A rat model of chronic kidney disease-mineral bone disorder
2008; Elsevier BV; Volume: 75; Issue: 2 Linguagem: Inglês
10.1038/ki.2008.456
ISSN1523-1755
AutoresSharon M. Moe, Neal X. Chen, Mark F. Seifert, Rachel M. Sinders, Dana Duan, Xianming Chen, Yun Liang, Jeff Radcliff, Kenneth E. White, Vincent H. Gattone,
Tópico(s)Genetic and Kidney Cyst Diseases
ResumoChronic Kidney Disease-Mineral Bone Disorder (CKD-MBD) is a newly defined syndrome encompassing patients with chronic kidney disease that have a triad of biochemical alterations in calcium, phosphorus and parathyroid hormone, vascular calcification, and bone abnormalities. Here we describe a novel Cy/+ rat model of slowly progressive kidney disease spontaneously developing the three components of CKD-MBD when fed a normal phosphorus diet. Since the renal disorder progressed ‘naturally’ we studied the effect of dietary manipulation during the course of the disease. Animals with early, but established, chronic kidney disease were fed a casein-based or a grain-based protein diet both of which had equivalent total phosphorus contents. The two different sources of dietary protein had profound effects on the progression of CKD-MBD, likely due to differences in intestinal bioavailability of phosphorus. Although both dietary treatments resulted in the same serum phosphorous levels, the casein-fed animals had increased urinary phosphorus excretion and elevated serum FGF23 compared to the grain-fed rats. This model should help identify early changes in the course of chronic kidney disease that may lead to CKD-MBD. Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD) is a newly defined syndrome encompassing patients with chronic kidney disease that have a triad of biochemical alterations in calcium, phosphorus and parathyroid hormone, vascular calcification, and bone abnormalities. Here we describe a novel Cy/+ rat model of slowly progressive kidney disease spontaneously developing the three components of CKD-MBD when fed a normal phosphorus diet. Since the renal disorder progressed ‘naturally’ we studied the effect of dietary manipulation during the course of the disease. Animals with early, but established, chronic kidney disease were fed a casein-based or a grain-based protein diet both of which had equivalent total phosphorus contents. The two different sources of dietary protein had profound effects on the progression of CKD-MBD, likely due to differences in intestinal bioavailability of phosphorus. Although both dietary treatments resulted in the same serum phosphorous levels, the casein-fed animals had increased urinary phosphorus excretion and elevated serum FGF23 compared to the grain-fed rats. This model should help identify early changes in the course of chronic kidney disease that may lead to CKD-MBD. Vascular calcification is common in patients with chronic kidney disease (CKD), appearing in 30–65% of patients with stages 3–5 CKD and 50–80% of patients with stage 5D CKD, and is associated with increased morbidity and mortality.1.Block G.A. Spiegel D.M. Ehrlich J. et al.Effects of sevelamer and calcium on coronary artery calcification in patients new to hemodialysis.Kidney Int. 2005; 68: 1815-1824Google Scholar, 2.Mehrotra R. Budoff M. Hokanson J.E. et al.Progression of coronary artery calcification in diabetics with and without chronic kidney disease.Kidney Int. 2005; 68: 1258-1266Google Scholar, 3.Moe S.M. Chen N.X. Mechanisms of vascular calcification in chronic kidney disease.J Am Soc Nephrol. 