W. Charles O'Neill

Novel inhibitors of alkaline phosphatase suppress vascular smooth muscle cell calcification.

We report three novel inhibitors of the physiological pyrophosphatase activity of alkaline phosphatase and show that these compounds are capable of reducing calcification in two models of vascular calcification (i.e., they suppress in vitro calcification by cultured Enpp1−/− VSMCs and they inhibit the increased pyrophosphatase activity in a rat aortic model).

Sonographic evaluation of renal failure

Sonography is a critical component of the evaluation of both acute and chronic renal failure; however, most nephrologists have a limited knowledge of this procedure. The acoustic properties, limited spectrum of pathological changes, and ease of visualization of the kidneys, coupled with the safety, simplicity, and low cost of sonography, make it the modality of choice for renal imaging. This review discusses the basics of sonography as they apply to the kidney and describes the findings encountered in the more common causes of renal failure. Although many sonographic findings are nonspecific, their diagnostic use is greatly enhanced by a familiarity with the clinical presentation and a thorough understanding of renal pathophysiological characteristics. Therefore, nephrologists should be knowledgeable about renal sonography and participate in its interpretation.

Pathophysiological Role of Vascular Smooth Muscle Alkaline Phosphatase in Medial Artery Calcification

Medial vascular calcification (MVC) is a pathological phenomenon that causes vascular stiffening and can lead to heart failure; it is common to a variety of conditions, including aging, chronic kidney disease, diabetes, obesity, and a variety of rare genetic diseases. These conditions share the common feature of tissue‐nonspecific alkaline phosphatase (TNAP) upregulation in the vasculature. To evaluate the role of TNAP in MVC, we developed a mouse model that overexpresses human TNAP in vascular smooth muscle cells in an X‐linked manner. Hemizygous overexpressor male mice (Tagln‐Cre+/–; HprtALPL/Y or TNAP‐OE) show extensive vascular calcification, high blood pressure, and cardiac hypertrophy, and have a median age of death of 44 days, whereas the cardiovascular phenotype is much less pronounced and life expectancy is longer in heterozygous (Tagln‐Cre+/–; HprtALPL/−) female TNAP‐OE mice. Gene expression analysis showed upregulation of osteoblast and chondrocyte markers and decreased expression of vascular smooth muscle markers in the aortas of TNAP‐OE mice. Through medicinal chemistry efforts, we developed inhibitors of TNAP with drug‐like pharmacokinetic characteristics. TNAP‐OE mice were treated with the prototypical TNAP inhibitor SBI‐425 or vehicle to evaluate the feasibility of TNAP inhibition in vivo. Treatment with this inhibitor significantly reduced aortic calcification and cardiac hypertrophy, and extended lifespan over vehicle‐treated controls, in the absence of secondary effects on the skeleton. This study shows that TNAP in the vasculature contributes to the pathology of MVC and that it is a druggable target.

Regulation by cell volume of Na+-K+-2Cl− cotransport in vascular endothelial cells: role of protein phosphorylation

SummaryNa+-K+-2Cl− cotransport in aortic endothelial cells is activated by cell shrinkage, inhibited by cell swelling, and is responsible for recovery of cell volume. The role of protein phosphorylation in the regulation of cotransport was examined with two inhibitors of protein phosphatases, okadaic acid and calyculin, and a protein kinase inhibitor, K252a. Both phosphatase inhibitors stimulated cotransport in isotonic medium, with calyculin, a more potent inhibitor of protein phosphatase I, being 50-fold more potent. Neither agent stimulated cotransport in hypertonic medium. Stimulation by calyculin was immediate and was complete by 5 min, with no change in cell Na + K content, indicating that the stimulation of cotransport was not secondary to cell shrinkage. The time required for calyculin to activate cotransport was longer in swollen cells than in normal cells, indicating that the phosphorylation step is affected by cell volume. Activation of cotransport when cells in isotonic medium were placed in hypertonic medium was more rapid than the inactivation of cotransport when cells in hypertonic medium were placed in isotonic medium, which is consistent with a shrinkage-activated kinase rather than a shrinkage-inhibited phosphatase. K252a, a nonspecific protein kinase inhibitor, reduced cotransport in both isotonic and hypertonic media. The rate of inactivation was the same in either medium, indicating that dephosphorylation is not regulated by cell volume. These results demonstrate that Na+-K+-2Cl− cotransport is activated by protein phosphorylation and is inactivated by a Type I protein phosphatase. The regulation of cotransport by cell volume is due to changes in the rate of phosphorylation rather than dephosphorylation, suggesting the existence of a volume-sensitive protein kinase. Both the kinase and the phosphatase are constitutively active, perhaps to allow for rapid changes in cotransport activity.

