Calcium Metabolism in Uremic Nephrocalcinosis: Preventive Effect of Verapamil

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Kidney International, Vol. 27 (1985), pp. 774—779

Calcium metabolism in uremic nephrocalcinosis: Preventive effect of verapamil

MICHAEL S. GOLIGORSKY, CIDJ0 CHAIM0vITz, JAYSON RAPOPORT, JED GOLDSTEIN, and RINA KOL

Departments of Nephrology and Pathology, Soroka University Hospital and Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-S heva, Israel

Calcium metabolism in uremic nephrocalcinosis: Preventive effect of traités au vérapamil. Nous concluons que l'administration chronique de verapamil. The aim of the present study was to examine calciumvérapamil améliore la néphrolcalcinose urémique. metabolism of the renal cortex in experimental chronic renal failure, together with morphologic criteria of nephrocalcinosis and to determine the effect of chronic verapamil administration on these parameters. In subtotally nephrectomized (SNX) rats 3 weeks after surgery, renal Uremic nephrocalcinosis, although a common complication cortical calcium content increased more than two-fold. 45Ca incorporation into renal cortical slices in SNX revealed a 35% increase,of chronic renal failure, remains an enigma [1—4].Since associated with a 50% increase in a lanthanum-resistant fraction of 45Ca parathyroidectomy almost completely prevents the develop- uptake. Radiocalcium wash-out curves in this group demonstratedment of uremic nephrocalcinosis, it has been suggested that abnormal retention of the isotope for up to 30 mm of incubation. In secondary hyperparathyroidism may play an important role in contrast, radiocalcium incorporation and wash-out in SNX rats chroni- its pathogenesis [51. cally treated with verapamil were similar to that obtained in the sham group. Verapamil administration significantly reduced, but did not The in vitro effect of parathyroid hormone (PTH) was studied normalize, renal cortical calcium content. Von Kossa staining demon- by Bone [6] and Bone and Uchikawa [7], who demonstrated strated the deposition of calcium in the renal parenchyma of SNX rats. that the addition of PTH to the incubation medium led to an Ultrastructurally, it was accompanied by mitochondrial disorganization almost three-fold increase in 45Ca incorporation into renal and calcification, as well as by the tubular basement membrane destruc- cortical cells. This enhanced calcium incorporation was attenu- tion and mineralization. These morphologic patterns of nephrocalcinosis were significantly ameliorated in SNX rats treated with verapamil. ated significantly following exclusion of phosphate and magne- We conclude that chronic verapami! administration results in ameliora- sium from the incubation medium; however, calcium incorpo- tion of uremic nephrocalcinosis. ration still remained elevated. In chronic experiments on rats Mtabolisme du calcium au cours de Ia néphro-calcinose urémique: with the phosphate-induced secondary hyperparathyroidism, effet préventif du vérapamil. Le but de cette étude a été d'examiner le Borle and Clark [8] demonstrated a marked stimulation of renal métabolisme calcique du cortex renal au cours d'une insuffisance rénale cell calcium metabolism. It was shown that secondary hyper- chronique expérimentale en méme temps que les critéresparathyroidism caused an intracellular accumulation of cal- morphologiques de nCphrocalcinose, et de determiner l'effet decium, consequently leading to nephrocalcinosis. l'administration chronique de vérapamil sur ces paramCtres. Chez des Since intracellular calcium accumulation plays a pivotal role rats nephrectomises partiellement (SNX), 3 semaines aprCs Ia chirurgie le contenu en calcium de Ia corticale rénale augmentait plus de deux in the expression of PTH-induced nephrocalcinosis, it follows fois. L'incorporation de 45Ca aux coupes de corticale rénale chez les that attenuation of intracellular calcium influx would be ex- SNX révélait une augmentation de 35%, associée a une élévation de pected to be of value in ameliorating this effect of PTH. 50% de Ia fraction lanthanum-résistante de Ia captation de 45Ca. Les Recently, calcium channel blockers have been shown to courbes de disparition du radiocalcium dans cc groupe démontraient une retention anormale de l'isotope jusqu'à 30 mm d'incubation. Ainhibit various specific effects of PTH on erythrocytes and l'opposé, l'incorporation et Ia disparition du radiocalcium chez les rats cultured myocardial cells [9—11]. Furthermore, attenuation of SNX traités chroniquement au vérapamil étaient identiques a celles calcium influx by verapamil has been demonstrated in the obtenues dans Ic groupe simulacre. L'administration de verapamil a various epithelial layers as well as in the renal brush border réduit significativement, mais n'a pas normalisé le contenu cortical renal en calcium. Une coloration de Von Kossa a démontré des depOts membrane preparation [12—14]. Thus, it might be suggested thai de calcium dans Ic parenchyme renal des rats SNX. Ultrastructuralle- the calcium channel blockers may prevent or attenuate uremic ment, cela a été accompagné d'une desorganisation et de calcifications nephrocalcinosis. The aim of the present study was to examine mitochondriales, et par une destruction et une minéralisation de Iathe renal calcium content, morphologic criteria of nephrocalci- membrane basale tubulaire. Ces aspects morphologiques denosis, and calcium kinetics in mild chronic renal insufficiency nCphrocalcinose Ctaient significativement amCliorCs chez les rats SNX during verapamil treatment. We found that nephrocalcinosis in mildly uremic rats is associated with an increased calciuni accumulation, severe mitochondrial disorganization, and min- Received for publication April 20, 1984, eralization of the tubular basement membrane. Chronic and in revised form November 12, 1984 verapamil administration to otherwise identical animals re- © 1985 by the International Society of Nephrology sulted in an almost complete prevention of nephrocalcinosis.

