Production and metabolic clearance of calcitriol in acute renal failure

Kidney International, Vol. 33 (1988), PP. 530—535

Production and metabolic clearance of calcitriol in acute renal failure CHEN H. Hsu, SANJEEV PATEL, ERIC W. YOUNG, and ROBERT U. SIMPSON Division of Nephrology, Departments of Internal Medicine and Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109, USA

Production and metabolic clearance of calcitriol in acute renal failure. Metabolic clearance rate (MCR) and production rate (PR) of calcitriol were studied three and seven days after ischemic acute tubular necrosis (ATN). Creatinine clearance was decreased three days after clamping the renal arteries (0.42 0.03 mlIminJlOO g, N = 6 in ATN vs. 0.68 0.09, N = 7 in sham controls; P < 0.001). Plasma concentrations (24.1 1.9 pg/mI) and PR of calcitriol (9.8 0.91 ng/kg/day) were signifi-

also depend on the metabolism of calcitriol by its target organs. In this experiment, we have utilized the technique of constant infusion of radiolabelled calcitriol to study the metabolic production and clearance of calcitriol in acute renal failure.

7.3 pg/mI, and 29.6

Male Sprague-Dawley rats (160 to 230 g) were fed with Purina rat chow containing 1.0% calcium and 0.8% phosphorus and tap

cantly lower in ATN rats three days after ischemic insult when compared to sham control rats, respectively (76.6 3.3

ng/kg/day; both P < 0.01). The MCRs of calcitriol were not

different between ATN (0.28 0.02 mI/mm/kg) and sham control rats (0.27 0.01) By the seventh day after ischemic injury, when creatinine

clearance of ATN rats returned to normal, both the PR and plasma concentrations of calcitriol also returned to normal values in these animals. In order to assess the effect of uremia on calcitriol metabolism, MCR and PR of calcitriol were measured in rats with reinfusion of their

urines for 24 hours. The PR of calcitriol was significantly decreased (9.42 1.21; vs. controls, 20.5 2.9 ng/kglday, P < 0.001) in uremic animals. Since decreased PR of calcitriol was also accompanied by decreased MCR of calcitriol, plasma concentrations of calcitriol of the uremic rats with intact kidneys remained within normal values. We

conclude that the PR of calcitriol is decreased early in ATN rats. Although the MCR was not decreased in mild ATN rats, it may decrease in severe acute renal failure.

Calcitriol is synthesized in the kidney by conversion of 25-hydroxyvitamin D3 by the enzyme lcs-hydroxylase [1]. 1cr-

hydroxylase is localized in the mitochondria of proximal tubules [21. Therefore, the biosynthesis of calcitriol requires metabolically normal renal tubule. Although patients with acute tubular necrosis and severe chronic renal failure are expected to have decreased plasma concentrations of calcitriol, the synthesis of this active vitamin D metabolite has not been measured in

acute renal failure. Moreover, the effect of uremia on the metabolic degradation of calcitnol has not been studied in acute

Methods

water throughout the experiment. Acute tubular necrosis was achieved by clamping bilateral renal arteries for 40 minutes through bilateral flank incisions as previously described [4]. Control animals had sham operations. Metabolic clearance of calcitriol was performed in two groups of animals: one group rt three days and the other at seven days after the ischemic insult of the kidney. The radiolabelled calcitriol was infused into animals on day 2 through day 3, and day 6 through day 7 for 20 hours, respectively, after ischemic injury. MCR of calcitnol was measured at 18, 19 and 20 hours after the isotope infusion as described below. The effect of uremia on metabolic clearance of calcitriol was also conducted in another group of animals (230 to 260 g) with normal kidneys. Acute uremia was created in this group of rats by reinfusion of their urines for 24 hours as we have previously described [5]. Briefly, this was accomplished by means of an electronic switching device that was connected to the urine container by two wire electrodes. Whenever the animal excreted 0.5 ml of urine into the container, the switching device was triggered to activate a pump that infused 0.5 ml of urine back into the animal through a femoral venous catheter. Control animals were infused with saline solution at a rate of 0.005 ml/min/l0O g. The radiolabelled calcitriol was infused into rats four hours after urine reinfusion or saline infusion. MCR was measured at 18, 19, and 20 hours after the isotope infusion as described below.

