Acute and 3-month effects of calcium carbonate on the calcification propensity of serum and regulators of vascular calcification: secondary analysis of a randomized controlled trial.

SUMMARY: Calcium supplements have been associated with increased cardiovascular risk, but the mechanism is unknown. We investigated the effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50). Changes in serum calcium were related to changes in T50. INTRODUCTION: Calcium supplements have been associated with increased cardiovascular risk; however, it is unknown whether this is related to an increase in vascular calcification. METHODS: We investigated the acute and 3-month effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50), and on three possible regulators of calcification: fetuin-A, pyrophosphate and fibroblast growth factor-23 (FGF23). We randomized 41 postmenopausal women to 1 g/day of calcium as carbonate, or to a placebo containing no calcium. Measurements were performed at baseline and then 4 and 8 h after their first dose, and after 3 months of supplementation. Fetuin-A, pyrophosphate and FGF23 were measured in the first 10 participants allocated to calcium carbonate and placebo who completed the study. RESULTS: T50 declined in both groups, the changes tending to be greater in the calcium group. Pyrophosphate declined from baseline in the placebo group at 4 h and was different from the calcium group at this time point (p = 0.04). There were no other significant between-groups differences. The changes in serum total calcium from baseline were significantly related to changes in T50 at 4 h (r = -0.32, p = 0.05) and 8 h (r = -0.39, p = 0.01), to fetuin-A at 3 months (r = 0.57, p = 0.01) and to pyrophosphate at 4 h (r = 0.61, p = 0.02). CONCLUSIONS: These correlative findings suggest that serum calcium concentrations modulate the propensity of serum to calcify (T50), and possibly produce counter-regulatory changes in pyrophosphate and fetuin-A. This provides a possible mechanism by which calcium supplements might influence vascular calcification.

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

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.

The fallacy of the calcium-phosphorus product.

Scattered through the practice of medicine are dogmas with little or no scientific basis. One of these is the product of the serum calcium and phosphorus concentrations, the so-called calcium-phosphorus product or Ca x P. The assumption that ectopic calcification will occur when the product of the serum calcium and phosphorus concentrations exceeds a particular threshold has become standard practice in nephrology even though there is little scientific basis. Experimental support is lacking, the chemistry underlying the use of the product is oversimplified and the concept that ectopic calcification is simply the result of supersaturation is biologically flawed. The evidence that the Ca x P is an independent risk factor for mortality and morbidity is also questionable. Although ectopic calcification can occur in many sites, this review will focus on vascular calcification, as it is the most common site and the site most likely to affect patient outcomes.

Vascular calcification: not so crystal clear.

Patients with renal failure are predisposed to calcification of the medial layer of arteries. This calcification is far more complex than simple precipitation of calcium and phosphate and involves multiple forms of calcium phosphate. Like bone, calcification in the vessels also involves biologic events. The two are necessarily linked and unraveling the pathophysiology will require an understanding of both.

Chemical and hormonal determinants of vascular calcification in vitro.

Vascular calcification is a complex process that is dependent not only on the physicochemical effects of Ca, PO(4), and pH, but also on smooth muscle factors that may be regulated by these ions as well as by 1,25-dihydroxyvitamin D(3) (calcitriol) and parathyroid hormone (PTH). These minerals and hormones were tested in a model of medial calcification in rat aorta maintained in culture for 9 days. Calcification was quantitated as incorporation of (45)Ca, alkaline phosphatase activity was measured in aortic homogenates, and osteopontin production was measured from immunoblots of culture medium. At 1.8 mM Ca (1.46 mM free), calcification occurred at or above 2.8 mM PO(4). At 3.8 mM PO(4), calcification occurred at or above 1.10 mM free [Ca]. At a constant [Ca] x [PO(4)], calcification varied directly with [Ca] and inversely with [PO(4)]. Calcification was directly related to pH between 7.19 and 7.50 but not altered by PTH or calcitriol. Alkaline phosphatase activity and osteopontin production were increased by Ca, PO(4), calcitriol, and PTH. We conclude that calcification of rat aorta in vitro requires elevation of both [Ca] and [PO(4)], and that [Ca] rather than [PO(4)] or the product of the two is the dominant determinant. The induction of alkaline phosphatase and osteopontin indicates that Ca and PO(4) have effects in addition to simple physicochemical actions. Although PTH and calcitriol did not increase calcification in vivo, they have effects on smooth muscle that could influence calcification in vivo. Calcification is enhanced by alkalinity within the range produced during hemodialysis.

