Renal 25-hydroxyvitamin D3-1-hydroxylase in patients with renal disease

Kidney International, Vol. 34 (1988), pp. 712—716

Renal 25-hydroxyvitamin D3-1-hydroxylase in patients with renal disease KENIcHI SATOMURA, Y0sHIKI SEIN0, KANJI YAMAOKA, Y0sHIYuKI TANAKA, MAKOTO ISHIDA, HYAKUJI YABUUCHI, YoKo TANAKA,1 and HECTOR F. DELUCA Department of Pediatrics, Osaka University Hospital, Osaka University School of Medicine, Osaka, Japan, and Department of Biochemistry, University of Wisconsin-Madison, College of Agricultural and Life Sciences, Madison, Wisconsin, USA

Renal 25-hydroxyvitamin D3-1-hydroxylase in patients with renal disease. Renal 25-hydroxyvitamin D3-l-hydroxylase (1-hydroxylase) activ-

ity has been measured in 20 patients with renal disease using the remaining portion of needle renal biopsy specimens taken for diagnostic

purposes and in five patients using kidney tissue removed during transplantation. The 1-hydroxylase activity of 12 patients with asymptomatic proteinuria and/or hematuria (group A) measured 83.2 37.7 pg/mg tissue/20 mm. Since these 12 patients did not show impaired mineral metabolism or pathological changes in the renal tubules, we have presumed that these results indicate normal activity in man. We also measured the 1-hydroxylase activity in four patients treated with prednisolone (group B). The 1-hydroxylase activity (81.1 27.1 pg/mg tissue/20 mm) of group B did not differ from that of group A. However,

the urinary excretion of calcium (ratio of calcium/creatinine) was 0.07 vs. 0.07 increased (0.18 0.03, P < 0.01) by prednisolone therapy. These data suggest that glucocorticoid-induced changes in

urinary calcium excretion are not the result of a direct effect of glucocorticoid on renal 1-hydroxylase. In the three patients with mild renal insufficiency (group C), the 1-hydroxylase activity (75.4 22.4 pg/mg tissue/20 mm) did not differ from that of group A. However, in five patients with severe renal insufficiency (group D), the 1-hydrox3.7 pg/mg tissue/20 mm) was significantly deylase activity (8,5 creased (P C 0.01).

ylase activity, they were not applicable to small biopsy specimens. Tanaka and DeLuca [81 have developed a technique for the measurement of mammalian renal 1-hydroxylase activity that uses substantial amounts of non-radioactive 25-OH-D3 substrate to saturate the inhibitor protein. More recently, we have developed a micro assay modification of that method for use with small biopsy specimens for clinical use [9]. In patients with renal disease, vitamin D and mineral metabolism may be impaired by the reduction of nephron mass and the effects of the administration of some drugs. Although serum levels of 1 ,25-(OH)2D have been studied in patients with renal disease, l-hydroxylase activity has not been measured. In this study we have measured 1-hydroxylase activity in patients with asymptomatic proteinuria and/or hematuria, in patients treated with prednisolone, and in patients with mild or severe renal insufficiency.

Methods

As shown in Table 1, we measured 1-hydroxylase activity in 12 patients with asymptomatic proteinuria and/or hematuria (group A, normal renal function), four patients taking prednisVitamin D undergoes sequential 25-hydroxylation in the liver olone therapy (group B, normal renal function, two had lupus and 1-hydroxylation in the kidney to become an active form that regulates calcium and phosphorus metabolism [1]. 25-Hydroxyvitamin D3- 1 -hydroxylase (1-hydroxylase) has been studied

nephropathy and the other had idiopathic nephrotic syndrome), three patients with mild renal insufficiency (group C, glomerular filtration rate (GFR); 50 to 70 ml/min/l .73 m2), and four patients

extensively in avian kidneys because the existence of 25- with severe renal insufficiency on chronic hemodialysis or hydroxyvitamin D3 (25-OH-D3) binding protein in mammalian kidney reduces the availability of substrate to the enzyme [2, 3]. Recently, 1-hydroxylase activity was detected in mammalian kidney homogenate [4], mitochondria [5], isolated cells [6], and slices [7]. These preparations required extensive washing with buffer to eliminate the inhibitor. Since these methods require a relatively large amount of kidney tissue to detect the I -hydrox-

continuous ambulatory peritoneal dialysis (CAPD; group D). The GFR in another patient of group D was 3.5 ml/min/l.73 m2. The patients of group A had not received any agents that affect calcium or phosphorus metabolism. The patients of group B were being treated with 15 to 20 mg of prednisolone daily at renal biopsy and had been treated with prednisolone for 45 to

105 days before biopsy. The four patients of group D on hemodialysis or CAPD had been treated with 1-hydroxyvitamin

Present address: VA Medical Center (ill), 113 Holland Avenue, Albany, NY 12208.