Vascular calcification is dependent on plasma levels of pyrophosphate

Plasma levels of pyrophosphate, an endogenous inhibitor of vascular calcification, are reduced in end-stage renal disease and correlate inversely with arterial calcification. However, it is not known whether the low plasma levels are directly pathogenic or are merely a marker of reduced tissue levels. This was tested in an animal model in which aortas were transplanted between normal mice and Enpp1−/− mice lacking ectonucleotide pyrophosphatase phosphodiesterase, the enzyme that releases extracellular pyrophosphate. Enpp1−/− mice had very low plasma pyrophosphate and developed aortic calcification by 2 months that was greatly accelerated with a high-phosphate diet. Aortas of Enpp1−/− mice showed no further calcification after transplantation into wild type mice fed a high phosphate diet. Aorta allografts of wild type mice calcified in Enpp1−/− mice but less so than the adjacent recipient Enpp1−/− aorta. Donor and recipient aortic calcium contents did not differ in transplants between wild type and Enpp1−/− mice, demonstrating that transplantation per se did not affect calcification. Histology revealed medial calcification with no signs of rejection. Thus, normal levels of extracellular pyrophosphate are sufficient to prevent vascular calcification and systemic Enpp1 deficiency is sufficient to produce vascular calcification despite normal vascular extracellular pyrophosphate production. This establishes an important role for circulating extracellular pyrophosphate in preventing vascular calcification.

Regulation of vascular smooth muscle cell calcification by extracellular pyrophosphate homeostasis: synergistic modulation by cyclic AMP and hyperphosphatemia

Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate (PP(i)), and regulated generation and homeostasis of extracellular PP(i) is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular PP(i) generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular PP(i) mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate (P(i)) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular PP(i). Attenuated PP(i) accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades PP(i). Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular P(i) alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated P(i) induced decreases in the ATP release pathway(s) that supports ENPP1 activity; this resulted in markedly reduced rates of PP(i) accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) alpha-actin, SM22alpha, and calponin. Notably, addition of exogenous ATP (or PP(i) per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular PP(i) generation mediated by both ANK- and ATP release-dependent mechanisms serves as a critical regulator of VSMC calcification.

Bedside renal biopsy: Ultrasound guidance by the nephrologist

The safety and efficacy of percutaneous biopsy of native kidneys performed entirely by nephrologists at the patients bedside was evaluated in 101 consecutive patients. The location and depth of the kidney were determined with a portable ultrasound machine, and biopsy was performed with a 15G, automatic, spring-loaded biopsy device without direct ultrasonographic guidance. Renal tissue was obtained in 99 patients, and all samples were adequate for diagnosis, with an average of 33 glomeruli and more than 10 glomeruli in 97%. The number of biopsy attempts was four or fewer in 80% of patients. Three patients developed symptomatic bleeding, all of whom had a risk factor for bleeding, but none required procedures to control the bleeding. Asymptomatic hematuria occurred in two other patients. Overall, the mean decrease in hematocrit was 1.5, with a decrease of 5.0 or greater in six patients. The results are similar to those of previous studies using automatic devices but under direct ultrasound guidance. A subset of 20 patients with abnormal platelet counts, coagulation times, or bleeding times accounted for four of the five patients with complications. We conclude that percutaneous biopsy of native kidneys can be adequately and safely performed in its entirety by nephrologists at the patients bedside. Furthermore, excellent results can be obtained without direct sonographic guidance. Hemorrhage occurs almost exclusively in those patients with abnormal platelet counts, coagulation times, or bleeding times.

