The Influence of Renal Prostaglandins on Urinary Calcium Excretion in Idiopathic Urolithiasis

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INFLUENCE OF RENAL PROSTAGLANDINS ON URINARY CALCIUM EXCRETION IN IDIOPATHIC UROLITHIASIS A. C. BUCK,* C. J. LOTE

AND

W. F. SAMPSON

From the Department of Urology, Welsh National School al Medicine, Cardilf, and the Department ol Physiology, the Medical School, Birmingham, United Kingdom

ABSTRACT

Hypercalciuria is well recognized as an important factor in the cause of idiopathic calcium stone disease. Identification of the exact mechanism for the renal tubular handling of calcium has proved elusive, hence, treatment methods to alter the concentration of urine calcium in hype.rcalciu:ric stone formers have hitherto been non-specific. It is now well established that .renal prostaglandins influence intrarenal hemodynamics and tubular electrolyte excretion. As the renal handling of sodium and calcium is intimately related, the possibility that the mechanism underlying hypercalciuria may be prostaglandin mediated was considered. Experiments were performed in conscious Sprague-Dawley rats (n = 10) to determine the changes in calcium excretion following prostaglandin synthetase inhibition with indomethacin, Calcium excretion was significantly .reduced (p < 0.01), compared with control animals (n = 10). Further experiments were performed in anesthetized monkeys (Macaca fascicularis) to see if the inhibitory effect of indomethacin was reversible. Exogenous prostaglandin (PGE2) infusion :resulted in a marked calciuretic response without producing changes in glome.rular filtration .rate or blood pressure. Forty-three hypercalciuric patients we.re treated with a prostaglandin inhibitor fo.r periods ranging from 2 to 4 weeks, and all showed a significant fall in urinary calcium excretion to within the normal range. This clinical and experimental study suggests that p:rostaglandin (PGE2} is a hormone which determines the renal handling of calcium by influencing renal tubular function. on a rat cake diet, with free access to water. Each rat was lightly anesthetized with ether and a flexible cannula implanted into the tail vein. The animal was placed in a perspex restraining cage and allowed to recover from the anesthetic. A constant infusion of NaCl 0.9 per cent was begun into the tail vein at a rate of 5.8 ml. hours- 1 and the infusion continued for 6 hours. Urine samples were collected at 2 hours and thereafter, at hourly intervals, the 1st 2-hour period was allowed for stabilization. Voiding was encouraged by gentle sensory stimulation. Experimental animals (n = 10) received indornethacin (10 mg.kg.- 1 body weight) in buffered saline via the tail vein given over 15 minutes, beginning at 3 hours 52 minutes, while control animals (n = 10) received buffered saline alone over this 15minute period. The buffered saline was prepared according to the method of Ganguli and associates5 and consisted of 0.1 ml. 0.5 M. Na 2 Co3 (in which indomethacin was dissolved for the experimental series), 0.2 ml. 0.05 M HCl, 0.4 Inl. H20, and 0.5 wJ. 0.05 M HCl in 0.9 per cent sodium chloride. Statistical analysis. The results are presented as the mean ± standard error. Significance of differences between the control period (2 to 4 hours) and the experimental (indomethacin) period, (4 to 6 hours) were assessed by the paired test. Differences between the control and experimental groups of animals were assessed by Student's t test.

The well recognized diathesis of hypercalciuria and hyperoxaluria, which is believed to favor the precipitation of calcium salts and stone growth in patients with idiopathic urolithiasis, is mainly attributed to an increased gastrointestinal absorption of dietary calcium. 1• 2 Less commonly, a urinary "leak" of calcium due to faulty calcium conservation by the distal nephron is said to account for hype:rcalciuria, leading secondarily to a normocalcemic hyperparathyroidism in some patients. 3 In a clinical study, hypercalciuric stone formers appeared to have significantly higher glome:mlar filtration rates (GFR) when compared with age matched normal subjects, and in addition, the dynamic renal isotope scintigram in patients with nephrocal.cinosis showed localized isotopic densities v;ith enhanced functional wcn,,n, indicative of increased blood flow.4 This led us to .__,v,c,s,,wc, the possibility that renal prostaglandins, which axe known to influence renal hemodynamics and electroalso be involved in the mechanism of altered rrlcrn»"''""' filtration. We herein 0

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Part II: Our experience in the treatment of hypercalciuria with a prostaglandin inhibitor in a selected group of recurrent stone formers.