Received for publication October 15, 1987 and in revised form June 6, 1988

© 1988 by the International Society of Nephrology

D5 (l.OH-D3) until one day before renal transplantation for prophylaxis of renal osteodystrophy. Except in group D, the kidney specimens were obtained by an ultrasound-guided needle biopsy of the lower pole of the kidney. In the five patients with severe renal insufficiency, 200 to 400 mg of kidney specimen was obtained from the outer layer during kidney transplan712

713

Satomura et a!: 25-OH-D3-1-hydroxylase activity in renal disease

Table 1. Clinical findings in patients Case no.

Group A

1

2 3 4 5 6 7 8 9

GroupD

9 8 9 10

7

12 13

14

14

17

15

14

16

16

17 18

22

11

GroupC

11

12 14 13 10 15 15

10

Group B

Age (year)

Sex M

10—20

+ +

F F

20—30

M

30—40 20—30

+ +

>100

2+

10—15

+ +

F F F F F

2—3

—

2+

90—100

M

10—20

+

F F F F

2—3

—

—

—

—

—

—

+ 2+

M M M

7 9

20

11

21 22 23

12

10

F

24

13

M

M M M

—

2—3

10—20

ND ND ND ND ND

ethanol). A control was always run in which 1 l of 95% ethanollmg tissue was added to a comparable tube. In severe renal insufficiency, the functional tubules might not be evenly distributed in the cortex. To get a representative sample, more than 100 mg of kidney tissue was homogenized and then one ml

of homogenate per tube (25 mg per tube) was used for the enzyme assay. We assumed that the 1-hydroxylase activity measured by this method represented the 1-hydroxylase activ-

ity of the kidney. Incubation was carried out at 37°C with

F

>100 >100

M

19

5

Urinalysis Protein RBC/HPF

homogenate. The test tubes were placed in a water bath shaker at 37°C and reaction was initiated by the addition of 25-OH-D3 dissolved in 95% ethanol (1 g of 25-OH-D3/mg tissue/id of 95%

—

+ + ND ND ND ND ND

Abbreviations are: RBC/HPF, red blood cell/high power field; ND, not determined.

tation. The parents of all children had given their informed consent for renal biopsy. Blood samples were obtained in the fasting state for serum analysis and determination of renal function. Serum calcium

shaking at 100 oscillations/mm for 20 minutes. The reaction was terminated by the addition of five volumes of methylene chloride. Next, 5000 dpm of l,25-(OH)2-[3H]D3 dissolved in 20 d of 95% ethanol was added to the assay tubes to monitor recovery of 1 ,25-(OH)2-D3 through extraction and chromatography. The mixture was vigorously vortexed and methylene chloride layer

was then carefully removed with a syringe. The remaining aqueous phase was re-extracted twice with the same volume of methylene chloride. The combined methylene chloride extracts from a total of three extractions were evaporated under nitrogen and the residue dissolved in 10% 2-propanol in hexane. Separation of 1 ,25-(OH)2-D3 was carried out by high performance liquid chromatography with a Model LC-3A Chromatograph (Shimazu Corp., Kyoto, Japan) equipped with a 4.5 mm x 25 cm Zorbax-Sil column (Dupont, Wilmington, Delaware, USA) and a 254 nm UV absorbance detector. Chromatography was carried out with 10% 2-propanol in hexane at 30 kg/cm2 and