2008; 19: 213-216Google Scholar In the coronary arteries, this calcification is typically intimal, within atherosclerotic plaques or as circumferential intimal lesions, whereas in the aorta, calcification occurs in both the intimal and medial layers of the vessel wall (atherosclerosis and Mönkeberg's medial calcific sclerosis). The pathogenesis of vascular calcification is complex, but in vitro studies support the concept of an initial transformation of vascular smooth muscle cells to chondrocyte/osteoblast-like cells in response to hyperphosphatemia, uremia, inflammation, and elevated glucose levels.4.Chen N.X. O'Neill K.D. Chen X. et al.Fetuin-A uptake in bovine vascular smooth muscle cells is calcium dependent and mediated by annexins.Am J Physiol Renal Physiol. 2007; 292: F599-F606Google Scholar These transformed cells then lay down a matrix of collagen and non-collagenous proteins and produce matrix vesicles that serve as the initial nidus for calcification, similar to the process of normal bone mineralization. This process is accelerated in the clinical setting of CKD, possibly because of hyperphosphatemia and hyperparathyroidism, the use of high dose calcium salts as phosphate binders that increase the overall calcium load, abnormal bone remodeling, and relative deficiencies of circulating and locally produced inhibitors of calcification. This interrelationship of vascular calcification with abnormal serum biochemistries and abnormal bone remodeling is the basis for the recently named syndrome, chronic kidney disease-mineral bone disorder (CKD-MBD).5.Moe S.M. Drueke T. Lameire N. et al.Chronic kidney disease-mineral-bone disorder: a new paradigm.Adv Chronic Kidney Dis. 2007; 14: 3-12Google Scholar The complexity of this interrelationship makes studies in humans difficult, as one cannot easily control one variable without impacting another. Thus, there is a clear need for an appropriate animal model in which to understand the progressive pathophysiology of CKD-MBD, as well as to test interventions. Several animal models of vascular calcification exist, including the adenine nephrotoxic model6.Katsumata K. Kusano K. Hirata M. et al.Sevelamer hydrochloride prevents ectopic calcification and renal osteodystrophy in chronic renal failure rats.Kidney Int. 2003; 64: 441-450Google Scholar, 7.Price P.A. Roublick A.M. Williamson M.K. Artery calcification in uremic rats is increased by a low protein diet and prevented by treatment with ibandronate.Kidney Int. 2006; 70: 1577-1583Google Scholar and the 5/6th nephrectomy models in rats.8.Cozzolino M. Dusso A.S. Liapis H. et al.The effects of sevelamer hydrochloride and calcium carbonate on kidney calcification in uremic rats.J Am Soc Nephrol. 2002; 13: 2299-2308Google Scholar, 9.Hirata M. Katsumata K. Endo K. et al.In subtotally nephrectomized rats 22-oxacalcitriol suppresses parathyroid hormone with less risk of cardiovascular calcification or deterioration of residual renal function than 1,25(OH)2 vitamin D3.Nephrol Dial Transplant. 2003; 18: 1770-1776Google Scholar In both models, animals develop severe hyperphosphatemia and hyperparathyroidism due to an acute kidney injury, which is followed by CKD. These models have provided useful information on the pathophysiology of vascular calcification. However, they represent advanced CKD, due to the severity of acute injury, with creatinine clearances that approximate late 4 or 5 stage human CKD. In both disease states, diet is an important factor. The 5/6th nephrectomy rat model is generally fed a high-phosphorus diet to induce hyperphosphatemia.8.Cozzolino M. Dusso A.S. Liapis H. et al.The effects of sevelamer hydrochloride and calcium carbonate on kidney calcification in uremic rats.J Am Soc Nephrol. 2002; 13: 2299-2308Google Scholar, 9.Hirata M. Katsumata K. Endo K. et al.In subtotally nephrectomized rats 22-oxacalcitriol suppresses parathyroid hormone with less risk of cardiovascular calcification or deterioration of residual renal function than 1,25(OH)2 vitamin D3.Nephrol Dial Transplant. 