RESULTS

Urine flow. During the initial 2-hour equilibration period,

Part I

the urine flow increased to match, and usually exceed, the infusion rate, 96.7 µLminute.- 1 In the 2 to 4 hour infusion period, urine flow was stable, and was not significantly different in the 2 groups of animals. However, in the 4 to 6 hour period, flow fell significantly in the indomethacin treated animals (p < 0.05, n = 10), but did not change in the control animals (n = 10). Calcium excretion. The time course of changes in calcium excretion are shown in figure l. The urinary calcium output in the 2-hour period after Indomethacin administration was significantly lower than the urine calcium excretion in the 2 hours

1) Experiments in Rats MATERIALS AND METHODS

Experiments were performed in male rats (Sprague-Dawley, weight 250 to 400 gm.) which had previously been maintained Accepted for publication November 24, 1982. * Requests for reprints: Welsh National School of Medicine, Department of Urology, Cardiff Royal Infirmary, Cardiff, United Kingdom. 421

422

BUCK, LOTE AND SAMPSON

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over a 15-minute period via the cannula in the femoral vein. One hour after the indomethacin infusion prostaglandin (PGE2) in a dose of 0.2 µg./kg.- 1 minute- 1 was infused intraarterially for a 30-minute period. Urine was collected for a further halfhour period after the infusion of prostaglandin (PGE2). Urine volume, calcium, sodium, potassium excretion and endogenous creatinine clearance were measured for each 30-minute period. Analytical procedures for urine. The volume of each urine sample was recorded. Urinary sodium and potassium were determined by flame photometry by means of a Beckman flame photometer with lithium internal standard. Urine calcium was measured by atomic absorption spectroscopy. Statistical analysis. As in the rat experiments, the results are presented as the mean± standard error. Significance of differences between the control and experimental periods was determined by means of the paired t test. Differences between control and experimental groups were determined by Student's t test.

6

Fm. 1. Urine calcium output in experimental rats (n = 10) and control rats (n = 10). Experimental animals received indomethacin (10 mg./kg.) at 3 hours 52 minutes. There was a significant fall in urinary calcium excretion in the 4 to 6 hour period in experimental animals compared with the 2 to 4 hour period (p < 0.01) and in comparison with the control animals (p < 0.05). Values reported are mean ± standard error.

before indomethacin (p < 0.01, paired t test); and also decreased in comparison with the urine calcium excretion in the 4 to 6 hour period in the control animals (p < 0.05, unpaired t test). The mean urinary calcium excretion for the 2-hour period before indomethacin in the experimental animals (n = 10) was 0.24 ± 0.04 µmol.minute-1; in the 2-hour period after indomethacin this was reduced to 0.16 ± 0.03 µmol.minute- 1 (p < 0.01).

Experiments in Cynomolgus Monkeys (Macaca Fascicularis) [2) Experiments in Cynomolgus Monkey 6} MATERIALS AND METHODS