renal failure. We have previously demonstrated that the production of calcitriol is decreased in chronic renal failure. The On the day of experiments, the rats were weighed and decreased production is attended by decreased metabolic clear- anesthetized with ether. Both femoral artery and femoral vein ance of calcitriol, suggesting that the degradation of calcitriol by were cannulated with polyethylene tubing (PE 50) for blood

various tissues in renal failure is also reduced [3]. These sampling and infusion purposes. A PE 50 tube was placed in the findings indicate that normal calcium homeostasis not only bladder for urine collection. After surgical instrumentation the requires normal plasma concentrations of calcitriol, but may animals were placed in individual restraining cages and allowed to recover from ether anesthesia. All animals were fasted during Received for publication February 24, 1987 and in revised form August 10, 1987

the clearance studies. Metabolic clearance rate (MCR) of calcitriol was measured by constant infusion method [6, 7]. Assuming that infused tracer 3H-calcitriol and endogenous

© 1988 by the International Society of Nephrology

calcitriol are metabolized identically in vivo, then it follows that

530

Hsu et a!: Calcitriol metabolism in renal failure

531

when the infused tracer 3H-calcitriol achieves a steady-state plasma concentration after a given period of infusion, the ratio of the infusion rate of 3H-calcitriol to the steady state concentration of 3H-calcitriol equals the ratio of the rate of calcitriol synthesis to the endogenous plasma concentration of calcitriol. Thus production rate (PR) of calcitriol can be calculated as

ments at 18, 19, and 20 hours averaged less than 7%. The

calcitriol (500 ng in 50 pi absolute ethanol) was added to

injury are summarized in Table 1. Body weights of sham

radioactivity of 3H-calcitriol extracted from plasma by HPLC ranged from 1300 to 2400 cpm/ml, which is 14% to 31% lower than the total plasma radioactivity. We have demonstrated that the straight phase HPLC system separates 1 ,25-dihydroxyvitamm D3 from 1,25,26-trihydroxyvitamin D3, calcitroic acid and follows: 1 ,24,25-trihydroxyvitamin D3. Furthermore, the peak of radioactivity comigrating with calcitriol on straight phase HPLC PR = Infusion rate of 3H-calcitriol/Mean steady-state plasma eluted as a single radioactive peak that co-eluted with standard concentration 3H-calcitriol x endogenous plasma concentration calcitriol on a reverse phase system (20% water in methanol) of calcitriol using a 5 s C-18 analytical column with excellent recovery (> and metabolic clearance rate of calcitriol (MCR) is defined as: 90%). The non-radioactive vitamin D metabolites were obtained from Dr. M. Uskokovic (Roche Institute). MCR = Infusion rate of 3H-calcitriol/Mean steady-state plasma At the end of the MCR study, arterial blood was obtained for concentration of 3H-calcitriol the measurements of pH, pCO2, ionized calcium, total plasma Radioactive calcitriol 1 a,25(26,27-3H)(OH)D3, specific activ- calcium, phosphorus, creatinine and calcitriol. Creatinine clearity 160 Ci/mmol (New England Nuclear, Boston, Massachu- ance was calculated using the 20 hours urine collection. Plasma setts, USA), in ethanol was mixed with 0.5 ml rat plasma and calcitriol was measured in duplicate using radioreceptor assay diluted with a sufficient volume of normal saline to infuse kits (Immuno Nuclear Corp., Stillwater, Minnesota, USA) intravenously at a rate of 0.0025 ml/min/lOO g. Approximately according to the method described by Reinhardt et al [8]. 0.005 pCi/hr of radioactive calcitriol was infused into each Analytical methods animal for 20 hours. Animals were fasted during the infusion Calcium was measured with atomic absorption spectrophoperiod. The total amount of radiolabelled calcitriol infused into each animal was approximately 3% of the estimated daily tometry (Model 306 Perkin Elmer, Norwalk, Connecticut). endogenous production of calcitriol. Arterial blood, 0.5 ml, was Phosphorus was determined by a method described previously withdrawn at 18, 19, and 20 hours. The blood was centrifuged [10]. Ionized calcium was measured by NOVA Model 6 calcium immediately, and plasma was separated and stored at —20°C electrode (NOVA Biomedical, Newton, Massachusetts, USA). until radioactive calcitriol was determined. The red blood cells Blood p1-I and pCO2 were measured by IL 713 pH/blood gas were mixed with saline equivalent to the plasma volume taken analyzer (Instrumentation Lab Inc., Lexington, Massachusetts, and returned to the animals. In our preliminary study, we have USA). Creatinine was determined by the Jaffe reaction. All data are expressed as mean SEM. Statistical analysis found that less than 1% (N 16) of the infused 3H-calcitriol tracer was excreted in 24 hours. Therefore, urine reinfusion into was performed using paired and unpaired Student (-tests wherrats does not change the infusion rate of 3H-calcitriol to the ever appropriate. A value of < 0.05 was considered significant. animals in this experiment. Results Plasma concentrations of radioactive calcitriol were meaRenal function and plasma concentrations of calcium, phossured as follows [8]: Two hundred microliters of thawed plasma were brought to a volume of 1 ml with normal saline. Cold phorus and blood gases of animals three days after ischemic controls (224 1.9 g) and animals with acute tubular necrosis (ATN; 229 5.8 g) were not different before the surgery. Three The supernatant was combined with 0.5 ml K2HPO4 (0.4 M, pH days after renal arterial clamping, the animals with ATN lost 10.6) and applied to a 3 ml LC-l8 Supelclean mini-column weight (216 3.0 g), whereas the weight of animals with sham 3.3 g). The creatinine (Supelco Inc., Bellefonte, Pennsylvania, USA) that had been operation remained the same (228 clearance of ATN rats was significantly decreased compared to equilibrated in methanol overnight. The column was eluted with the controls. Plasma concentrations of calcium and phosphorus 5 ml of double distilled water, 3 ml of 70% methanol and 6 ml of