774 Verapamil in uremic nephrocalcinosis 775

Methods 45Ca uptake Studies were performed on male Charles River rats weighing Renal cortical slices, each weighing approximately 100 mg, 250 to 300 g. Animals were fed with regular rat laboratory chowwere preincubated in a metabolic shaker (Dubnoff) for 60 mm at and allowed water ad lib. Experimental animals were subjected37°C in oxygenated Krebs-Henseleit solution buffered with 5 to 5/6 nephrectomy (SNX) performed via flank incisions in twomM Hepes, pH 7.4. Calcium uptake and wash-out was studied steps separated by 1 week, and using intraperitoneal pentobar-according to the slightly modified technique of Uchikawa and bital anesthesia. Another group of identical rats underwentBorIc [151. Briefly, following the equilibration period, slices sham operation consisting of flank incisions and kidneywere placed in plastic tubes containing 10 ml of the above decapsulation. All animals were allowed 4 to 5daysto recovermedium and 45Ca, 2 CiIml, was added. After the 60-mm from surgery and then SNX and sham-operated rats wereincubation period, slices were removed rapidly for uptake randomized into four groups: (1) 10 SNX rats treated for 3study. Slices were placed over a Millipore 0.45 p. filter and weeks by verapamil, 10 tg/l00 g body weight twice a day,rinsed at 4°C twice with 5 ml 45Ca-free buffer. Slices were then administered intramuscularly (SNX+V); (2) 9 SNX rats receiv-dry-weighed and digested with 5 ml Aquasol. The 45Ca uptake ing intramuscular injections of 0.9% sodium chloride, 0.1 mlwas measured in a scintillation counter (Packard Instruments, twice a day for 3 weeks (SNX); (3) 9 sham-operated rats treatedDowners Grove, Illinois, USA). identically to the SNX+V group (C+V); (4) 11 sham-operated In a separate series of experiments, a Lanthanum (La)- rats treated for 3 weeks with the saline injections, as in group 2resistant fraction of calcium uptake was studied, according to (C). the slightly modified technique of Godfraind [161. Following a At the end of the 3-week period, animals were slightly60-mm incubation period with radiocalcium (as previously anesthetized with ether and sacrificed by exsanguination. Blooddescribed), incubation medium was replaced rapidly by Krebs- samples were analyzed for plasma concentration of creatinine,Henseleit buffer supplemented with 30 mivi LaCI3. After an BUN, calcium, and inorganic phosphorus. Following sacrifice,additional 5-mm incubation, the reaction was stopped by filtra- the kidney remnants were removed immediately, and scarredtion; slices were dry-weighed and digested, and radioactivity tissue was excised carefully. The renal cortex was sectionedwas counted in the same fashion as in the 45Ca uptake series. and used for the following studies: (1) hematoxylin-eosin and Von Kossa staining for light microscopic examination to ensure 45Ca wash-out the completeness of removal of scarred parenchyma and to Similarly preloaded with 45Ca, slices were incubated in 10 ml verify the presence of nephrocalcinosis; (2) electron micros-of Krebs-Henseleit solution free of 45Ca for an additional 60 mm copy and x-ray microprobe analysis; (3) determination of tissueat 37°C. The incubation medium was exchanged every 5 mm. At calcium content and (4) 45Ca uptake and wash-out study. 2, 5, 10, 30, and 60 mm, samples were removed quickly, rinsed twice over a Millipore filter, dry-weighed, and digested with Metabolic study Aquasol. Radioactivity was measured by a scintillation counter (Packard Instruments). In each experiment, five slices were Another 20 rats (five in each group) were placed into meta-used at each point. Results were expressed as counts per bolic cages for 3 weeks and their daily food intake was studied.minute per milligram of dry weight. Weight gain was examined on a weekly basis. After 1 week on an unrestricted diet sham-operated animals were pairfed with Calcium content of the renal cortex SNX rats. The metabolic study was continued for an additional The renal cortex was weighed and dried for 24 hr in an oven 2 weeks. at 105°C. Reweighed tissue was hydrolyzed with 0.5 ml of concentrated hydrochloric acid. The residue was then neutral- Electron microscopy ized with sodium hydroxide solution to pH 7.0, and lanthanum Renal cortical samples were fixed with 2% glutaraldehyde inchloride was added to each sample to a final concentration of cocadylate buffer, and then transferred to osmium tetroxide1%. Calcium concentration was determined by atomic absorp- fixative. After dehydration in ethanol, samples were embeddedtion spectrophotometry. in araldite 502. Sections (0.8 )werecut with an ultramicro- Statistical comparisons between the groups were performed tome (LKB-III, Sweden) and stained with uranyl-citrate andby means of the Student's non-paired t test. Radiocalcium lead citrate. Preparations were examined by means of transmis-wash-out curves were obtained by least square fitting with the sion electron microscopy (Philips 201, Holland) and with energyaid of MINIPACK-l code [17]. dispersive x-ray microanalysis on a STEM Jeol 120 XC with ED Results spectrum analyzer (Proxan, Elscint, Israel). Preparations were tilted to 45°; an acceleration voltage of 80 kv was used. In the By 3 weeks after surgery, experimental animals were in preliminary experiments, stained 0.8-p and unstained 2- sec-stable renal insufficiency with creatinine and BUN plasma tions were examined, and no significant difference in theconcentrations of 1.30.3 mg% versus 1.2 0.3 mg% and 48 sensitivity of detection of calcium in stained and unstained 5 mg% versus 464 in SNX animals and SNX treated with sections was found. Because of the obvious resolutional advan-verapamil (SNX+V), respectively. In control animals of groups tages in studying stained sections, we subsequently used this3 and 4, creatinine and BUN levels were 0.50.1 mg% versus kind of preparation for x-ray microprobe analysis. For this0.5 0.15 mg% and 18 3 mg% versus 19 4 mg%, reason, the elemental spectrum of the stained sections alsorespectively. No significant alterations in calcium and inorganic contained osmium and lead peaks. phosphorus plasma concentration were detected in SNX and 776 Goligorsky et al

