A rat model of chronic kidney disease-mineral bone disorder

Abstract

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.

Figures

Figure 1. Biochemical changes over time

Cy/+ rats were placed on the study diet at 20 weeks of age. The 38 week old animals had an intermediate blood draw at 34 weeks for phosphorus, parathyroid hormone, and calcium for a total of three time points, whereas BUN was measured at 20 weeks and 38 weeks. The graphs show changes in plasma levels over time for BUN (A), phosphorus (B), intact parathyroid hormone (C), and calcium (D). The data demonstrate that CKD animals fed a 0.7% phosphorus diet had progression of CKD as assessed by BUN, hyperphosphatemia, and hyperparathyroidism compared to the normal littermates. Feeding a low phosphorus diet (0.2%) ameliorated these changes. As detailed in the text, these changes over time were significant (p

Figure 1. Biochemical changes over time

Cy/+ rats were placed on the study diet at 20 weeks of age. The 38 week old animals had an intermediate blood draw at 34 weeks for phosphorus, parathyroid hormone, and calcium for a total of three time points, whereas BUN was measured at 20 weeks and 38 weeks. The graphs show changes in plasma levels over time for BUN (A), phosphorus (B), intact parathyroid hormone (C), and calcium (D). The data demonstrate that CKD animals fed a 0.7% phosphorus diet had progression of CKD as assessed by BUN, hyperphosphatemia, and hyperparathyroidism compared to the normal littermates. Feeding a low phosphorus diet (0.2%) ameliorated these changes. As detailed in the text, these changes over time were significant (p

Figure 1. Biochemical changes over time

Cy/+ rats were placed on the study diet at 20 weeks of age. The 38 week old animals had an intermediate blood draw at 34 weeks for phosphorus, parathyroid hormone, and calcium for a total of three time points, whereas BUN was measured at 20 weeks and 38 weeks. The graphs show changes in plasma levels over time for BUN (A), phosphorus (B), intact parathyroid hormone (C), and calcium (D). The data demonstrate that CKD animals fed a 0.7% phosphorus diet had progression of CKD as assessed by BUN, hyperphosphatemia, and hyperparathyroidism compared to the normal littermates. Feeding a low phosphorus diet (0.2%) ameliorated these changes. As detailed in the text, these changes over time were significant (p

Figure 1. Biochemical changes over time

Cy/+ rats were placed on the study diet at 20 weeks of age. The 38 week old animals had an intermediate blood draw at 34 weeks for phosphorus, parathyroid hormone, and calcium for a total of three time points, whereas BUN was measured at 20 weeks and 38 weeks. The graphs show changes in plasma levels over time for BUN (A), phosphorus (B), intact parathyroid hormone (C), and calcium (D). The data demonstrate that CKD animals fed a 0.7% phosphorus diet had progression of CKD as assessed by BUN, hyperphosphatemia, and hyperparathyroidism compared to the normal littermates. Feeding a low phosphorus diet (0.2%) ameliorated these changes. As detailed in the text, these changes over time were significant (p

Figure 2. Thoracic aorta calcification

At the time of sacrifice, the thoracic aorta was removed and its calcium content determined biochemically. The results demonstrate that there is increased arterial calcification in the normal (0.7%) phosphorus treated CKD animals at 38 weeks, but not at 34 weeks. This implies that hyperphosphatemia and hyperparathyroidism precede arterial calcification. Interestingly, the low phosphorus diet (0.2%), despite producing modest hyperphosphatemia and hyperparathyroidism, did not increase calcification in CKD animals compared to the normal littermates. Solid black bars= normal littermates; open white bars = CKD (Cy/+) animals fed a 0.7% phosphorus casein-based diet; hatched bars = CKD animals fed a 0.2% phosphorus casein-based diet. Symbols: * different than normal littermates (p

Figure 3. Histologic 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. B: C and D. one from a CKD animal fed 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.

Figure 3. Histologic 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. B: C and D. one from a CKD animal fed 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.