To determine whether the inhibitory effect of indomethacin on calcium excretion was reversible by exogenous prostaglandin replacement, experiments were performed on anesthetised monkeys (Macaca fascicularis, n = 4, weighing 4.8 to 6.4 kg.). The animals were anesthetised with pentobarbitone sodium (25 mg./kg.) administered intravenously and maintained in a light plane of surgical anesthesia. An intravenous injection of 1 mCi of 99m Tc DTP A (Sn) was given and the renal clearance of the isotope continuously monitored throughout the experiment by • means of a cadmium telluride detector placed over the chest. The femoral artery and vein were cannulated; the tip of the arterial cannula was sited in the aorta to lie proximal to the origin of the renal arteries. Saline 0.9 per cent was infused through the intravenous cannula at a rate of 3.5 ml./minute throughout the course of the experiment. Saline 0.9 per cent was infused through the arterial cannula by means of an infusion pump set to deliver infusate at a rate of 0.3 ml./minute. The intraarterial cannula was coupled to an Elcomatic EM 750 pressure transducer for the continuous measurement of blood pressure which was recorded on an RAF patient monitoring system Type IV with heart rate and BP meter. A midline abdominal incision was made and the ureters cannulated for urine collections. The experiment was begun when 3 5-minute urine volumes were stable within 0.5 ml. from each kidney. The study comprised 5 infusion periods of 30 minutes duration each, urine samples were collected at the end of each period; the 1st half-hour collection was taken as the control. Midway through each period venous blood samples were obtained for the measurement of creatinine clearance. Normal saline was infused into the femoral vein and aorta. Buffered indomethacin (10 mg./kg. body weight) was given

RESULTS

Calcium excretion. The time course of the effect of endogenous prostaglandin inhibition with Indomethacin followed by exogenous prostaglandin infusion are shown in figure 2. Urine calcium output in the 1st half-hour control period was 2.83 ± 0.20 µmmol.minute 1 • The intravenous infusion ofindomethacin produced a significant fall in urinary calcium to 2.00 ± 0.39 µmol.minute- 1 at a half-hour and to 2.07 ± 0.21 mol.minute- 1 at 1 hour after indomethacin infusion (p < 0.001). The intraarterial infusion of PGE2 resulted in a marked calciuretic response with a significant increase in calcium output to 4.93 ± 0.24 µmol.minute- 1 above the control level (p < 0.001). There was a rapid return to normal levels in the final half hour control period; 2.76 ± 0.13 µmol.minute.- 1 Electrolyte and water excretion. The time course of changes in sodium and potassium excretion to indomethacin and prosINHIBITION AND STIMULATION OF PROSTAGLANDIN MEDIATED CALCIUM EXCRETION IN THE MONKEY (MACACA FASCICULARIS) (N:4)

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423

PROSTAGLANDINS AND URINARY CALCIUM EXCRETION

taglandin infusion are shown in figure 3, A, B and C. The decrease in sodium and potassium excretion at the half and 1 hour period after indomethacin was not statistically significant. However, PGE2 infusion significantly increased both the sodium and potassium output (p < 0.001). Urine output for the control period was 38.25 ± 2.92 ml./30 minutes; this fell to 29.96 ± 1.62 ml./30 minute at 1 hour after indomethacin (p < 0.05). PGE2 infusion produced a marked diuresis with the urine output increasing to 91.19 ± 4.17 ml./30 minute above that of the control period (p < 0.001). GFR. Although there was some fluctuation in creatinine clearance throughout the course of the experiment, a significant fall in creatinine clearance (GFR) was apparent only at one hour after indomethacin compared with the other infusion periods. However, GFR measured by means of 99m Tc DTPA (Sn) isotope clearance was stable throughout the experiment with no significant changes in any of the infusion periods (fig. 4).

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Part II

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PATIENTS AND METHODS 0

On the basis of the results of these animal experiments we studied the effect of prostaglandin inhibitors in a selected group of 43 hypercalciuric recurrent stone formers: indomethacin (28 patients), Flurbiprofen (15 patients). Urinary calcium excretion, creatinine clearance GFR and urine volume was measured over a 24-hour period on 3 occasions before and after a course of treatment with indomethacin (25 mg. t.i.d.) or Flurbiprofen (50 mg. t.i.d.) ranging from 2 to 4 weeks.