Autocrine ATP release coupled to extracellular pyrophosphate accumulation in vascular smooth muscle cells

Extracellular inorganic pyrophosphate (PP(i)) is a potent suppressor of physiological calcification in bone and pathological calcification in blood vessels. Ectonucleotide pyrophosphatase/phosphodiesterases (eNPPs) generate PP(i) via the hydrolysis of ATP released into extracellular compartments by poorly understood mechanisms. Here we report that cultured vascular smooth muscle cells (VSMC) from rat aorta generate extracellular PP(i) via an autocrine mechanism that involves ATP release tightly coupled to eNPP activity. The nucleotide analog beta,gamma-methylene ATP (MeATP or AMPPCP) was used to selectively suppress ATP metabolism by eNPPs but not the CD39-type ecto-ATPases. In the absence of MeATP, VSMC generated extracellular PP(i) to accumulate >or=600 nM within 2 h while steadily maintaining extracellular ATP at 1 nM. Conversely, the presence of MeATP completely suppressed PP(i) accumulation while increasing ATP accumulation. Probenecid, which inhibits PP(i) efflux dependent on ANK, a putative PP(i) transporter or transport regulator, reduced extracellular PP(i) accumulation by approximately twofold. This indicates that autocrine ATP release coupled to eNPP activity comprises >or=50% of the extracellular PP(i)-generating capacity of VSMC. The accumulation of extracellular PP(i) and ATP was markedly attenuated by reduced temperature but was insensitive to brefeldin A, which suppresses constitutive exocytosis of Golgi-derived secretory vesicles. The magnitude of extracellular PP(i) accumulation in VSMC cultures increased with time postplating, suggesting that ATP release coupled to PP(i) generation is upregulated as cultured VSMC undergo contact-inhibition of proliferation or deposit extracellular matrix.

Recent progress in the treatment of vascular calcification

Vascular calcification is common in patients with advanced chronic kidney disease and is associated with poorer outcomes. Although the pathophysiology is not completely understood, it is clear that it is a multifactorial process involving altered mineral metabolism, as well as changes in systemic and local factors that can promote or inhibit vascular calcification, and all of these are potential therapeutic targets. Current therapy is closely linked to strategies for preventing disordered bone and mineral metabolism in advanced kidney disease and involves lowering the circulating levels of both phosphate and calcium. The efficacy of compounds that specifically target calcification, such as bisphosphonates and thiosulfate, has been shown in animals but only in small numbers of humans, and safety remains an issue. Additional therapies, such as pyrophosphate, vitamin K, and lowering of pH, are supported by animal studies, but are yet to be investigated clinically. As the mineral composition of vascular calcifications is the same as in bone, potential effects on bone must be addressed with any therapy for vascular calcification.

How echogenic is echogenic? Quantitative acoustics of the renal cortex

The echogenicity of the cortex is an important parameter in interpreting renal sonograms that suggest changes in cortical structure. Echogenicity is currently measured qualitatively, and no attempts have been made at quantification. We developed a method to quantify renal cortical echogenicity in reference to the liver and evaluated its reproducibility, dependence on scanning variables, and potential utility. Sonograms of the right kidney were digitized, and the mean pixel density of regions of the renal cortex and liver was measured and normalized to the gray scale. Echogenicity was expressed as the ratio of the brightness (inverse of mean pixel density) of the cortex to that of the liver. The mean coefficient of variation among measurements performed on multiple sonograms from the same study was 2.8%, and the coefficient of variation among multiple measurements performed on the same kidney over 1 year was 1.8%. The correlation between measurements obtained by two different individuals on identical images was 0.92, with a mean variation of 3.0%. Echogenicity was not significantly affected by type of scanner or probe frequency, but varied inversely with gain. However, the effect of gain was very small within the useful range. Water loading after an overnight fast increased echogenicity in all cases, with a mean increase of 6.4%. Echogenicity of normal kidneys was significantly less than that of the liver (range, 0.810 to 0.987), and in clinical sonograms analyzed retrospectively but blindly, echogenicity correlated with the qualitative gradations of echogenicity originally assigned. The most echogenic kidneys were 62% brighter than normal kidneys, many times greater than the variability of the measurement. We conclude that quantification of renal cortical echogenicity is feasible and reproducible and may be useful in detecting and following renal disease. Echogenicity of the renal cortex is less than that of the liver in healthy subjects and is influenced by the state of diuresis.