Early chronic kidney disease-mineral bone disorder stimulates vascular calcification

Abstract

The chronic kidney disease-mineral and bone disorder (CKD-MBD) syndrome is an extremely important complication of kidney diseases. Here we tested whether CKD-MBD causes vascular calcification in early kidney failure by developing a mouse model of early CKD in a background of atherosclerosis-stimulated arterial calcification. CKD equivalent in glomerular filtration reduction to human CKD stage 2 stimulated early vascular calcification and inhibited the tissue expression of α-klotho (klotho) in the aorta. In addition, osteoblast transition in the aorta was stimulated by early CKD as shown by the expression of the critical transcription factor Runx2. The ligand associated with the klotho-fibroblast growth factor receptor complex, FGF23, was found to be expressed in the vascular media of sham-operated mice. Its expression was decreased in early CKD. Increased circulating levels of the osteocyte-secreted proteins, FGF23, and sclerostin may have been related to increased circulating klotho levels. Finally, we observed low-turnover bone disease with a reduction in bone formation rates more than bone resorption. Thus, the CKD-MBD, characterized by cardiovascular risk factors, vascular calcification, increased circulating klotho, FGF23 and sclerostin levels, and low-turnover renal osteodystrophy, was established in early CKD. Early CKD caused a reduction of vascular klotho, stimulated vascular osteoblastic transition, increased osteocytic secreted proteins, and inhibited skeletal modeling producing the CKD-MBD.

Figure 1. A Schematic Drawing of the Experimental Design Defining the Various Animal Groups

WT, wild type C57B6J mice were used to establish normative parameters; SHAM, ldlr−/− mice on a high fat diet (d) undergoing sham operations (SO) are the control mice for the effects of CKD; CKD-2, ldlr−/− high fat fed mice undergoing mild renal injury (EC) and contralateral nephrectomy (NX) with euthanasia at 22 weeks; CKD-2-28, same as CKD-2 but euthanasia at 28 weeks, (six additional weeks of slowly progressive CKD); CKD-3, ldlr−/− high fat fed mice undergoing moderate renal injury (EC*) and contralateral nephrectomy (NX) with euthanasia at 22 weeks; CKD-3-28, same as CKD-3 but euthanasia at 28 weeks.

Figure 2. Inulin clearances in ldlr−/− high fat fed mice

Sham operated mice had inulin clearances of 0.006mls/min/g. The mild renal injury group (CKD-2) had reductions in inulin clearance (GFR) up to 40% (mean 33%) of the sham operated control levels,. The BUN levels were not different between sham operated and CKD-2 mice, More severe renal injury (CKD-3) produced inulin clearance reductions of 75% in CKD-3 mice and elevations in BUN levels to the 45mg/dl range. Inulin clearances and BUN levels were determined at 22 weeks of age.

Figure 3. Longitudinal analysis of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) levels in ldlr−/− high fat fed mice with CKD-2 and CKD-2-28, and FGF23 levels in ldlr−/− high fat fed mice with CKD-2-28 and CKD-3-28. A and B

CKD was established at 14 weeks of age as described in methods and Fig.1. Plasma was obtained at 15, 22, and 28 weeks. FGF23 was measured using an intact hormone assay (Kainos). C and D, Two different Elisa assays for FGF23, an intact hormone assay and a C-terminal assay, as described in methods, were used to determine the effects of CKD on FGF23 levels in the circulation. FGF23 levels in C57Bl6J wild type mice were measured to establish the reference range. FGF23 levels measured using the intact hormone assay were increased in CKD-3-28 mice compared to CKD-2-28, but not when the C-terminal assay was used.

Figure 3. Longitudinal analysis of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) levels in ldlr−/− high fat fed mice with CKD-2 and CKD-2-28, and FGF23 levels in ldlr−/− high fat fed mice with CKD-2-28 and CKD-3-28. A and B

CKD was established at 14 weeks of age as described in methods and Fig.1. Plasma was obtained at 15, 22, and 28 weeks. FGF23 was measured using an intact hormone assay (Kainos). C and D, Two different Elisa assays for FGF23, an intact hormone assay and a C-terminal assay, as described in methods, were used to determine the effects of CKD on FGF23 levels in the circulation. FGF23 levels in C57Bl6J wild type mice were measured to establish the reference range. FGF23 levels measured using the intact hormone assay were increased in CKD-3-28 mice compared to CKD-2-28, but not when the C-terminal assay was used.

Figure 4. Aortic calcium levels are increased by CKD stage 2-28 in ldlr−/− high fat fed mice

A, alizarin red staining revealed questionable early aortic calcium deposition along elastic laminae (arrows) in ldlr−/− high fat fed mice with CKD-2-28 compared to sham operated mice. Von Kossa staining of adjacent sections (not shown) was negative. B, CKD-2-28 significantly increased aortic Ca content.

Figure 5. Osteoblastic transition and detection of FGF-23 and klotho expression in aortas of CKD -2-28 ldlr−/− mice

Real time PCR (A &B) and western blotting were used to measure Runx2, klotho (A) and FGF 23 (B) expression in aortas of sham and CKD-2-28 mice. Immunolocalization of aortic FGF23 and klotho (C). Plasma levels of circulating klotho (c klotho) (D). A, CKD-2-28 increased Runx2 expression and decreased α-Klotho expression compared to sham operated controls determined by real time RT-PCR (left) and by Western (right). n = 5 for RT-PCR. The representative Westerns were uniform for five CKD-2-28 mice. FGF23 was expressed in the sham operated control mice and decreased with induction of CKD-2-28. n = 5 for RT-PCR. The representative Westerns were uniform for five CKD-2-28 mice. C, Antibodies to FGF23 and Klotho, as described in Methods, were used for detection of expression in the aortas of the various groups of mice. FGF23 was expressed in occasional cells in the media (two representative sham mice are shown) which was absent in CKD-2-28 mice. A strong nonspecific advential reaction was increased in the CKD-2-28 mice of unknown nature. α-Klotho was strongly expressed in the aortic media of wild type and sham operated mice, but severely reduced in the stage 2 CKD mice. IHC for α-Klotho was uniform in five WT, Sham, and CKD-2 mice. Use of a nonspecific IgG in place of the primary antibody produced nonspecific positivity by both the secondary antibodies for both FGF23 and klotho. D, Plasma c klotho levels were increased several fold in the CKD-2 mice at 15 and 22wks.