Growth factors stimulate the Na-K-2Cl cotransporter NKCC1 through a novel Cl(-)-dependent mechanism.

The Na-K-2Cl cotransporter NKCC1 is an important volume-regulatory transporter that is regulated by cell volume and intracellular Cl(-). This regulation appears to be mediated by phosphorylation of NKCC1, although there is evidence for additional, cytoskeletal regulation via myosin light chain (MLC) kinase. NKCC1 is also activated by growth factors and may contribute to cell hypertrophy, but the mechanism is unknown. In aortic endothelial cells, NKCC1 (measured as bumetanide-sensitive (86)Rb(+) influx) was rapidly stimulated by serum, lysophosphatidic acid, and fibroblast growth factor, with the greatest stimulation by serum. Serum increased bumetanide-sensitive influx significantly more than bumetanide-sensitive efflux (131% vs. 44%), indicating asymmetric stimulation of NKCC1, and produced a 17% increase in cell volume and a 25% increase in Cl(-) content over 15 min. Stimulation by serum and hypertonic shrinkage were additive, and serum did not increase phosphorylation of NKCC1 or MLC, and did not decrease cellular Cl(-) content. When cellular Cl(-) was replaced with methanesulfonate, influx via NKCC1 increased and was no longer stimulated by serum, whereas stimulation by hypertonic shrinkage still occurred. Based on these results, we propose a novel mechanism whereby serum activates NKCC1 by reducing its sensitivity to inhibition by intracellular Cl(-). This resetting of the Cl(-) set point of the transporter enables the cotransporter to produce a hypertrophic volume increase.

Contractile regulation of the Na(+)-K(+)-2Cl(-) cotransporter in vascular smooth muscle.

Vasoconstrictors activate the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 in rat aortic smooth muscle, but the mechanism is unknown. Efflux of (86)Rb(+) from rat aorta in response to phenylephrine (PE) was measured in the absence and presence of bumetanide, a specific inhibitor of NKCC1. Removal of extracellular Ca(2+) completely abolished the activation of NKCC1 by PE. This was not due to inhibition of Ca(2+)-dependent K(+) channels since blocking these channels with Ba(2+) in Ca(2+)-replete solution did not prevent activation of NKCC1 by PE. Stimulation of NKCC1 by PE was inhibited 70% by 75 microM ML-9, 97% by 2 microM wortmannin, and 70% by 2 mM 2,3-butanedione monoxime, each of which inhibited isometric force generation in aortic rings. Bumetanide-insensitive Rb(+) efflux, an indication of Ca(2+)-dependent K(+) channel activity, was reduced by ML-9 but not by the other inhibitors. Stretching of aortic rings on tubing to increase lumen diameter to 120% of normal almost completely blocked the stimulation of NKCC1 by PE without inhibiting the stimulation by hypertonic shrinkage. We conclude that activation of the Na(+)-K(+)-2Cl(-) cotransporter by PE is the direct result of smooth muscle contraction through Ca(2+)-dependent activation of myosin light chain kinase. This indicates that the Na(+)-K(+)-2Cl(-) cotransporter is regulated by the contractile state of vascular smooth muscle.

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.

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.

Bedside renal biopsy: ultrasound guidance by the nephrologist.

The safety and efficacy of percutaneous biopsy of native kidneys performed entirely by nephrologists at the patient's 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 patient's 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.