NS A P<0.05 La 0.001 C P<0.0O1 P<0.001 40 120 30 -I- 80 • SNX + V 20 .-1-, } I C+v • 40 I10 i.

0 0 C C+V SNX SNX+V C C+V SNX SNX+V 0.00 0.20 0.40 0:60 0.80 1 Hr C+La SNX+La Incubation time Fig. IA. Calcium content of the renal cortex in experimental groups: C, control; C+V, control rats chronically treated with verapamil; SNX, subtotally nephrectomized rats; SNX+V, subtotally nephrectomized rats treated chronically with verapamil. Vertical bars represent mean SD. B45Ca incorporation into renal cortical slices in four experimental groups (designation here and below as in Fig. lA). Dotted bars represent La-resistant fraction of 45Ca uptake. C 5Ca wash-out curves in four experimental groups (in percent to control postloading values). Every curve is constructed from five time points of 2, 5, 10, 30, and 60 mm (five slices for each point) by least square fitting.

SNX±V rats (9.60.6and 8.40.4mg% versus 9.4 0.5and 8.60.5mg%), as compared to control and control+V animals (9.8 0.4and 8.2 0.3 mg% versus 9.7 0.6and 8,2 0.5 mg%). Food consumption in sham-operated animals on the liberal diet averaged 24 2glday, resulting in a weight gain of 40.3 3.7glweek. In SNX and SNX+V rats daily food intake was 18 1.6and 19 2g of rat chow, respectively. Weekly weight gain in both groups was also similar, averaging 12.62.8and 13.1 1.5g. Sham-operated animals pairfed with SNX rats revealed a weekly weight gain of 13 2g: the value was not statistically different from both experimental groups. Hence, verapamil-treated and untreated SNX rats were similar in terms of their blood chemistry and protein and caloric intake. Thus, these mildly azotemic animals offer a convenient model to studyFig. 2A. Von Kossa staining of kidney section obtained from SNX rat. the effect of chronic verapamil administration, unrelated to(Magnification x400) B Von Kossa staining of kidney section from SNX changes in the plasma level of BUN, creatinine, calcium andrat treated with verapamil. (Magnification X400) phosphorus, and in the metabolic state. In Figure 1A, renal cortical calcium content in the four experimental groups is depicted. The average calcium contentfrom SNX rats is due to intracellular accumulation of radi- in SNX rats was 32.3 5.8mmoles/kg dry weight, a valueocalcium. It is noteworthy that 45Ca incorporation into renal considerably higher (P <0.001)than 14.0 2.6mmoles/kg drycortical slices from SNX+V animals was similar to that ob- weight and 12.5 2.5mmoles/kg dry weight seen in sham-tained in control animals of both verapamil-treated and un- operated untreated and verapamil-treated animals, respec-treated groups. Radiocalcium wash-out curves obtained from tively. In verapamil-treated SNX rats, renal cortical calciumboth control groups and verapamil-treated SNX animals were content (21.3 4.4mmoles/kg dry weight) was significantlyidentical (Fig. lC). In the SNX group, the radioactivity of slices lower (P <0.001) than that seen in SNX rats, but still higher (Premained elevated (P <0.05)as compared to sham and <0.001) than control values. SNX+V groups up to 30 mm of incubation. 45Ca incorporation into kidney cortical slices from SNX rats Von Kossa-stained sections of kidney remnants obtained (Fig. 1B) revealed a 35% increase (P <0.05)as compared tofrom SNX rats showed a delicate punctate calcification of renal control values, as well as a substantial elevation (30%), com-tubular cells in the region of the tubular basement membrane pared to SNX animals treated with chronic verapamil adminis-(Fig. 2A). In contrast, calcification was demonstrated to an tration (P <0.05).La-resistant fraction of radiocalcium uptakeobviously lesser extent in sections of verapamil-treated SNX exhibited even more profound differences between control andanimals (Fig. 2B). SNX animals. 