monitor recovery. Plasma protein was precipitated with 1 ml acetonitrile followed by vigorous vortexing and centrifugation.

acetonitrile [81. The final fraction, containing hydroxylated and the values of blood gases were not different between the vitamin D metabolites, was dried under nitrogen. The residue two groups of animals. The production rate of calcitriol and its metabolic clearance was dissolved in 500 pi of HPLC solvent containing 88% in rats with acute renal failure are presented in Table 2. The hexane, 10% isopropanol and 2% methanol [91 and injected into individual MCRs and the mean MCRs of calcitriol of the two a HPLC solvent delivery system (Spectra Physics SP 8710, San groups of animals at 18, 19, and 20 hours after the infusion of Jose, California, USA) equipped with a 5 s silica analytical isotope are depicted in Figure 1. The MCR of each group of column (Alltech 605 SI, Deerfield, Illinois, USA). Solvent flow

rate was 2 ml/min, and 1 ml fractions were collected concurrently with the appearance of the cold calcitriol peak on a 254 nm optical detector (Spectra Physics 8300). Calcitriol recovery averaged 75%. Collected fractions were mixed with an organic phase scintillation liquid and counted (Beckman LS 8100 Scintillation Counter, Beckman Instruments, Fullerton, California, USA). Coefficients of variation for the plasma tracer measure-

animals achieved a steady state after 18 hours of infusion, since the mean MCRs measured at the above time intervals were not

different within each group of animals (paired f-test). The average MCRs of the three time intervals of sham controls and ATN rats are presented in Table 2. Plasma concentrations of calcitriol were significantly lower in the ATN animals three days after the ischemic insult than in the controls. Therefore, the calculated production rates of calcitriol were also signifi-

532

Hsu et a!: Calcitriol metabolism in renal failure

Table 1. Renal function and plasma coneentrations of calcium, phosphorus and blood gases t hree days after ischemic injury of kidneys Body wt

g

Sr

CCr

PCa

PCa

P,

ml/min/JOO g

mg/dl

mg/d!

0.68

10.30

4.73

mg/dl 7.05

Sham Controls

228

mg/di 0.42

ATN

216

0.66

0,42

10.26

4.75

<0.02

<0.001

<0.001

NS

NS

pCO2

pH 7.39

mmHg 34.5

7.59

7.39

34.4

NS

NS

NS

N= 6 N=7

P values

Abbreviations are: SCr, serum creatinine; Ccr, creatinine clearance; P and PCa+, total plasma calcium and ionized calcium; Ps,, plasma phosphorus; and ATN, acute tubular necrosis.

cantly lower in ATN rats. MCR, however, was not different between the two groups of animals. The coefficient of variation for the measurements of plasma tracer averaged less than 7%, suggesting that the plasma concentrations of 3H-calcitriol had reached steady state.

