± 0.83 309.3a ± 51.54 3.07a ± 0.33 55.06 ± 2.30 3.84 ± 0.74 250.6d ± 34.70 3.58d ± 0.33 47.76e ± 1.88 PTx +Nx + LP 24.72 ± 2.41 0.83a ± 0.16 2.12 ± 0.18 10.78 ± 1.65 13.83 ± 1.97 7.33 ± 1.28 Sham + LP 26.77 ± 1.72 0.34 ± 0.11 2.05 ± 0.24 5.47 ± 1.38 10.37 ± 1.44 2.69 ± 0.60 PTx + Nx + HP 16.64a ± 1.58 2.31 ± 1.00 3.04 ± 0.60 17.12 ± 4.39 13.04 ± 2.21 10.57 ± 2.80 Sham + HP 16.99d ± 1.66 0.62 ± 0.19 2.16 ± 0.09 6.72 ± 1.93 11.86 ± 2.20 4.26 ± 1.65

TbTh l/m TbN /mm TbSp l/m OcS/BS % ObS/BS % ES/BS % OS/BS % OTh l OV/BV % BV/TV %

Table 3. Histomorphometric variables of trabecular bone in the distal femur

MS/BS %

MAR l/day

BFR/BS (l3 /l2 /day

MLT day

Neves et al: Hyperphosphatemia in rats with renal failure

phosphorus-induced hypertrophy could be another factor contributing to high cardiovascular mortality in patients with CRF. Many factors that may influence the progression of renal failure have been identified, predominant among which are glomerular and hemodynamic factors, systemic hypertension, and high dietary levels of protein and phosphorus. Experimental evidence has been found that excess phosphorus plays a role in the progression of renal failure in partially Nx rats [18]. However, these authors did not employ a pair-feeding protocol and intake of dietary protein was not controlled. Therefore, some of the beneficial effects may have been the result of protein restriction that accompanied the phosphorus restriction. In addition, PTH probably differed between rats fed a HP diet and those fed a LP diet. Other reports have documented the injurious effect of an HP diet on the kidney. It is assumed that calcium phosphate deposition resulting from hyperphosphatemia incites tissue injuries. However, there have been no studies discounting the influence of PTH on the pathogenesis of renal dysfunction. In this study, we used physiologic doses to restore PTH to appropriate levels and thereby demonstrated that kidney dysfunction could be aggravated by HP diet alone. We also found that, when dietary phosphorus was restricted (even at constant PTH levels), kidney function was protected. The worsening of kidney function was apparently unrelated to nephrocalcinosis, since we found only sparse calcification in Nx animals (PTx + Nx + LP and PTx + Nx + HP, data not shown). It is probable that we were unable to induce nephrocalcinosis due to the lower PTH levels and to the fact that our rats were not fed a high calcium diet. Cozzolino et al [19] analyzed the impact of different phosphate binders on kidney tissue. The authors found higher kidney calcium deposition in both untreated rats (with high levels of phosphorus and PTH) and in calcium salt–treated rats (with control of phosphorus and PTH levels, but with a high calcium load). As in our study, these authors were not able to identify vascular calcification in their experimental model and suggested that Ca × P product-related induction of calcification is tissuespecific and time-dependent. Regarding bone effects of HP diets, we found lower trabecular bone volume in rats fed an HP diet. In addition, a loss of connectivity, unrelated to uremia, was a significant finding in such rats (PTx + Nx + HP and sham + HP). This is the first study to address the role of dietary phosphorus and hyperphosphatemia in bone remodeling through histomorphometry. Szabo´ [20] performed an experimental study that evaluated whether physiologic doses of either PTH or calcitriol were able to normalize osteoclast and osteoblast function in uremic rats. The authors found that administration of PTH and calcitriol, in physiologic doses that raised calcium serum levels, failed to normalize the percentage of osteoclast surface. However, they were

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Table 4. Univariate analysis Heart weight/100 g Tail-cuff body weight plethysmyography Hematocrit Creatinine −0.67 <0.001