45Ca incorporation into La-treated renal cortical Ultrastructurally, there is an obvious swelling and disorgani- slices from SNX rats revealed a 50% increase (P <0.001), zation of mitochondria in uremic animals both treated and compared to similarly treated slices in the sham group. Thisuntreated (Fig. 3). However, the prevalence of disorganized data indicates that the increase in 45Ca incorporation into slicesforms in the SNX group, as compared to SNX+V animals is Verapamil in uremic nephrocalcinosis 777

Fig. 3. Detailed view of mitochondrial population in control, SNX, and SNX+ V rats. (Magnification x30,000)

P<0.05

80 P<0.05

+1 -C C.) 40

20 Fig. 5. X-ray microprobe patterns of background (A), intact mitochondria (B), and disorganized mitochondria (C). Peaks of osmium and lead are the result of staining procedures. Note the appearance of a peak characteristic of calcium in the disorganized mitochondria. 0. 2 3 1 2 3 SNX SNX+V tion of the internal structure. The comparative analysis of Fig.4. Comparative semiquantitative analysis of mitochondrial dam- mitochondrial ultrastructure in SNX and SNX+V animals is age in verapamil-trea ted and untreated SNX animals. The numbers depicted in Figure 4. Verapamil treatment is associated with an represent: 1, percent of intact mitochondria; 2, partially disorganized increase (P < 0.05) in the fraction of intact mitochondria and a mitochondria; 3, percent of mitochondria with completely distorted decrease (P < 0.05) in the subpopulation of completely dis- internal structure. torted forms. The typical x-ray microanalysis patterns of intact and dis- prominent. Mitochrondria of SNX rats revealed swelling oforganized mitochondria are depicted in Figure 5 (B and C, condensed forms, electron density, herniation of internal mem-respectively). The disorganized mitochondria reveal a small but brane, destruction of cristae and the complete loss of internalconsistent peak characteristic of calcium (5C),which com- structure (Fig. 3B). These findings are in obvious contrast to theprised 7.3 0.5% of the total elemental content. In contrast, in relatively intact state of mitochondrial structure in SNX ratsintact mitochondria calcium content was beyond the lower limit treated with verapamil (Fig. 3C). of detection. To semiquantitate the extent of mitochrondrial disorganiza- The tubular basement membrane represents another site of tion in this model of chronic uremia, 500 randomly selectedsevere structural alteration in SNX rats. As depicted in Figure objects were examined in each group of treated and untreated6B, the tubular basement membrane in this condition appears animals. Mitochrondrial ultrastructure was arbitrarily subdi-irregular and non-homogenous. X-ray microanalysis of such a vided as following: (1) intact mitochondria; (2) partial dis-membrane (Fig. 6C2) shows the consistent patterns of calcium, organization (destruction of at least one-third of the cristaephosphorus, aluminum, and silicon, indicating that structural and/or herniation of internal membrane); (3) complete destruc-abnormalities are associated with the linear mineralization of 778 Goligorskyet a!

