Upregulation of alkaline phosphatase and pyrophosphate hydrolysis: potential mechanism for uremic vascular calcification

Abstract

Pyrophosphate is a potent inhibitor of medial vascular calcification where its level is controlled by hydrolysis via a tissue-nonspecific alkaline phosphatase (TNAP). We sought to determine if increased TNAP activity could explain the pyrophosphate deficiency and vascular calcification seen in renal failure. TNAP activity increased twofold in intact aortas and in aortic homogenates from rats made uremic by feeding adenine or by 5/6 nephrectomy. Immunoblotting showed an increase in protein abundance but there was no increase in TNAP mRNA assessed by quantitative polymerase chain reaction. Hydrolysis of pyrophosphate by rat aortic rings was inhibited about half by the nonspecific alkaline phosphatase inhibitor levamisole and was reduced about half in aortas from mice lacking TNAP. Hydrolysis was increased in aortic rings from uremic rats and all of this increase was inhibited by levamisole. An increase in TNAP activity and pyrophosphate hydrolysis also occurred when aortic rings from normal rats were incubated with uremic rat plasma. These results suggest that a circulating factor causes pyrophosphate deficiency by regulating TNAP activity and that vascular calcification in renal failure may result from the action of this factor. If proven by future studies, this mechanism will identify alkaline phosphatase as a potential therapeutic target.

Figure 1. Alkaline phosphatase activity in aortas from uremic (filled bars) and control pair-fed rats (open bars)

(a) Hydrolysis of p-nitrophenylphosphate in homogenates of aortas from adenine-fed rats, expressed as mg of protein. (b) Hydrolysis of p-nitrophenylphosphate in homogenates of aortas from nephrectomized rats, expressed as mg protein. (c) Hydrolysis of p-nitrophenylphosphate by intact, freshly isolated aortas from adenine-fed rats, expressed as mg dry weight. Results are the means+s.e. of the number of animals indicated in parentheses. *P<0.0001; **P<0.002 vs control.

Figure 2. Immunoblot of extracts from control and uremic aortas using a polyclonal antibody against TNAP

Also shown are bone extracts from wild-type (TNAP+/+) mice and mice lacking TNAP. Each sample was 50 μg of protein.

Figure 3. Hydrolysis of PPi by rat aorta

(a) Time course showing representative results from a single uremic rat (5/6 nephrectomy) and a control rat. (b) Hydrolysis rate in aortas from uremic (filled bars) and control pair-fed rats (open bars). Results are the means+s.e. of the number of animals indicated in parentheses. *P<0.0001; **P<0.005 vs control.

Figure 4. Effect of levamisole on alkaline phosphatase and PPi hydrolysis in intact rat aorta

(a) Alkaline phosphatase activity in aortic rings from normal rats at different concentrations of levamisole, expressed per mg dry weight. (b) Hydrolysis of PPi by aortas from uremic and control pair-fed rats in the absence (black bars) and presence (gray bars) of 1 mm levamisole. Results are the means+s.e. of seven control and five uremic aortas.

Figure 5. Hydrolysis of PPi in aortas from mice lacking tissue-nonselective alkaline phosphatase (Akp2−/− mice; solid circles) from heterozygote mice (open circles), and wild-type mice (triangles)

Each line represents a single aorta.

Figure 6. Effect of uremic plasma on cultured rat aortas

Aortic rings were cultured for 9 days in 10% plasma from control (open bars) or uremic (filled bars) rats. (a) Alkaline phosphatase activity in homogenates expressed per mg protein. (b) Hydrolysis of PPi expressed per dry weight. Results are the means+s.e. of the number of animals indicated in parentheses. *P<0.0001; **P<0.005 vs control.