2003; 18: 1770-1776Google Scholar The adenine model has a more rapid onset and severe course of kidney disease as well as hyperparathyroidism and arterial calcification than the 5/6th nephrectomy model. Curiously, the adenine model develops more consistent arterial calcification on a low-protein diet.7.Price P.A. Roublick A.M. Williamson M.K. Artery calcification in uremic rats is increased by a low protein diet and prevented by treatment with ibandronate.Kidney Int. 2006; 70: 1577-1583Google Scholar The type of calcium deposition may also differ in these models.10.Verberckmoes S.C. Persy V. Behets G.J. et al.Uremia-related vascular calcification: more than apatite deposition.Kidney Int. 2007; 71: 298-303Google Scholar In mice, spontaneous calcification in the setting of surgically induced acute kidney injury followed by CKD, is not found unless there is a concomitant genetic abnormality that is proatherogenic, such as ablation of the low-density lipoprotein receptor or the ApoE genes.11.Davies M.R. Lund R.J. Hruska K.A. BMP-7 is an efficacious treatment of vascular calcification in a murine model of atherosclerosis and chronic renal failure.J Am Soc Nephrol. 2003; 14: 1559-1567Google Scholar, 12.Massy Z.A. Ivanovski O. Nguyen-Khoa T. et al.Uremia accelerates both atherosclerosis and arterial calcification in apolipoprotein E knockout mice.J Am Soc Nephrol. 2005; 16: 109-116Google Scholar Although useful, none of these rodent models provide the opportunity to study a slowly progressive CKD, nor earlier stages of CKD-MBD. This is important because studies demonstrate that patients in earlier stages of CKD also have coronary artery calcification1.Block G.A. Spiegel D.M. Ehrlich J. et al.Effects of sevelamer and calcium on coronary artery calcification in patients new to hemodialysis.Kidney Int. 2005; 68: 1815-1824Google Scholar, 13.Mehrotra R. Adler S. Coronary artery calcification in nondialyzed patients with chronic kidney diseases.Am J Kidney Dis. 2005; 45: 963Google Scholar, 14.Russo D. Miranda I. Ruocco C. et al.The progression of coronary artery calcification in predialysis patients on calcium carbonate or sevelamer.Kidney Int. 2007; 72: 1255-1261Google Scholar suggesting that the process begins before beginning dialysis. Therefore, there is a clear need for additional animal models to provide the opportunity to study slowly progressive CKD to better understand the triad of CKD-MBD: (1) abnormal serum biochemistries, (2) abnormal bone remodeling, and (3) vascular calcification. In this report, we describe a novel model of progressive CKD-MBD, the Cy/+ rat, and demonstrate its usefulness for evaluating early pathogenesis and the impact of different dietary regimens on the course of CKD-MBD. To assess the magnitude of uremia, we compared blood urea nitrogen (BUN), creatinine, weight, and hematocrit in the 38-week Cy/+ CKD animals each treated for 18 weeks with the casein protein-based diet with either 0.7% (normal) or 0.2% phosphorus (low), comparing these CKD animals to normal littermates. The 38-week animals with CKD had elevated BUN at 20 weeks, which persisted (Figure 1a ). The animals in the CKD groups also had elevated serum creatinine values (normal, 0.62±0.01 mg/100 ml; CKD/0.7% Pi, 3.05±0.27 mg/100 ml; CKD/0.2% Pi, 1.93±0.25 mg/100 ml; P<0.01). The low-phosphorus diet attenuated the progressive CKD as assessed by BUN (Figure 1a). In the 38-week end-point groups, the CKD animals in both treatment groups weighed less than normal littermate animals (at week 38: normal, 588±9 g; CKD/0.7% Pi, 471±17 g; CKD/0.2% Pi, 537±8 g; P<0.01, between normal and both CKD groups). Lastly, animals in the