Table 2. Production and metabolic clearance of calcitriol three days after ischemic injury of kidneys Plasma calcitriol

Renal function and plasma concentrations of calcium and Sham phosphorus as well as blood gases of animals seven days after Controls

the ischemic insult are summarized in Table 3. The mean N= 7 plasma concentrations of creatinine five days after clamping the ATH N=6 renal arteries was 0.75 0.03 mgldl. This value was significantly higher than that of control sham-operated animals (0.49 .02 mgldl). By the seventh day after ischemic injury, the plasma concentrations of creatinine and creatinine clearances had already returned to normal values. Body weight of rats

prior to the surgery were not different between the sham controls (173

3.5 g) and ATN rats (172

76.6

MCR mi/mm/kg 0.27

PR ng/kg/day 29.6

24.1

0.28

9.8


NS

<0.01

pg/mi

P values

C.V.

% 5.13 6.85

NS

Abbreviations are: MCR, metabolic clearance rate (the value represents the average of three determinations performed at 18, 19, and 20 hours after the isotope infusion); PR, production rate; C.V., coefficient of variation for the plasma tracer measurements at 18, 19, and 20 hours.

2.4 g). Both groups

of animals had gained weight during the seven days after surgery, and body weights were not different (Table 3). Plasma

Discussion

concentrations of calcium and phosphorus and blood gases were also not different between the two groups of animals. The production rate and the MCR of calcitriol of rats seven

The kidney is the major organ capable of converting 25hydroxyvitamin D3 [25(OH)D31 to caicitriol. Thus, plasma days after the initial ischemic injury and control rats are concentrations of calcitriol are decreased during the oliguric presented in Table 4. The individual as well as the mean MCRs phase in rhabdomyolysis-induced acute renal failure [11]. In of calcitriol are depicted in Figure 2. The plasma concentrations patients with gram-negative sepsis and acute renal failure, of calcitriol and the production rates of calcitriol of ATN rats plasma levels of calcitriol are also reported to be lower [121. In had returned to normal, and were no longer different from those that report several patients may have developed peripheral tissue resistance to calcitriol. Calcitriol is the most potent of the of the control sham-operated rats. In order to examine whether severe uremia has any influence vitamin D metabolites in modulating intestinal absorption of on calcitriol metabolism in an animal with intact kidneys, calcium [1]. Intestinal absorption of calcium is impaired in renal another group of animals were made uremic by reinfusion of failure [13—151. Thus decreased calcium absorption in renal their urines for 24 hours, and calcitriol metabolism was studied failure could be due to either lower plasma concentrations of near the end of urine reinfusion. Plasma concentrations of calcitriol or decreased intestinal responsiveness to calcitriol creatinine and phosphorus of urine reinfused rats were signifi- stimulation. It is, therefore, important to characterize not only cantly elevated at the end of urine reinfusion as compared to the the production of calcitriol but also its metabolic clearance, controls. Total plasma concentrations of calcium were lower in since the latter may provide information about the degradation of calcitriol by its target organs in renal failure. uremic animals; however, plasma concentrations of ionized Acute tubular necrosis occurs following renal ischemia. The calcium were not different between the two groups of animals. proximal tubular changes include necrosis of tubular epitheThe animals with urine reinfusion became severely acidotic as hum, marked vacuolization of tubule cells, and swelling and the blood pH had decreased to 7.15 (Table 5). disruption of mitochondria [16]. Since the enzyme la-hydroxThe results of the effect of uremia on calcitriol metabolism in animals with normal kidneys are summarized in Table 6 and Figure 3. The plasma concentrations of calcitriol in uremic rats tended to be lower but were not different from the controls.