JNK is a volume-sensitive kinase that phosphorylates the Na-K-2Cl cotransporter in vitro.

Cell shrinkage phosphorylates and activates the Na-K-2Cl cotransporter (NKCC1), indicating the presence of a volume-sensitive protein kinase. To identify this kinase, extracts of normal and shrunken aortic endothelial cells were screened for phosphorylation of NKCC1 fusion proteins in an in-the-gel kinase assay. Hypertonic shrinkage activated a 46-kDa kinase that phosphorylated an NH2-terminal fusion protein, with weaker phosphorylation of a COOH-terminal fusion protein. This cytosolic kinase was activated by both hypertonic and isosmotic shrinkage, indicating regulation by cell volume rather than osmolarity. Subsequent studies identified this kinase as c-Jun NH2-terminal kinase (JNK). Immunoblotting revealed increased JNK activity in shrunken cells; there was volume-sensitive phosphorylation of NH2-terminal c-Jun fusion protein; immunoprecipitation of JNK from shrunken cells but not normal cells phosphorylated NKCC1 in gel kinase assays; and treatment of cells with tumor necrosis factor, a known activator of JNK, mimicked the effect of hypertonicity. We conclude that JNK is a volume-sensitive kinase in endothelial cells that phosphorylates NKCC1 in vitro. This is the first demonstration of a volume-sensitive protein kinase capable of phosphorylating a volume-regulatory transporter.

Vasoconstrictors and nitrovasodilators reciprocally regulate the Na+-K+-2Cl- cotransporter in rat aorta.

Little is known about the function and regulation of the Na+-K+-2Cl- cotransporter NKCC1 in vascular smooth muscle. The activity of NKCC1 was measured as the bumetanide-sensitive efflux of 86Rb+ from intact smooth muscle of the rat aorta. Hypertonic shrinkage (440 mosmol/kgH2O) rapidly doubled cotransporter activity, consistent with its volume-regulatory function. NKCC1 was also acutely activated by the vasoconstrictors ANG II (52%), phenylephrine (50%), endothelin (53%), and 30 mM KCl (54%). Both nitric oxide and nitroprusside inhibited basal NKCC1 activity (39 and 34%, respectively), and nitroprusside completely reversed the stimulation by phenylephrine. The phosphorylation of NKCC1 was increased by hypertonic shrinkage, phenylephrine, and KCl and was reduced by nitroprusside. The inhibition of NKCC1 significantly reduced the contraction of rat aorta induced by phenylephrine (63% at 10 nM, 26% at 30 nM) but not by KCl. We conclude that the Na+-K+-2Cl- cotransporter in vascular smooth muscle is reciprocally regulated by vasoconstrictors and nitrovasodilators and contributes to smooth muscle contraction, indicating that alterations in NKCC1 could influence vascular smooth muscle tone in vivo.

Physiological significance of volume-regulatory transporters.

Research over the past 25 years has identified specific ion transporters and channels that are activated by acute changes in cell volume and that serve to restore steady-state volume. The mechanism by which cells sense changes in cell volume and activate the appropriate transporters remains a mystery, but recent studies are providing important clues. A curious aspect of volume regulation in mammalian cells is that it is often absent or incomplete in anisosmotic media, whereas complete volume regulation is observed with isosmotic shrinkage and swelling. The basis for this may lie in an important role of intracellular Cl- in controlling volume-regulatory transporters. This is physiologically relevant, since the principal threat to cell volume in vivo is not changes in extracellular osmolarity but rather changes in the cellular content of osmotically active molecules. Volume-regulatory transporters are also closely linked to cell growth and metabolism, producing requisite changes in cell volume that may also signal subsequent growth and metabolic events. Thus, despite the relatively constant osmolarity in mammals, volume-regulatory transporters have important roles in mammalian physiology.

A volume-sensitive, IP3-insensitive Ca2+ store in vascular endothelial cells.