Control 30

DISCUSSION

Parts I and II The kidney is a crucial organ for the regulation of calcium and phosphate metabolism and in recent years a considerable amount of research has focused on the renal handling of these elements. Tubular calcium reabsorption prevents the large amounts of calcium filtered at the glomerulus (about 8 to 10 gm./d) from being excreted in the urine, thus both the filtered load of calcium and tubular re-absorption are important interrelated aspects of renal function in hypercalciuria. Approximately 60 per cent of filtered calcium is reabsorbed in the proximal tubule, both by coupled transport with sodium and by passive movement. 6 ' 7 Although only a minor portion of filtered calcium is reabsorbed in the distal nephron, particularly the thick acending limb of Henle's loop and distal convoluted

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RESULTS

Urine calcium excretion. The mean pretreatment 24-hour urine calcium excretion in the indomethacin treated group of patients was 11.1 ± 0.53 mmol. d. -I This fell significantly to 6.19 ± 0.25 mmol. d- 1 with indomethacin (p < 0.0005) (fig. 5). The mean 24-hour urine calcium excretion before treatment with Flurbiprofen was 10.89 ± 0.49 mmol. d- 1 and this fell to 6.28 ± 0.49 mmol. d- 1 with treatment (p < 0.001) (fig. 6). Creatinine clearance (GFR). The creatinine clearance GFR in 22/28 patients treated with indomethacin was 121 ± 3.57 ml.minute- 1 and decreased to 104 ± 6.6 ml.minute- 1 after treatment (p < 0.0005). The creatinine clearance GFR in Flurbiprofen treated patients fell from 124.36 ± 4.59 ml.minute- 1 to 106.24 ± 6.00 ml.minute- 1 (p < 0.05). The decrease in creatinine clearance GFR was statistically significant in both groups of patients but not clinically significant when corrected for age. Urine volume. The mean urine output over 24 hours for the 3 days before indomethacin administration was 2293 ± 154 ml. d- 1 and this decreased to 1807 ± 106 ml. d- 1 after indomethacin (p < 0.01). In the group of patients treated with Flurbiprofen the pretreatment urine output was 2080.6 ± 89.5 ml. d- 1 and after treatment was 1966.6 ± 106.4 ml. d- 1 (not significant).

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424

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EFFECT OF INDOMETHACIN ON URINE CALCIUM EXCRETION IN 28 HYPERCALCIURIC STONE FORMERS

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of renal calculi has been concerned with the physicochemical aspects of crystal aggregation upon a matrix nucleus, promoted or prevented by the absence or presence of inhibitors of crystallisation in the urine. 10• 11 The matrix-nucleation-crystallization and the inhibitor-absence theories fail to account for the gross and microscopic foci of calcium phosphate deposition which are seen to occur within the substance of the kidney, principally affecting the renal medulla and papilla. 12' 13 Histologic studies have clearly demonstrated the precipitation of calcium salts to be within the interstitial cells, the epithelial tubular cells of Henle's loop and the collecting duct cells, often surrounded by an inflammatory infiltrate. 14 This pathologic lesion which is seen to affect all age groups but which is 2 to 3 times more common in males than females, is generally regarded as the precursor of the true calcium oxalate calculus, which begins to form when, as a result of necrosis of the tubular epithelium, the calcium concretion breaks through to establish communication with the tubular lumen and is exposed to urine super-saturated with crystalloid (fig. 7). 12' 15 These events suggest that the pathogenesis of stone formation begins in the interstitial tissues and the renal tubular cell. A valid and acceptable mechanism for stone formation must explain both intranephronic focal calcification and urinary crystal aggregation, or else provide a link between these 2 anatomical and physicochemical concepts of calculogenesis. Investigations into the actions of renal prostaglandins indicate that their major effect on kidney function in man, as in other species, is concerned with the regulation of renal blood flow and electrolyte excretion by the kidney. 16' 17 Several pathologic conditions are accompanied by an immediate release of renal prostaglandins. However, the increase in prostaglandin synthesis resulting in changes in intrarenal hemodynamics which is seen to occur following acute and chronic ureteral obstruction, 18• 19 is particularly relevant to renal stone disease. Hypercalciuric stone formers with a higher incidence of obstructing calculus as well as the presence of a stone or nephrocalcinosis and perhaps even intranephronic calcification causing renal tubular obstruction could conceivably provide the local stimulus for prostaglandin synthesis reflected in the hemody-