Fig. 6. Electron micrographs of the tubular basement membrane in SNX+V(A) and SNX (B) rats. Note the loss of homogeneity and integrity of the tubular basement membrane in the SNX rat, as compared to intact membrane in SNX+V animal. (Magnification x30,000) C represents an x-ray microprobe analysis of the tubular basement membrane in SNX+V (1) and SNX (2) rats. The latter reveals calcium, phosphorus, aluminum, and silicon patterns characteristic of linear mineralization of the tubular basement membrane (2). This phenomenon is ameliorated in SNX+V rats (I).

the tubular basement membrane. Chronic verapamil treatmentrecently demonstrated in vivo [18], we cannot say at this stage. of SNX rats leads to a structural sparing of the tubular base-Much controversy exists at present concerning the possibility ment membrane (Fig. 6A) and considerably reduced mineraliza-of a direct effect of verapamil on calcium influx in epithelial tion (Fig. 6C1). cells. Our data, together with others [12—14], does not exclude such a possibility. However, whatever the precise mechanism Discussion of action of verapamil, the observation that reduced tissue This study offers good evidence that intracellular calciumcalcium uptake after verapamil therapy was associated with an accumulation plays a pivotal role in the pathogenesis of uremicamelioration of nephrocalcinosis indicates that abnormal cel- nephrocalcinosis, and that the calcium channel blockerlular accumulation of calcium represents an important step in verapamil reduces nephrocalcinosis via normalization of cel-the pathogenesis of uremic nephrocalcinosis. In this context, lular calcium metabolism. the study of Nayler, Mas-Dliva, and Williams [19] on the Both histology and elevated calcium content of the renalisolated perfused heart is of interest. During low-perfusion parenchyma indicate that nephrocalcinosis already occurs inprotocol, myocardial cells displayed mitochondrial calcification mildly uremic rats. The radiocalcium study revealed that thiswhich was ameliorated by verapamil. Since PTH was excluded condition is associated with an increased incorporation of theby the experimental design, in this study at least, verapamil isotope by the renal cortical slices. Increased La-resistantappeared to have a direct membrane effect. Recent data on fraction of 45Ca uptake in SNX rats further indicates thatPTH-induced changes in the membrane phospholipid composi- intracellular accumulation is responsible for enhanced incorpo-tion which modulate membrane permeability to calcium [14, 20, ration of radiocalcium into renal cortical slices. Ultrastructur-211, may provide the missing link between secondary hyper- ally, this is accompanied by mitochondrial disorganization, asparathyroidism and nephrocalcinosis. In this sequence altera- well as irregularity and destruction of the tubular basementtion of the cellular permeability to calcium would occupy the membrane. Disorganized mitochondria retain calcium and thecentral role. tubular basement membrane contains mineral deposits in ure- This view is supported by evidence from another model of mic nephrocalcinosis, as revealed by x-ray microprobe. nephrocalcinosis, namely, that induced by PTH. Secondary We have further found that chronic verapamil administrationhyperparathyroidism in rats fed a high-phosphate diet was to similarly azotemic rats was associated with much lesser Vonassociated with renal cellular calcium accumulation within 24 hr Kossa staining patterns and reduced calcium content of renal[8]. Accumulation of calcium within the mitochondrial compart- tissue. Ultrastructural analysis revealed morphologic complete-ment was prominent in this model of nephrocalcinosis. Simi- ness and normal mineral content of the tubular basementlarly, repeated injections of PTH in mice resulted in nephrocal- membrane. Normal radiocalcium kinetics in renal cortical slicescinosis. Mitochondrial disorganization was detected in the early was found in this experimental group. Whether this preventivestage of the process, even before the appearance of nephrocal- effect of verapamil on the development of nephrocalcinosis wascinosis [22, 23]. It is of interest that in nephrocalcinosis pro- mediated via a direct effect on inward calcium flux, or whetherduced by vitamin D overdose, mitochondria are also the pri- it was due to verapamil-induced suppression of PTH secretion.mary site of the cellular damage [24]. When 2,4-dinitrophenol Verapamil in uremic nephrocalcinosis 779 was inoculated together with vitamin D, the dosage of the latter extraosseous calcification in the rat. C/in Sci 61:621—626, 1981 required to induce nephrocalcinosis was reduced significantly. 