The mechanism by which cell swelling and other physical forces increase the intracellular Ca2+ concentration ([Ca2+]i) is poorly defined. In vascular endothelial cells, the increase in [Ca2+]i after hypotonic swelling was independent of external Ca2+ and membrane potential, was not blocked by La3+ or Gd3+, and was prevented by thapsigargin, indicative of intracellular release. This release also occurred after depletion of agonist-sensitive Ca2+ stores. In cells in which the plasma membrane was permeabilized with saponin, hypotonic medium stimulated release of 45Ca2+ from a thapsigargin-sensitive site. Isosmotic substitutions with sucrose or urea revealed that this release was due specifically to swelling and not to changes in osmolarity or ion concentrations. This volume-sensitive release was inhibited by high concentrations of La3+ and Gd3+ in a time-dependent manner, suggesting inhibition from within the storage compartment. Release was not inhibited by ruthenium red or by prior stimulation with inositol 1,4,5-trisphosphate (IP3), indicating that the volume-sensitive storage site is distinct from mitochondria and from stores sensitive to ryanodine or IP3. The results suggest the presence of a novel, stretch-activated Ca2+ store in endothelial cells that could contribute to their mechanosensitivity.

Shrinkage-induced activation of Na+/H+ exchange: role of cell density and myosin light chain phosphorylation.

Previously, we suggested that myosin light chain kinase (MLCK) is involved in shrinkage-induced activation of the Na+/H+ exchanger in rat astrocytes. Here we have studied the effects of hyperosmotic exposure in C6 glioma cells, a common model for astrocytes. Shrinkage-induced activation of the Na+/H+ exchanger in C6 cells is directly proportional to the degree of shrinkage, results in an alkaline shift in the pK' of the exchanger, is dependent on ATP, and is inhibited by ML-7 (an MLCK inhibitor) and by various calmodulin inhibitors. Cell shrinkage also results in increased phosphorylation of myosin light chain (MLC). Interestingly, shrinkage-induced activation of the exchanger does not occur in subconfluent C6 cells. However, phosphorylation of MLC still occurs in subconfluent cultures of C6 cells on shrinkage, suggesting that the lack of activation in these cells occurs at a point between MLC phosphorylation and Na+/H+ exchange activation. The lack of activation of Na+/H+ exchange in subconfluent C6 cells can be utilized to further elucidate the shrinkage-induced activation pathway.

Volume-sensitive myosin phosphorylation in vascular endothelial cells: correlation with Na-K-2Cl cotransport.

To identify protein kinases that are regulated by cell volume, we examined protein phosphorylation in hypertonically shrunken aortic endothelial cells. Shrinkage reversibly increased, and swelling decreased, phosphorylation of a 19-kDa cytoskeletal protein identified as myosin light chain (MLC) by immune precipitation and immunoblotting. Shrinkage also increased MLC phosphorylation in human umbilical vein endothelial cells, rat aortic smooth muscle cells, and human dermal fibroblasts. Phosphorylation was blocked by ML-7, an inhibitor of MLC kinase (MLCK). Neither inhibition of protein kinase C nor inhibition of myosin phosphatase (with calyculin) altered MLC phosphorylation. Peptide mapping of MLC indicated phosphorylation by MLCK. Na-K-2Cl cotransport activation paralleled MLC phosphorylation in hypertonic medium. Na-K-2Cl was stimulated by low concentrations of ML-7 with no further stimulation by hypertonic shrinkage and was inhibited by higher concentrations, paralleling inhibition of MLC phosphorylation. Shrinkage-induced phosphorylation of the cotransporter was not blocked by ML-7. We conclude that cell volume regulates MLC phosphorylation by MLCK. MLCK influences Na-K-2Cl cotransport but independently of cotransporter phosphorylation. These data suggest an important link between cell volume, volume-regulatory transporters, and the contractile state of the cytoskeleton.

Flow-mediated NO release from endothelial cells is independent of K+ channel activation or intracellular Ca2+.

