The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification

Abstract

Ectopic calcification is a frequent complication of many degenerative diseases. Here we identify the serum protein alpha2-Heremans-Schmid glycoprotein (Ahsg, also known as fetuin-A) as an important inhibitor of ectopic calcification acting on the systemic level. Ahsg-deficient mice are phenotypically normal, but develop severe calcification of various organs on a mineral and vitamin D-rich diet and on a normal diet when the deficiency is combined with a DBA/2 genetic background. This phenotype is not associated with apparent changes in calcium and phosphate homeostasis, but with a decreased inhibitory activity of the Ahsg-deficient extracellular fluid on mineral formation. The same underlying principle may contribute to many calcifying disorders including calciphylaxis, a syndrome of severe systemic calcification in patients with chronic renal failure. Taken together, our data demonstrate a critical role of Ahsg as an inhibitor of unwanted mineralization and provide a novel therapeutic concept to prevent ectopic calcification accompanying various diseases.

Figure 1

A mineral/vitamin D–rich diet leads to ectopic calcification in Ahsg–/– mice. Ahsg+/+ and Ahsg–/– mice (mixed genetic background C57BL/6-129/Sv) were fed a mineral/vitamin D–rich diet for 4 months and analyzed for the presence of ectopic calcification. (a) Alizarin red staining of skeletal preparations. Note that calcified deposits depict small vessels in muscles along the vertebra in Ahsg–/– mice, but not in Ahsg+/+ mice. (b) von Kossa staining and counterstaining with safranin O of kidney and lung. Calcified lesions are stained black in renal tubules and interstitium and pulmonary alveoli of Ahsg–/– mice, but not in Ahsg+/+ mice.

Figure 2

Ahsg deficiency in DBA/2 mice leads to severe ectopic calcification on a normal diet. (a) Radiological analysis of 9-month-old male DBA/2-Ahsg+/+ and DBA/2-Ahsg–/– littermate mice. Note the punctuate soft tissue calcification of the thorax, kidneys, and testes in DBA/2-Ahsg–/– mice. (b) Histological analysis of 7-month-old DBA/2-Ahsg+/+ and DBA/2-Ahsg–/– mice. Plastic-embedded tissues were sectioned and prepared with von Kossa staining and H&E counterstaining. Note the positive staining of calcified lesions in DBA/2-Ahsg–/–, but not in littermate DBA/2-Ahsg+/+ mice. Lesions were extracellular in dilated tubules of kidney cortex tissue. Lesions were often associated with atrophied glomeruli. The myocardium (myocard) contained lesions with a fibrosis capsule. Lung tissue contained numerous spherical, scale-like, calcified deposits blocking alveoli. Subdermal adipose tissue contained large calcified nodules. (c) Electron micrograph of kidney tubule interstitium (top). The electron-dense mineral deposits were first detected in foam cell–like phagocytes infiltrating the extracellular space between tubular cells and the basement membrane. Energy dispersive x-ray analysis of the electron dark mineral deposit in these cells identified Ca and P as the major elements present in this sample (bottom).

Figure 3

The ectopic calcification in DBA/2-Ahsg–/– mice leads to high blood pressure, renal failure, and secondary hyperparathyroidism. (a) Blood pressure was recorded in 4- to 5-month-old Ahsg+/+ and Ahsg–/– mice on a C57BL/6 (B6) and a DBA/2 (D2) genetic background. Values are presented as mean ± SE in mmHg. Note that systolic blood pressure (white bars) and diastolic blood pressure (black bars) are significantly elevated (**P < 0.0001) in DBA/2-Ahsg–/– mice (n = 12) compared with pressures in DBA2/Ahsg+/+ (n = 13), C57BL/6-Ahsg–/– (n = 20), and C57BL/6-Ahsg+/+ mice (n = 20). (b) Macroscopic view of the kidney at 9 months of age shows severe calcification and hydronephrosis in DBA/2-Ahsg–/– mice but not in DBA2/Ahsg+/+ littermates (left). Urinary albumin was measured in Ahsg+/+ and Ahsg–/– mice on a C57BL/6 and a DBA/2 genetic background (right). Severe albuminuria was observed in DBA/2-Ahsg–/– mice. (c) Determination of serum concentrations of intact parathyroid hormone (iPTH) and the bone parameters bone volume per total volume (BV/TV), osteoblast number (ObN), and osteoclast number (OcN) in 9-month-old DBA/2-Ahsg–/– and DBA/2-Ahsg+/+ littermates (*P < 0.05). Note the presence of hyperparathyroidism, osteopenia, and an increased number of osteoclasts in DBA/2-Ahsg–/– mice (n = 6 for all parameters).

Figure 4

Calcification of Ahsg-deficient DBA/2 mice precedes kidney failure. (a) Plastic-embedded tissues of 1-month-old mice were sectioned and stained by von Kossa staining and counterstained with H&E. Calcification was detected in soft tissues including tongue, lung, myocardium, and kidney pelvis, but not yet in the kidney cortex. Note that even at 9 months of age, WT DBA/2 mice showed no calcification (see Figure 2b). (b) Radiological analysis detected progressive calcification of the kidneys and the skin. Note that the mice were alive at the time of radiography and therefore calcified heart and lung tissue was blurred due to movement. (c) Determination of serum phosphate, calcium, creatinine, and intact parathyroid hormone indicated that kidney failure in DBA/2-Ahsg–/– mice occurred after 4 months of age and therefore after the onset of soft tissue calcification (1 month, n = 4; 3–4 months, n = 6; over 6 months, n = 4). *P < 0.05, **P < 0.001.

Figure 5

Serum chemistry of Ahsg-deficient mice. (a) Serum electrolytes are given as the mean ± SE of at least six mice for each measurement (**P < 0.01). Where there are no error bars, the SEs were too small to be visible. (b) Sera from C57BL/6-Ahsg+/+ (filled circles) and DBA/2-Ahsg+/+ mice (filled squares) both inhibited the de novo formation of BCP from supersaturated solutions of calcium and phosphate with an IC₅₀ of 0.8% serum. Serum from C57BL/6-Ahsg–/– (open circles) and DBA/2-Ahsg–/– (open squares) mice inhibited at a much reduced rate (IC₅₀ 5.2% and 6.8% serum, respectively). (c) Reconstitution of BCP precipitation inhibition. Note that the reduced inhibition of BCP precipitation by sera from Ahsg-deficient mice could be restored to the WT level by adding back purified mouse serum Ahsg. (d) Serum electrolyte concentrations in normal and calciphylaxis patients (*P < 0.05). (e) Inhibition of BCP precipitation by sera from three healthy subjects (filled circles) and eight calciphylaxis patients (open circles). Note that the inhibition of BCP precipitation is greatly reduced in all calciphylaxis patients. (f) Serum reconstitution by purified human Ahsg in serum of a calciphylaxis patient. Note that the reduced inhibition of BCP precipitation by serum from the calciphylaxis patient could be restored to normal levels by adding back purified human serum Ahsg.