6. BORLE AB: Kinetic analysis of calcium movements in cell cultures. It was thus postulated by Scarpelli [241 that mitochondria exert III. Effects of calcium and parathyroid hormone in kidney cells. J Gen Physiol 55:163—186, 1970 a protective effect against nephrocalcinosis, and their oblitera- 7. BORLE AB, UCHIKAWA T: Effects of parathyroid hormone on the tion accelerates the development of renal calcification. distribution and transport of calcium in cultured kidney cells. In this context, it is noteworthy that in subtotally nephrecto- Endocrinology 102:1725—1732, 1978 mized rats, the bulk of the mitochondrial population of proximal 8. BORLE AB, CLARK I: Effects of phosphate-induced hyperparathyroidism and parathyroidectomy on rat kidney calcium in vivo. tubular epithelial cells showed swelling, disorganization with Am J Physiol 241:E136—E141, 1981 the appearance of condensed forms, loss of integrity of the 9. BOGIN E, MAssEY SG, HARARY I: Effect of parathyroid hormone external membrane, and complete loss of cristae (Figs. 3B and on rat heart cells. J C/in Invest 67:1215—1227, 1981 4). In these disorganized mitochondria calcium is discernible by10. KATON Y, KLEIN KL, KAPLAN RA, SANBORN WG, KUROKAWA means of x-ray microanalysis. This phenomenon has recently K: Parathyroid hormone has a positive inotropic action in the rat. Endocrinology 109:2252—2254, 1981 been considered a manifestation of mitochondrial damage [25]. 11. BOGJN E, MASSRY SG, LEVI J, DJALDETI M, BRISTOL G, SMITH J: The latter could be a result of ischemia with secondary altera- Effect of parathyroid hormone on osmotic fragility of human tion of a calcium-accumulating capacity [26], or, alternatively, erythrocytes. J C/in Invest 69:1017—1025, 1982 could be caused by calcium overload [19, 271. In our experi- 12. WROBEL J, MICHALSKA L: The effect of verapamil on intestinal calcium transport. Eur J Pharmacol 45:385—387, 1977 mental setting an improvement in the mitochondrial state fol- 13. HUMES HD, SIMMONS CF, BRENNER BM: Effect of verapamil on lowing chronic verapamil administration might indicate that in the hydroosmotic response to antidiuretic hormone in toad urinary subtotally nephrectomized rats an elevation of intracellular bladder. Am J Physiol 239:F250—F257, 1980 calcium influx contributes to mitochondrial damage. However,14. SOMERMEYER MG, KNAUSS TC, WEINBERG JM, HUMES HD: Characterization of Ca2 transport in rat renal brush-border mem- this remains a speculation at present. branes and its modulation by phosphatidic acid. Biochem J In this study, we did not explore another possible mode of 214:37—46, 1983 action of verapamil in chronic uremia, namely, that mediated 15. UCHJKAWA T, BORLE AB: Studies of calcium-45 desaturation from via vascular smooth muscle tone. Recently, this mechanism has kidney slicesin flow-through chambers. Am J Physiol been shown essential in prevention of norepinephrine-induced 234:R34—R38, 1978 16. GODFRAIND T: Calcium exchange in vascular smooth muscle, acute renal failure [28]. We have shown [29] that chronic action of Noradrenaline and Lanthanum. J Physiol 260:21—35, 1976 verapamil administration to SNX rats resulted in an ameliora- 17. MORE JJ, GARBOW BS, HILL5TROM KE: Uses guide for Minpack-1. tion of arterial hypertension. It is hitherto unclear what impact ANL-80-74 Argoune National Laboratory, 1980, pp 1—28 this may have on the extent of uremic nephrocalcinosis. It is of18. HOVE K, SAND 0: Evidence for a role of calcium influx in the interest that renal calcium content in patients with arterial stimulation-secretion coupling in parathyroid cells, in Hormonal Control of Calcium Metabolism, Amsterdam, Excerpta Medica, hypertension is elevated [30]. The relationship between hyper- 1981, p 346 tension and nephrocalcinosis remains to be established. 19. 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