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FIG. 7. Biopsy of kidney showing focal calcification in the interstitial tissues and tubules, with inflammatory infiltrate, in the renal medulla (von Kossa stain for calcium phosphate).

namic effects of an increased GFR and the scintigraphic appearances of focal areas of increased functional activity which we have observed in our clinical study. 4 There is good circumstantial evidence to suggest that prostaglandins may influence calcium reabsorption by the kidney, as prostaglandins have been shown to alter sodium chloride and calcium transport across frog skin, which can be regarded as a model epithelium for renal tubules. 20 More significantly, indomethacin has been effective in normalizing the severe hyassociated with Ba.rtter's syndrome and congenital tubulopathies. 21 · 22 The argument is strengthened by the observation that frusemide, a drug shown to depress calcium and sodium reabsorption in experimental animals,7 produces both natriuresis and hypercalciuria in man. 23 Recent studies have shown that frusemide enhances prostaglandin production, 24 and inhibits prostaglandin degTadation. 25 This therefore, could be a mechanism for frusemide induced hypercalciuria. Haylor and Lote 26 demonstrated a significant reduction in water and sodium excretion following prostaglandin synthesis inhibition with indornethacin without producing changes in GFR and renal plasma flow. The present experiments in conscious rats confirm these findings and in addition show that calcium excretion was significantly reduced when endogenous prostaglandin was inhibited with indomethacin. The striking calciuretic response with PGE2 in ""~'""vr~ independent of changes in GFR and blood pressure suggest that this renal could determine the renal handling of calcium controlling tubular nu1ct:1m1, in a similar manner to its known effect on sodium and po;;a,ss1urn excretion which has been demonstrated intraa1-terial infusions of PG E2 and prostaglandin precursors 27 in the The marked reduction in urinary calcium excretion with indomethacin and ,ms.. ~-,n,,~~ in the hypercalciuric patients we have studied is further evidence that renal prostaglandin is an important factor in the mechanism of hypercalciuria which hitherto has not been recognized. This experimental and clinical study suggests that PGE2 could be an important determinant of the renal handling of calcium. The subcellular distribution of cyclo-oxygenase enzyme catalysing the production of these hormones indicates that PGE2 bio-synthetic systems are highly concentrated in the collecting duct epithelial cells and interstitial tissues. The question that arises is: What might be the predisposing factors in nephrolithiasis which interact to produce the pathophysiologic changes resulting in increased prostaglandin synthesis? First, it appears that phospholipase A2 enzyme in cells is activated by an increase in free intracellular calcium ions. 28 In other locations prostaglandins have been shown to behave as calcium ionophores and may

facilitate the Inovement of calcium into and out of cell membranes.29 Moreover, elevated calcium concentration within the renal tubular cell and interstitial tissues, resulting from an increased delivery of calcium to the excretory system, can interfere with key enzymes of intermediary metabolism, such as Na-K-ATPase of cell membranes and cause cellular death. 3032 The precipitation of calcium salts within damaged interstitial tissues and tubular epithelial collecting duct cells is a common finding in the kidneys of patients with a history of renal stones. 12' 15 The cellular injury associated with such a lesion in conjunction with a secondary inflammatory response could undoubtedly be the challenging event stimulating the synthesis and release of prostaglandins, as indeed prostaglandins are known to be important mediators of inflammation in other tissues. This study would indicate that prostaglandins formed in the kidney have other important physiologic and pathologic roles, such that PGE2 formed by the collecting ducts function in the calcium re-absorption process and perhaps cortical PGI2 has a predominantly hemodynamic action. The importance of these observations in the mechanism ofhypercalciuria and the pathogenesis of renal calculus disease cannot be underestimated and is the subject of further research. REFERENCES 1. Robertson, W. G., Peacock, M. and Nordin, B. E. C.: Activity

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14. 15.

16.

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