two CKD treatment groups were anemic by 38 weeks, with more severe anemia occurring in the animals on the normal (0.7%) phosphorus diet (hematocrit in week-38 end-point animals: normal, 0.50±0.00%; CKD/0.7% Pi, 0.29±0.01%; CKD/0.2% Pi, 0.36±0.02%; P<0.01, between each of the groups). The CKD animals in the 38-week end-point groups were sicklier, particularly those with severe hyperparathyroidism/hyperphosphatemia in Group 2 (CKD/0.7% phosphorus diet). Of the 43 animals in the CKD/0.7% phosphorus diet group, 18 either died (n=11) or became morbidly ill requiring euthanasia (n=7), whereas only 1 of 31 normal animals died, and 3 of 29 animals in the CKD/0.2% Pi group died. Thus, by all indices (BUN, creatinine, body weight, hematocrit), the Cy/+ animals on both the normal (0.7% phosphorus) and low (0.2% phosphorus) diets had worse kidney disease when compared to the normal littermates, but the low-phosphorus diet attenuated these abnormalities and prevented early morbidity and mortality. To assess the biochemical parameters of CKD-MBD, plasma parathyroid hormone (PTH), phosphorus, and calcium were assessed at the start of treatment (20 weeks), at 34 weeks, and again at 38 weeks (killing). The CKD animals fed with the normal 0.7% phosphorus diet developed progressive hyperphosphatemia and hyperparathyroidism. Figure 1b–d demonstrates the change over time for these measures in the 38-week animals. These differences in end-point measurements for phosphorus (P=0.03) and PTH (P=0.006) remained significant even if adjusted for baseline values of phosphorus or PTH (respectively), baseline BUN, end-point BUN, or change in BUN using a mixed model, suggesting that the observed differences were not solely due to changes in kidney function and that the premature death did not alter our findings. In fact, the excessive animal deaths in the normal-phosphorus diet group would have increased the observed differences. In contrast, the serum calcium was slightly increased only in the 38-week CKD/0.2% phosphorus-treated animals compared to the CKD/0.7% Pi-treated animals (P=0.04 when adjusted for baseline calcium and BUN; Figure 1d). In the second group of animals, killed at 34 weeks, biochemical assessments were made at baseline (week 20), mid point (week 29), and end point (week 34). Similar to the 38-week treatment group, the 34-week animals had progressive hyperphosphatemia and hyperparathyroidism (Table 1 ).Table 1Biochemical findings in 34-week animalsN=12–17GroupBaseline (week 20)Week 29Week 34BUN (mg/100 ml)Normal20.9±0.5ND15.8±1.2CKD/0.7% Pi47.0±1.1ND106.7±13.5*P<0.05 for difference from baseline.CKD/0.2% Pi47.8±1.3ND52.7±3.9Phosphorus (mg/100 ml)Normal6.9±0.16.5±0.26.1±0.4CKD/0.7% Pi7.4±0.28.5±0.311.6±1.0*P<0.05 for difference from baseline.CKD/0.2% Pi7.5±0.16.4±0.28.0±0.9Calcium (mg/100 ml)Normal10.9±0.311.9±0.411.0±0.6CKD/0.7% Pi11.1±0.411.6±0.59.8+0.3CKD/0.2% Pi11.9±0.210.5±0.311.8±1.0PTH (pg/ml)Normal91±1498±10177±52CKD/0.7% Pi218±731351±246*P<0.05 for difference from baseline.3543±648*P<0.05 for difference from baseline.CKD/0.2% Pi114±18110±14560±163*P<0.05 for difference from baseline.Weight (g)Normal487±7560±10585±12*P<0.05 for difference from baseline.CKD/0.7% Pi481±5535±6535±11*P<0.05 for difference from baseline.CKD/0.2% Pi483±6510±9519±10*P<0.05 for difference from baseline.BUN, blood urea nitrogen; CKD, chronic kidney disease; ND, not done; PTH, parathyroid hormone.Mean±s.e.m.