Both the production rate and the MCR of calcitriol were significantly decreased in uremic rats 24 hours after urine reinfusion.

ylase resides in the mitochondria of proximal tubule [2], it is not surprising that the synthesis of calcitriol is reduced in animals with ATN. Although metabolic acidosis [17—191 and retention of

phosphate and hyperphosphatemia [6, 20], which suppress calcitriol synthesis, may occur in severe acute renal failure, they were absent in the animals with ATN in this study. In ischemic as well as various etiologies of ATN, glomerular

Hsu et a!: Calcitriol metabolism in renal failure 3-Day control

3-Day ATN

Means

0.37

0.37 T

• •______•

0.37

0.32

0.32

LD

0.32

0.27

E

0.22

0

0.17

0.12

9::=

20 18 0.27 0.22

0.17

0.27

0.22

Fig. 1. Individual and mean metabolic clearance rats of caicitriol (MCR) at 18, 19, and 20 hours after infusion of radiolabeiled calcitriol in rats 3 days after ischemic injury of kidneys. Symbols are in Means: open,

0.17

0.12

0.12

20

19

18

19

533

20

18

19

Time, hours

control; closed, ATN.

Table 3. Renal function and plasma concentrations of calciurn, phosphorus, and blood gases seven days after ischemic injury of kidneys Body Wt g

Sham Controls

199

±5.5

N= 7 ATN

N=

8

SCr

Ccr mi/mini

mgidl

100 g

0.49 ±0.01

Pc

Pc** mg/dl

0.56 ±0.02

mg/dl 10.50

4.70

mg/dl 7.87

±0.11

±0.05

±0.16

10.39

P,

198

0.48

±3.5

±0.01

0.56 ±0.01

±0.12

4.73 ±0.08

7.54 ±0.27

NS

NS

NS

NS

NS

NS

P values

pH 7.42 ±0.02 7.45

±0.02 NS

pCO2 mm Hg 36.7

±2.5 35.2 ±1.1

NS

Abbrevi ations are in Table I.

7-Day control

Means

7-Day ATN

0.37

0.37

. 0.32

0.32

0.27

0.27

0.22

0.22

0.22

0.17

0.17

0.17

E

0.12

0.37

a__ •—

19

20

0.27

0.12

0.12 18

0.32

18

19

20

18

Time, hours

Sham

MCR

pg/mi

mi/mm/kg

115.8

0.26 0.01

0.27 ±0.01 NS

±2.6

ATN

105.0

N= 8 N =7

±4.9

seventh day after ischemic tubular necrosis, therefore, suggests

that normal production of calcitriol does not require fully regenerated tubular cells. Alternatively, compensatory mecha-

Plasma calcitriol

±8.9

20

control; (•) ATN.

Table 4. Production and metabolic clearance of calcitr jol seven days after ischemic injury of kidneys

Controls

19

Fig. 2. Individual and mean metabolic clearance rates of calcitriol (MCR) at 18, 19 and 20 hours after infusion of radiolabelied caicitriol in rats 7 days after ischemic injury of kidneys. Symbols are in Means: ()

%

nisms of calcitriol synthesis may also be involved in the recovery phase of ATN. Calcitriol could be predominately

42.1

4.9

±2.1

synthesized by the mildly injured and completely recovered

± 1.08

tubular cells. A compensatory mechanism of calcitriol synthesis occurs in acute unilaterally nephrectomized rats [221. Plasma concentrations of calcitriol remain within normal values in rats

PR ng/kg/day

41.2

C.V.

6.7

±1.5 NS

48 hours after unilateral nephrectomy. Parathyroid hormone, which may be elevated after nephrectomy, does not appear to Abbrev iations are in Table I. be the factor maintaining the normal levels of calcitriol [22]. On the other hand, plasma concentrations of calcitriol are greater than normal values during recovery phase in rhabdomyolysisfiltration rates usually return to near normal values approxi- induced acute renal failure [10]. The elevated levels correlate mately seven days after the initial insult [4, 21], yet tubular positively with the PTH concentrations. regeneration is often incomplete and lags behind recovery of In the present study, the MCR of calcitriol of animals with renal function [4, 211. The recovery of calcitriol synthesis on the ATN was not decreased, suggesting that the degradation of P values

NS

NS

Hsu et a!: Calcitriol metabolism in renal failure

534

Control

Uremia

Means

0.37

0.37

0.37 T

0.32

0.32

0.32

0.27

0.27

—a-—-——.