Final weight Heart weight/100 g body weight

0.56 <0.001 0.41 <0.01

Heart weight Food intake Tail-cuff plethysmyography Hematocrit

0.45 <0.01 −0.32 =0.07

Ionized calcium

Phosphorus

Ca × P

BV/TV

−0.71 <0.0001 0.75 <0.0001

0.70 <0.0001 −0.76 <0.0001

−0.74 <0.0001 0.76 <0.0001

−0.69 <0.001 0.76 <0.0001

0.36 <0.05

−0.43 <0.05 0.54 <0.005 −0.61 <0.001

0.63 <0.0001 −0.39 <0.05 0.57 <0.001 −0.84 <0.0001

−0.49 <0.001 0.43 <0.05 −0.67 <0.0001 0.94 <0.0001 −0.89 <0.0001

−0.48 <0.01 0.56 <0.001 −0.55 <0.01 0.81 <0.0001 −0.76 <0.0001 0.88 <0.0001

Creatinine Ionized calcium Phosphorus

Table 5. Multivariate analysis of predictors of myocardial hypertrophy, renal function, and trabecular bone volume

Heart weight/100 g body weight Hematocrit Tail-cuff plethysmyography Phosphorus Creatine Tail-cuff plethysmyography Phosphorus BV/TV Calcium Phosphorus

B

Confidence index

P value

0.004 0.001 0.005

0, 0.008 0.0008, 0.0012 0.003, 0.007

0.059 0.03 0.00001

0.003 0.037

−0.001, 0.005 0.017, 0.057

0.052 0.002

6.9 0.93

2.87, 10.93 0.1, 1.72

0.002 0.03

BV/TV, trabecular bone volume to bone volume; B, standardized regression coefficient (b).

−0.34 0.06 0.52 <0.01 −0.29 0.11

CONCLUSION We have demonstrated that isolated hyperphosphatemia was associated with myocardial hypertrophy, worsening of renal function, and negative effects on bone remodeling. Although no vascular calcification was observed in this model, we cannot rule out an adverse effect of hyperphosphatemia on the vascular (arterial) bed. Our finding corroborates the importance of phosphorus control in reducing the morbidity and mortality described in CRF patients. The molecular basis of the effect of hyperphosphatemia on target organs should be elucidated in further studies. ACKNOWLEDGMENTS

unable to determine if the lack of responsiveness of the bone cells at the surface was due to resistance to PTH, calcitriol, both, or some other unidentified uremic factor. In our study, dietary phosphorus probably played an important role in the pathogenesis of the bone lesion, even in the absence of hypocalcemia, since animals that ate an HP diet and had normal serum calcium levels (sham + HP) also developed osteoporosis-like bone lesions. Bover et al [21] also found that uremia and phosphorus each had separate and major effects on skeletal resistance to PTH. Our results show that restoration of PTH to appropriate physiologic levels did not normalize bone remodeling. This may be due to the fact that hyperphosphatemia decreases bone response to PTH, and that the HP diet was also an important determinant of bone lesion. It is important to note that adynamic bone lesions were not found. Another factor that could be related to the lower trabecular bone volume is undernourishment, since we found, in the univariate analysis, a positive correlation between BV/TV and final body weight.

This study was performed with grants from Funda¸cao ˜ de Aux´ılio a` Pesquisa do Estado de Sao ˜ Paulo (FAPESP, grant: 01/01789–0) and was presented, in part, at the 36th Annual Meeting of the American Society of Nephrology, San Diego, CA, 2003. The authors would like to acknowledge the assistance given by Mariana Cury in the statistical analysis, by Jefferson D. Boyles in the translation and editing of the text, and by Rozidete Bizerra and Rosimeire Aparecida Bizerra in technical assistance. Reprint requests to Vanda Jorgetti, M.D., University of S˜ao Paulo— ´ Nephrology Department, Laboratorio de Fisiopatologia Renal (LIM16), Av. Dr. Arnaldo, 455, s/3342, 01246–903, S˜ao Paulo, SP, Brazil. E-mail: [email protected]

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