* P<0.05 for difference from baseline. Open table in a new tab BUN, blood urea nitrogen; CKD, chronic kidney disease; ND, not done; PTH, parathyroid hormone. Mean±s.e.m. To determine the extent and magnitude of arterial calcification, the calcium content of the thoracic aorta was examined by quantitative biochemistry and semiquantitative histology. The thoracic aorta was more calcified in the CKD animals treated with the normal (0.7% phosphorus) diet than either the normal animals or the CKD animals treated with the low (0.2%) phosphorus diet (Figures 2 , 3a and b ), but calcification was quantifiable at levels above normal animals only at 38 weeks. The magnitude of the calcification correlated with the phosphorus level (r=0.67, P<0.001), but not PTH or calcium. Histologic evaluation of the 38-week animals at identical levels of the upper thoracic aorta revealed that 60% (6 of 10) of the CKD/0.7% phosphorus-treated animals had significant medial calcification (3 animals with 3+ calcification, 2 animals with 2+ calcification, and 1 animal with 1+ calcification). In contrast, none of the normal animals or CKD/0.2% phosphorus-treated animals had calcification. No histologic evidence of calcification was observed at 34 weeks. Scanning electron microscopy with energy dispersive X-ray microanalysis demonstrated that the mineral deposits were composed of calcium, phosphorus, and oxygen indicative of brushite or hydroxyapatite (Figure 3e).Figure 3Histologic evaluation of vascular calcification and bone. (a) A thoracic aorta from a normal (non-CKD) 40-week old animal stained with MacNeal's stain demonstrating no calcification. (b) Thoracic aorta from a CKD animal fed with a normal (0.7%) phosphorus diet demonstrates calcification (in black) in the medial layer with a vascular smooth muscle cell visible within the calcified area. (c, d) Bone from a CKD animal fed with a normal (0.7%) phosphorus diet demonstrating osteitis fibrosa cystica with osteoclasts (arrowhead), active osteoblasts (arrows), and in (d), peritrabecular fibrosis (arrow). (e) Scanning electron microscopy with EDS X-ray microanalysis of the aortic calcifications. Backscatter imaging allowed the identification of the calcification (white region) in the aorta and energy dispersive spectrometry was used to assess the elemental composition. Compared with the non-calcified region (pink arrow and top spectrum), the calcified region (green arrow and bottom spectrum) had increased amounts of calcium, oxygen, and phosphorus (compared to carbon and sodium peaks) indicating a calcium phosphate material such as hydroxyapatite or brushite.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Static histomorphometry was performed in the CKD animals treated with normal (0.7%) and low (0.2%) phosphorus diets in the 34-week and 38-week animals and compared to the normal animals in each time group. The bone changes in the CKD animals were indicative of secondary hyperparathyroidism (Table 2 ; Figure 3c and d: increased fibrosis, osteoblasts, and osteoclasts) in the CKD animals treated with the 0.7% phosphorus diet, and a mineralization defect (increased osteoid volumes and osteoid surface) in the animals treated with 0.2% phosphorus diet. The bone volume and trabecular thickness were not different between the two groups, but the trabecular number was higher and the trabecular separation lower in the CKD animals treated with 0.7% phosphorus, consistent with excessive and uncontrolled hyperparathyroidism (Table 1). The differences from 34 to 38 weeks reflected continued progression of severe secondary hyperparathyroidism in the Cy/+ animals treated with the 0.7% phosphorus diet, and stabilization or slight improvement of the mineralization defect in the 0.2% phosphorus-treated animals.Table 2Static bone histomorphometric parameters in normal and CKD ratsGroup 1: normalGroup 2: CKD Cy/+ 0.7% Pi dietGroup 3: CKD Cy/+ 0.2% Pi dietParameter N=5–8 per group34 weeks38 weeks34 weeks38 weeks34 weeks38 weeksTurnover: Fibrosis (%)NoneaGroup 1 or 3 different