The role of K+ channels and intracellular [Ca2+] in flow-induced nitric oxide (NO) production was investigated in bovine aortic endothelial cells in culture. NO release (measured as nitrite production) and K+ channel activity (measured as 86Rb+ efflux) were measured in cells grown on collagen-coated microcarrier beads and perfused in a column. An eightfold increase in flow produced a rapid (within 1 min), sustained, and reversible sixfold increase in NO release. Efflux of 86Rb+ also increased but rapidly returned to baseline and then transiently decreased when flow was decreased. This was probably due to boundary layer washout rather than to K+ channel activation, because an identical pattern was seen for release of [3H]ouabain. Neither tetraethylammonium nor increasing medium [K+] to block K+ currents prevented flow-induced NO release. Removal of medium Ca2+ or chelation of intracellular Ca2+ also did not block flow-mediated NO release. The results demonstrate that flow rapidly increases NO release from endothelial cells but that this increase in NO release is not dependent on activation of K+ channels or changes in intracellular [Ca2+].

Shrinkage-induced activation of Na+/H+ exchange in primary rat astrocytes: role of myosin light-chain kinase.

Primary rat astrocytes exposed to hyperosmotic solutions undergo Na(+)-dependent amiloride-sensitive alkalinization of 0.36 U [measured with the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein], suggesting that shrinkage-induced alkalinization is due to activation of Na+/H+ exchange (NHE). Alkalinization is maintained for at least 20 min, and is readily reversible and ATP dependent. Hyperosmotic solutions produced no increase of intracellular Ca2+ or adenosine 3',5'-cyclic monophosphate (cAMP). Loading cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a Ca2+ chelator, or depleting cells of protein kinase C (PKC) had no effect on activation of NHE. Thus shrinkage-induced activation of NHE does not involve cAMP, Ca2+, or PKC. However, ML-7, an inhibitor of myosin light-chain kinase (MLCK), inhibited shrinkage-induced activation with a half-maximal inhibition of 56 microM. This activation was also inhibited by 500 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, 100 microM chlorpromazine, and 50 microM trifluoperazine, all calmodulin inhibitors. Shrinkage increased the phosphorylation of an 18-kDa protein that colocalizes with myosin light chain. Our data suggest that shrinkage-induced activation of NHE in astrocytes occurs via a novel pathway involving activation of calmodulin-dependent MLCK and phosphorylation of myosin light chain.

Functional coupling of Na(+)-K(+)-2Cl- cotransport and Ca(2+)-dependent K+ channels in vascular endothelial cells.

To determine whether the activation of Na(+)-K(+)-2Cl- cotransport by Ca(2+)-mobilizing agonists is a direct effect of Ca2+ or is secondary to activation of Ca(2+)-dependent K+ channels [via cell shrinkage or decreased intracellular Cl- concentration ([Cl-]), we measured K+ fluxes in aortic endothelial cells in response to ATP and bradykinin. With either agonist there was an immediate bumetanide-insensitive efflux inhibitable by the K+ channel blockers tetrabutylammonium (TBA, 23 mM) and quinidine (1 mM), followed several minutes later by increased bumetanide-sensitive efflux or influx (Na(+)-K(+)-2Cl- cotransport). ATP induced a loss of cell K+ that was prevented by TBA and augmented by bumetanide. Both TBA and quinidine prevented the stimulation of cotransport by agonists but not by hypertonic shrinkage. Raising medium [K+] to prevent K+ loss also blocked activation of cotransport by agonists. The results indicate that the stimulation of Na(+)-K(+)-2Cl- cotransport by Ca2+ is not direct but instead is indirect via activation of Ca(2+)-dependent K+ channels and a resulting decrease in cell volume and intracellular [Cl-]. This suggests that at least one role of Na(+)-K(+)-2Cl- cotransport in endothelial cells is to maintain cell volume and intracellular [Cl-] during agonist stimulation.