0.27 0.22

0.22

0.17

0.17

0.12

0.12 19

18

20

0.22

A_....—.A.,.O

AX

0.17

Fig. 3. individual and mean metabolic

0.12

19 20 Time, hours

18

18

g

Controls

N=

8

245

Scr mg/dl 0.4k

PCa

Ca

mg/dl mgldl mg/di 10.0 4.93 7.04

pH

Hg

7.38

37.2 ±1.1 33.4

4.87 12.0

7.15

±10.2 ±0.08 ±0.18 ±0.04 ±0.91 ±0.03

P values

NS <0.001 <0.001 NS <0.001 <0.001 NS

N= 8

8.78

Plasma calcitriol pg/mi

mm

Urine Reinfusion

2.17

Table 6. The effect of uremia on calcitriol metabolism in urine reinfused rats

pCO2

±11.4 ±0.01 ±0.20 ±0.03 ±0.17 ±0.01 250

19

in Means: (K;') control (•) uremia.

Table 5. Plasma concentrations of creatinine, calcium, phosphorus, and blood gases in animals with urine reinfusion for 24 hours Body wt

clearance rates of calcitriol (MCR) at 18, 19 and 20 hours after infusion of radiolabelled calcitriol in urine reinfused rats. Symbols are

I

±1.6

Abbreviat ions are in Table I.

Control

MCR mi/mm/kg

N=7

±7.0

Urine Reinfusion

±6.3

0.26 ±0.01 0.17 ±0.01

NS

<0.001

N= 8

P values

54.8 48.8

PR ng/kg/day 20.5

C.V.

% 5.3

±2.9

±0.9

9.42 ±1.21

±1.1

<0.001

NS

6.4

Abbreviati ons are in Tab Ic 2.

endorgan resistance to calcitriol may develop in severe uremic conditions. In the intestine calcitriol receptors mediate intestinecrotic kidney, the kidney is not considered a major organ of nal calcium transport [26]. The endorgan resistance to calcitnol calcitriol catabolism [23]. The loss of renal 24-hydroxylase could result from decreased calcitriol receptor sites or deapparently did not affect the metabolic degradation of calcitriol creased calcitriol binding affinity by receptors in the intestine. in ATN rats. We have demonstrated that unilaterally nephrec- The MCR of calcitriol of rats with urine reinfusion was signiftomized animals with a comparable degree of renal failure for icantly decreased in comparison to control animals. Since MCR three weeks had a significantly lower MCR of calcitriol com- of calcitriol occurs to a large extent in the intestine [27] and calcitriol is not altered in mild acute renal failure. Although loss

of the degradation enzyme, 24-hydroxylase, may occur in

pared to sham controls [31. Thus metabolic degradation of perhaps in the liver and bone as well [23], the decreased calcitj-iol could be decreased if the mild acute renal failure is metabolic degradation of calcitriol could account for the lower response of calcitriol-stimulated intestinal transport of calcium prolonged. The production of calcitriol was significantly reduced in the in uremic rats 24 hours after bilateral nephrectomy [25]. rats with uremia and normal kidney tissue. The animals developed severe uremia, acidosis and hyperphosphatemia. Both Acknowledgments metabolic acidosis [17—19] and hyperphosphatemia [20] have This work was supported by a grant-in-aid from the National Dairy

been shown to suppress calcitriol synthesis. It is not clear

Board administered in cooperation with the National Dairy Council and

whether uremic serum also suppresses la-hydroxylase. Since a grant-in-aid from the American Heart Association, the decreased production was associated with a concomitant Reprint requests to Chen H. Hsu, M.D., internal Medicine (Nephrolreduction of metabolic degradation of calcitriol, the plasma ogy), 3914 Taubman Health Care Center, University of Michigan, Ann concentrations of calcitriol remained within normal values in Arbor, Michigan 48109-0364, USA. these animals. The reasons for decreased MCR of calcitriol in these animals are not clear. Acute metabolic acidosis has been References shown to increase rather than decrease the MCR of calcitriol [17]. Phosphate is known to regulate metabolic production, but 1. DELuCA HF: The kidney as an endocrine organ for the production of 1 ,25-hydroxyvitamin D3, a calcium-mobilizing hormone. N Engi not metabolic clearance of calcitriol [6, 24]. Increased dietary J Med 289:359—365, 1973

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