from Group 2 at same time point, P<0.05.NoneaGroup 1 or 3 different from Group 2 at same time point, P<0.05.8.5±2.74.9±1.8NoneaGroup 1 or 3 different from Group 2 at same time point, P<0.05.NoneaGroup 1 or 3 different from Group 2 at same time point, P<0.05. Ob.S/BS (%)1.8±0.5aGroup 1 or 3 different from Group 2 at same time point, P<0.05.2.5±0.6aGroup 1 or 3 different from Group 2 at same time point, P<0.05.18.5±3.414.9±3.710.2±1.5aGroup 1 or 3 different from Group 2 at same time point, P<0.05.,bGroup 3 different from Group 1 at same time point, P<0.05.4.9±1.4aGroup 1 or 3 different from Group 2 at same time point, P<0.05. N.Ob/B.Pm (n/mm)1.4±0.3aGroup 1 or 3 different from Group 2 at same time point, P<0.05.1.7±0.4aGroup 1 or 3 different from Group 2 at same time point, P<0.05.12.4±2.310.1±2.67.1±1.0aGroup 1 or 3 different from Group 2 at same time point, P<0.05.,bGroup 3 different from Group 1 at same time point, P<0.05.3.5±0.9aGroup 1 or 3 different from Group 2 at same time point, P<0.05.,bGroup 3 different from Group 1 at same time point, P<0.05. Oc.S/BS (%)7.6±0.7aGroup 1 or 3 different from Group 2 at same time point, P<0.05.10.3±0.8aGroup 1 or 3 different from Group 2 at same time point, P<0.05.16.5±1.722.1±4.5aGroup 1 or 3 different from Group 2 at same time point, P<0.05.23.8±2.3aGroup 1 or 3 different from Group 2 at same time point, P<0.05.,bGroup 3 different from Group 1 at same time point, P<0.05.10.2±1.5aGroup 1 or 3 different from Group 2 at same time point, P<0.05. N.Oc/B.Pm (n/mm)1.5±0.1aGroup 1 or 3 different from Group 2 at same time point, P<0.05.2.3±0.1aGroup 1 or 3 different from Group 2 at same time point, P<0.05.3.2±0.34.1±0.64.3±0.3aGroup 1 or 3 different from Group 2 at same time point, P<0.05.,bGroup 3 different from Group 1 at same time point, P<0.05.2.2±0.2aGroup 1 or 3 different from Group 2 at same time point, P<0.05.Mineralization OV/BV (%)0.2±0.10.29±0.1aGroup 1 or 3 different from Group 2 at same time point, P<0.05.0.9±0.31.0±0.31.8±0.4bGroup 3 different from Group 1 at same time point, P<0.05.1.2±0.6 OS/BS (%)6.3±3.04.2±0.86.3±3.06.4±1.314.9±1.8bGroup 3 different from Group 1 at same time point, P<0.05.9.8±2.4Volume BV/TV (%)11.5±1.48.4±0.910.8±1.610.6±1.34.8±1.06.9±1.5 Tb.Th (μm)51.4±2.3aGroup 1 or 3 different from Group 2 at same time point, P<0.05.48.9±2.848±3.1aGroup 1 or 3 different from Group 2 at same time point, P<0.05.41.8±2.839.0±2.6aGroup 1 or 3 different from Group 2 at same time point, P<0.05.,bGroup 3 different from Group 1 at same time point, P<0.05.40.8±2.7 Tb.Sp (μm)427±50aGroup 1 or 3 different from Group 2 at same time point, P<0.05.571±44433±55405±611027±211aGroup 1 or 3 different from Group 2 at same time point, P<0.05.689±107aGroup 1 or 3 different from Group 2 at same time point, P<0.05. Tb.N (n/mm)2.2±0.21.6±0.1aGroup 1 or 3 different from Group 2 at same time point, P<0.05.2.2+0.22.5±0.21.2±0.21.6±0.2aGroup 1 or 3 different from Group 2 at same time point, P<0.05. Cross-sectional area (mm2)6.8±0.16.6±0.18.1±0.9aGroup 1 or 3 different from Group 2 at same time point, P<0.05.6.7±0.16.1+0.16.1±0.2aGroup 1 or 3 different from Group 2 at same time point, P<0.05.Static parameters: BV/TV, trabecular bone volume; Fb.V/TV, intertrabecular fibrosis; Ob.S/BS and Oc.S/BS, osteoblast and osteoclast surface; N.Ob/B.Pm and N.Oc/B.Pm, osteoblast and osteoclast number per millimeter bone perimeter; OV/BV, osteoid volume; OS/BS, osteoid surface; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Tb.N, trabecular number.Mean+s.e.m.a Group 1 or 3 different from Group 2 at same time point, P<0.05.b Group 3 different from Group 1 at same time point, P<0.05. Open table in a new tab Static parameters: BV/TV, trabecular bone volume; Fb.V/TV, intertrabecular