High phosphate intake induces bone loss in nephrectomized thalassemic mice

Abstract

Although patients with either β-thalassemia or chronic kidney disease (CKD) clinically correlate with severe osteoporosis, the mechanism by which CKD exposed to high phosphate affects bone turnover has not been characterized in β-thalassemia. We aimed to determine the effects of renal insufficiency on high phosphate intake induced changes in bone metabolism after 5/6th nephrectomy in hemizygous β-globin knockout (BKO) mice. Male BKO mice manifested severe anemia and osteopenia. Nephrectomy induced renal fibrosis and reduced renal function as assessed by increased serum urea nitrogen levels. Moreover, nephrectomy increased bone turnover leading to bone loss in wild type (WT) but not BKO mice. In nephrectomized BKO, PBS in drinking water induced hyperphosphatemia, and hypercalcemia along with osteopenia in both cancellous and cortical bone. Histomorphometric analysis confirmed reduced cancellous bone volume due to decreased bone formation rate, osteoblast number and osteoclast number. The mRNA levels for Alpl, Sp7, Kl, Tnfsf11, and Tnfsf11/Tnfrsf11b were decreased in nephrectomized BKO mice drinking PBS. Interestingly, Fgf23, a bone-derived hormone produced by osteocytes and osteoblasts in response to hyperphosphatemia, were remarkably increased in nephrectomized BKO mice following PBS intake. Serum FGF23 and erythropoietin levels were markedly elevated in BKO mice. Nephrectomy decreased serum erythropoietin but not FGF23 levels. Hyperphosphatemia in BKO mice increased serum erythropoietin, FGF23, and PTH levels, nominating these factors as candidate mediators of bone loss in thalassemic mice with CKD during phosphate retention.

Fig 1. High phosphate enhances renal fibrosis in nephrectomized WT and BKO mice.

(A) Kidneys stained with Masson’s Trichrome (n = 3 per group) from sham WT, nephrectomized WT, nephrectomized WT+PBS, sham BKO, nephrectomized BKO, and nephrectomized BKO+PBS mice. Blue color indicates fibrosis with an accumulation of collagen. (B) Violin plot showing IFTA scores in the kidneys. ^ap < 0.05 versus sham WT, ^bp < 0.05 versus nephrectomized WT, ^cp < 0.05 versus nephrectomized WT+PBS, ^dp < 0.05 versus sham BKO, and ^ep < 0.05 versus nephrectomized BKO. Scale bar: 100 μm.

Fig 2. Nephrectomy increases serum urea nitrogen and high phosphate enhances serum phosphorus and calcium in nephrectomized BKO mice.

(A) Serum urea nitrogen from sham WT (n = 8), nephrectomized WT (n = 8), nephrectomized WT+PBS (n = 8), sham BKO (n = 7), nephrectomized BKO (n = 8), and nephrectomized BKO+PBS mice (n = 5). (B) Serum creatinine from sham WT (n = 5), nephrectomized WT (n = 8), nephrectomized WT+PBS (n = 4), sham BKO (n = 6), nephrectomized BKO (n = 8), and nephrectomized BKO+PBS mice (n = 3). (C) Serum phosphorus from sham WT (n = 8), nephrectomized WT (n = 8), nephrectomized WT+PBS (n = 8), sham BKO (n = 7), nephrectomized BKO (n = 8), and nephrectomized BKO+PBS mice (n = 6). (D) Serum calcium from sham WT (n = 8), nephrectomized WT (n = 8), nephrectomized WT+PBS (n = 8), sham BKO (n = 7), nephrectomized BKO (n = 8), and nephrectomized BKO+PBS mice (n = 6). ^ap < 0.05 versus sham WT, ^bp < 0.05 versus nephrectomized WT, ^cp < 0.05 versus nephrectomized WT+PBS, ^dp < 0.05 versus sham BKO, and ^ep < 0.05 versus nephrectomized BKO.

Fig 3. High phosphate induces cancellous bone loss in nephrectomized BKO mice.

(A) Representative μCT images of cancellous bone. (B) μCT analysis of cancellous bone as violin plots. ^ap < 0.05 versus sham WT, ^bp < 0.05 versus nephrectomized WT, ^cp < 0.05 versus nephrectomized WT+PBS, ^dp < 0.05 versus sham BKO, and ^ep < 0.05 versus nephrectomized BKO.

Fig 4. High phosphate increases cortical bone loss in nephrectomized BKO mice.

(A) Representative μCT images of cortical bone. (B) μCT analysis of cortical bone as violin plots. ^ap < 0.05 versus sham WT, ^bp < 0.05 versus nephrectomized WT, ^cp < 0.05 versus nephrectomized WT+PBS, ^dp < 0.05 versus sham BKO, and ^ep < 0.05 versus nephrectomized BKO.

Fig 5. Bone histomorphometric analysis of femurs in BKO mice and WT controls after nephrectomy and PBS exposure.

^ap < 0.05 versus sham WT, ^bp < 0.05 versus nephrectomized WT, ^cp < 0.05 versus nephrectomized WT+PBS, ^dp < 0.05 versus sham BKO, and ^ep < 0.05 versus nephrectomized BKO.

Fig 6. Nephrectomy of WT with or without PBS exposure upregulates osteoblast-related gene expression in femurs.

Violin plots for qPCR analysis of osteoblast-related genes (n = 4–5 per group). ^ap < 0.05 versus sham WT, ^bp < 0.05 versus sham BKO, ^cp < 0.05 versus nephrectomized WT, ^dp < 0.05 versus nephrectomized BKO, and ^ep < 0.05 versus nephrectomized WT+PBS.

Fig 7. Nephrectomy in WT with or without PBS exposure upregulates osteoclast-related gene expression in femurs.

Violin plots for qPCR analysis of osteoclast-related genes (n = 4–5 per group). ^ap < 0.05 versus sham WT, ^bp < 0.05 versus sham BKO, ^cp < 0.05 versus nephrectomized WT, ^dp < 0.05 versus nephrectomized BKO, and ^ep < 0.05 versus nephrectomized WT+PBS.

Fig 8. High phosphate increases serum erythropoietin, FGF23, and PTH levels in nephrectomized BKO mice.

(A) Serum erythropoietin from sham WT (n = 7), nephrectomized WT (n = 5), nephrectomized WT+PBS (n = 8), sham BKO (n = 5), nephrectomized BKO (n = 4), and nephrectomized BKO+PBS mice (n = 3). (B) Serum FGF23 from sham WT (n = 7), nephrectomized WT (n = 7), nephrectomized WT+PBS (n = 8), sham BKO (n = 5), nephrectomized BKO (n = 5), and nephrectomized BKO+PBS mice (n = 6). (C) Serum PTH from sham WT (n = 8), nephrectomized WT (n = 9), nephrectomized WT+PBS (n = 7), sham BKO (n = 6), nephrectomized BKO (n = 8), and nephrectomized BKO+PBS mice (n = 5). ^ap < 0.05 versus sham WT, ^bp < 0.05 versus nephrectomized WT, ^cp < 0.05 versus nephrectomized WT+PBS, ^dp < 0.05 versus sham BKO, and ^ep < 0.05 versus nephrectomized BKO.