Figure 5. Osteoblastic transition and detection of FGF-23 and klotho expression in aortas of CKD -2-28 ldlr−/− mice

Real time PCR (A &B) and western blotting were used to measure Runx2, klotho (A) and FGF 23 (B) expression in aortas of sham and CKD-2-28 mice. Immunolocalization of aortic FGF23 and klotho (C). Plasma levels of circulating klotho (c klotho) (D). A, CKD-2-28 increased Runx2 expression and decreased α-Klotho expression compared to sham operated controls determined by real time RT-PCR (left) and by Western (right). n = 5 for RT-PCR. The representative Westerns were uniform for five CKD-2-28 mice. FGF23 was expressed in the sham operated control mice and decreased with induction of CKD-2-28. n = 5 for RT-PCR. The representative Westerns were uniform for five CKD-2-28 mice. C, Antibodies to FGF23 and Klotho, as described in Methods, were used for detection of expression in the aortas of the various groups of mice. FGF23 was expressed in occasional cells in the media (two representative sham mice are shown) which was absent in CKD-2-28 mice. A strong nonspecific advential reaction was increased in the CKD-2-28 mice of unknown nature. α-Klotho was strongly expressed in the aortic media of wild type and sham operated mice, but severely reduced in the stage 2 CKD mice. IHC for α-Klotho was uniform in five WT, Sham, and CKD-2 mice. Use of a nonspecific IgG in place of the primary antibody produced nonspecific positivity by both the secondary antibodies for both FGF23 and klotho. D, Plasma c klotho levels were increased several fold in the CKD-2 mice at 15 and 22wks.

Figure 5. Osteoblastic transition and detection of FGF-23 and klotho expression in aortas of CKD -2-28 ldlr−/− mice

Real time PCR (A &B) and western blotting were used to measure Runx2, klotho (A) and FGF 23 (B) expression in aortas of sham and CKD-2-28 mice. Immunolocalization of aortic FGF23 and klotho (C). Plasma levels of circulating klotho (c klotho) (D). A, CKD-2-28 increased Runx2 expression and decreased α-Klotho expression compared to sham operated controls determined by real time RT-PCR (left) and by Western (right). n = 5 for RT-PCR. The representative Westerns were uniform for five CKD-2-28 mice. FGF23 was expressed in the sham operated control mice and decreased with induction of CKD-2-28. n = 5 for RT-PCR. The representative Westerns were uniform for five CKD-2-28 mice. C, Antibodies to FGF23 and Klotho, as described in Methods, were used for detection of expression in the aortas of the various groups of mice. FGF23 was expressed in occasional cells in the media (two representative sham mice are shown) which was absent in CKD-2-28 mice. A strong nonspecific advential reaction was increased in the CKD-2-28 mice of unknown nature. α-Klotho was strongly expressed in the aortic media of wild type and sham operated mice, but severely reduced in the stage 2 CKD mice. IHC for α-Klotho was uniform in five WT, Sham, and CKD-2 mice. Use of a nonspecific IgG in place of the primary antibody produced nonspecific positivity by both the secondary antibodies for both FGF23 and klotho. D, Plasma c klotho levels were increased several fold in the CKD-2 mice at 15 and 22wks.

Figure 5. Osteoblastic transition and detection of FGF-23 and klotho expression in aortas of CKD -2-28 ldlr−/− mice

Real time PCR (A &B) and western blotting were used to measure Runx2, klotho (A) and FGF 23 (B) expression in aortas of sham and CKD-2-28 mice. Immunolocalization of aortic FGF23 and klotho (C). Plasma levels of circulating klotho (c klotho) (D). A, CKD-2-28 increased Runx2 expression and decreased α-Klotho expression compared to sham operated controls determined by real time RT-PCR (left) and by Western (right). n = 5 for RT-PCR. The representative Westerns were uniform for five CKD-2-28 mice. FGF23 was expressed in the sham operated control mice and decreased with induction of CKD-2-28. n = 5 for RT-PCR. The representative Westerns were uniform for five CKD-2-28 mice. C, Antibodies to FGF23 and Klotho, as described in Methods, were used for detection of expression in the aortas of the various groups of mice. FGF23 was expressed in occasional cells in the media (two representative sham mice are shown) which was absent in CKD-2-28 mice. A strong nonspecific advential reaction was increased in the CKD-2-28 mice of unknown nature. α-Klotho was strongly expressed in the aortic media of wild type and sham operated mice, but severely reduced in the stage 2 CKD mice. IHC for α-Klotho was uniform in five WT, Sham, and CKD-2 mice. Use of a nonspecific IgG in place of the primary antibody produced nonspecific positivity by both the secondary antibodies for both FGF23 and klotho. D, Plasma c klotho levels were increased several fold in the CKD-2 mice at 15 and 22wks.

Figure 6. Cortical bone microCT in ldlr−/− high fat fed mice with CKD stage 2

There was a decline in TV and in bone mineral density (BMD) in the CKD-2 mice. n = 10