fibrosis; Ob.S/BS and Oc.S/BS, osteoblast and osteoclast surface; N.Ob/B.Pm and N.Oc/B.Pm, osteoblast and osteoclast number per millimeter bone perimeter; OV/BV, osteoid volume; OS/BS, osteoid surface; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Tb.N, trabecular number. Mean+s.e.m. Thus, the Cy/+ animal model spontaneously reflects all three components of CKD-MBD: biochemical abnormalities, vascular calcification, and bone abnormalities. The biochemical and bone abnormalities show a progressive change over time, whereas the vascular calcification was only apparent in the older animals, perhaps due to more severe hyperphosphatemia and hyperparathyroidism. We previously noted (unpublished observations) that when our male Cy/+ CKD animals were treated with standard rat chow, in which protein content was not casein based, the magnitude of hyperparathyroidism was not as severe as with a casein-based diet. Therefore, we treated another group of animals on a grain-based diet. This was a commercially available diet (Purina 5002) that paralleled the protein and phosphorus content of the casein diet. Comparing the results from Cy/+ animals on the two diets (Table 3 ) demonstrated a marked difference in magnitude of hyperphosphatemia, hyperparathyroidism, and severity of CKD compared to the casein-based, 0.7% phosphorus diet-fed animals (whose biochemical data is also shown in Figure 1).Table 3Effect of dietary protein source in Cy/+ male animals with CKDCasein protein sourceGrain protein source34 weeks38 weeks34 weeks38 weeksBUN (mg/100 ml)107±13.4140±16.262±3.086±6.9Phosphorus (mg/100 ml)11.6±1.014.9±1.55.12±0.37.2±0.6Calcium (mg/100 ml)9.9±0.39.8±0.68.86±0.38.9±0.3Intact PTH (pg/ml)3543±6485151±1067303±116421±97BUN, blood urea nitrogen; PTH, parathyroid hormone.Mean±s.e.m., casein different than grain in all categories and all time points, P<0.01. Open table in a new tab BUN, blood urea nitrogen; PTH, parathyroid hormone. Mean±s.e.m., casein different than grain in all categories and all time points, P<0.01. To better understand how the protein source could cause such a marked difference, we conducted 1-week-long metabolic studies in 20-week-old male Cy/+ CKD rats. At baseline, before the diet intervention, the BUN levels were 38.0 mg/100 ml±1.4 in the animals on the casein diet, and 42.1 mg/100 ml±1.8 in the animals on the grain diet, which represents an approximate 50–60% reduction in GFR. After 1 week of diet intervention, there were no differences in serum phosphorus, calcium, or PTH values between the two groups, but the fibroblast growth factor 23 (FGF23) levels were higher in the CKD animals fed with a casein diet versus those fed a grain diet (P<0.001; Table 4 ). There was also increased urinary phosphate excretion in the casein compared to the grain diet, and an increase in the urine calcium/creatinine ratio (Table 4). Grain diets typically contain much of the phosphate source as phytate, which is poorly absorbed from the gastrointestinal tract. We thus assessed the diets for phytate content, demonstrating that 60% of the phosphate in the grain-based diet was in the non-bioavailable phytate form; whereas the casein diet was devoid of phytate, so all of the phosphate was bioavailable.Table 4Comparison of impact of diets in 20-week animalsCy/+ CKD animalsCasein (n=15)Grain (n=9)P-valueSerum Calcium (mg/100 ml)9.6±0.48.8±0.3NS Phosphorus (mg/100 ml)5.4±0.25.4±0.3NS Intact PTH (pg/ml) (median 25/75%)469±87347±56NS FGF23 (pg/ml) (median 25/75%)1254 (1183–1411)869 (799–903)<0.001Urine FE phosphorus (%) (median 25/75%)80.1 (67.2–116.724.2 (21.0–49.3)<0.001 Phosphorus (mg/24 h) (median 25/75%)76.3 (58.3–87.8)44.3 (39.4–49.0)<0.001 Calcium/creatinine ratio (mg/mg)0.21±0.020.12±0.020.013 Calcium (mg/24 h)3.2±1.12.9±1.2NSStool Calcium (mg/24 h)49.7 (38.9–44.8)74.0 (54.3–78.7)NS Phosphate (mg/24 h)33.1 (18.4–44.8)30.6 (12.4–44.3)NSCKD, chronic kidney disease; FE, fractional excretion; NS, not significant; PTH, parathyroid hormone.Mean±s.e.m. unless otherwise indicated. Open table in a new tab CKD, chronic kidney disease; FE, fractional excretion; NS, not significant; PTH, parathyroid hormone. Mean±s.e.m. unless otherwise indicated. In this study, we describe a novel animal model of spontaneous CKD-MBD. In previous rodent models of kidney disease, vascular calcification developed only on a high-phosphorus diet greater than 1%.6.Katsumata K. Kusano K. Hirata M. et al.Sevelamer hydrochloride prevents ectopic calcification and renal osteodystrophy in chronic renal failure rats.Kidney Int. 2003; 64: 441-450Google Scholar, 8.Cozzolino M. Dusso A.S. Liapis H. et al.The effects of sevelamer hydrochloride and calcium carbonate on kidney calcification in uremic rats.J Am Soc Nephrol. 2002; 13: 2299-2308Google Scholar, 15.Hirata M. Makibayashi K. Katsumata K. et al.22-Oxacalcitriol prevents progressive glomerulosclerosis without adversely affecting calcium and phosphorus metabolism in subtotally nephrectomized rats.Nephrol Dial Transplant. 2002; 17: 2132-2137Google Scholar The advantage of the Cy/+ rat includes the ability to induce all three components of CKD-MBD with normal (0.7%) dietary phosphorus concentration. The biochemical and bone abnormalities demonstrate a progressive course, whereas the calcification was only observed late in the disease course. The aorta vascular calcification was spontaneous and medial in location, paralleling findings in patients with CKD. Studies in humans demonstrate that abnormal biochemical changes16.Levin A. Bakris G.L. Molitch M. et al.Prevalence of abnormal serum vitamin D, PTH, calcium, and phosphorus in patients with chronic kidney disease: results of the study to evaluate early kidney disease.Kidney Int. 2007; 71: 31-38Google Scholar begin when GFR is around 40–50 ml/min (CKD 3–4), whereas bone17.Sprague S.M. The role of the bone biopsy in the diagnosis of renal osteodystrophy.Semin Dial. 2000; 13: 152-155Google Scholar and vascular calcification disorders1.Block G.A. Spiegel D.M. Ehrlich J. et al.Effects of sevelamer and calcium on coronary artery calcification in patients new to hemodialysis.Kidney Int. 2005; 68: 1815-1824Google Scholar, 13.Mehrotra R. Adler S. Coronary artery calcification in nondialyzed patients with chronic kidney diseases.Am J Kidney Dis. 2005; 45: 963Google Scholar, 14.Russo D. Miranda I. Ruocco C. et al.The progression of coronary artery calcification in predialysis patients on calcium carbonate or sevelamer.Kidney Int. 2007; 72: 1255-1261Google Scholar are more prevalent with CKD stages 4–5D. Thus, any effort to prevent CKD-MBD must first treat the biochemical abnormalities at this early stage. This Cy/+ rat model develops vascular calcification at 38 weeks, suggesting the presence of very severe and/or protracted hyperphosphatemia, and hyperparathyroidism are required to induce arterial calcification. CKD-MBD with arterial calcification in rodents is a rare finding. Taken together, this model allows a characterization of the progressive onset and course of CKD-MBD. This model also allows us to test interventions early and chronically to ensure that prevention of CKD-MBD can be done safely. To our knowledge, this represents the first animal model of CKD-MBD that occurs spontaneously (no surgery or drug) on a normal-phosphorus diet. Our study also demonstrates the importance of diet on CKD-MBD. Specifically, a low-phosphorus diet can s
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