a flow rate of 2 mllmin. The elution fraction containing

1 ,25-(OH)2D3 was collected, dried under nitrogen, and dissolved in 500 .tI of 95% ethanol. One aliquot was used for [10], inorganic phosphorus [11], and creatinine [12] levels were determination of [3H] to calculate recovery of I ,25-(OH)2D3. determined by automated methods. Urinary calcium, phospho- Duplicate aliquots were assayed for 1 ,25-(OH)2D3, which was rus, and creatinine were determined by an autoanalyzer from a measured by competitive protein binding assay [14], using twenty-four hour collection. The creatinine clearance, cor- chicken duodenal cytosol (Yamasa I ,25-(OH)2D3 receptor [15]; rected for 1.73 m2, was used to express the GFR. The tubular Yamasa Shoyu Co., Japan) as the binding protein. The intrathreshold for phosphate (Tmp/GFR) was determined on the assay coefficient of variation was 7.1%, the inter-assay coeffimorning of the serum analyses and derived according to the cient of variation was 14.8%, and the sensitivity was 2 pg/tube. Bijvoet nomogram [13]. The apparent l,25-(OH)2D3 detected in the tubes incubated The biopsy specimen was placed in a petri dish with ice-cold 15 mM Tris-acetate buffer (pH 7.4 at room temperature) con- without substrate was subtracted from the value determined taining 0.19 M sucrose, 2 mM EGTA, and 2 mivi dithiothreitol with samples incubated with substrate. Renal 1-hydroxylase activity was measured in pig prepara(DTT). It was examined directly by a stereomicroscopy. Since tions. Pig kidney was removed from immature (60 lb.) pigs and the medulla had a parallel linear striated appearance and the cortex had a tortuous structure, the cortex was easily distin- placed in the same ice-cold buffer as described above. The guished from medulla. In this study, we did not use specimens specimens were taken from the outer layer of the kidney. The that contained the medulla or the cortical-medullary junction. enzyme assay was performed as described above. Data are presented as mean SD. Wilcoxon rank test was The cortical specimen was divided into two portions for histoused to compare differences between values obtained. logical analysis and enzyme assay. After blotting the excess water by paper, the specimen for enzyme assay was placed in a preweighed aluminum foil boat containing a small amount of the same buffer. The weight without the specimen was subtracted from the weight with the specimen. A 2.5% (wt/vol) homoge-

nate was prepared in the same buffer using a micro grinder (American Scientific Products, McGraw Park, Illinois, USA). A 75 msi succinate solution was also prepared in the same buffer. The homogenate and the succinate solution were divided into two test tubes. The succinate solution was always added to the homogenate in an amount equal to one-half the volume of the

Results

The production of 1 ,25-(OH)2D3 is linearly related to the weight of the pig kidney specimen (Fig. 1; r = 0.988, P <0.01). The weight of kidney tissue of the patients used per tube ranged from 1.5 mg to 4.3 mg in groups A, B, and C, respectively. The clinical description of the patients is shown in Table 1, while the laboratory findings are shown in Table 2.

714

Satomura et at: 25-OH-DI-hydroxylase activity in renal disease

concentration of calcium and inorganic phosphorus did not differ from those of group A. Serum levels of 25-OH-I) and l,25-(OH)2flwere 12.8 2.8ng/mland77.9 4.1 pg/mlanddid

1500

not differ from those of group A. On the other hand, Tmp/GFR in group C was significantly lower (3.45 0.43 vs. 4.30 0.45 mg/dl 100 ml OF, P C 0.05) than that of group A (Table 2). Histological study by light microscopy showed there was focal atrophy in the tubules (Table 3). The l-hydroxylase activity did not differ from that of group A (Fig. 2).

E 1000 a, 1.,

V 0

0 0

r 0.988 Pc 0.01

I 500

N

4.0

8.0

12.0

16.0

20.0

Weight, mg

Fig. 1. The correlation between the production of I,25-(OH)2D3 and wet pig kidney weight.

Group D The histological study of group D showed end-stage kidney failure including extensive tubular atrophy (Table 3). The renal 3.7 pg/mg tissue/20 mm) was 1-hydroxylase activity (8.5 significantly lower (P < 0.01) than that of both groups A and C (Fig. 2). Although serum level of calcium and 25-OH-D did not differ from group A, serum levels of phosphorus (8.1 1.5 mg/dl vs. 4.7 0.4 mg/dl, P <0.01) and l,25-(OH)2D (41.4 18.3 pg/mI vs. 66.5 19.5 pg/mI, P C 0.05) were significantly different from those of group A (Table 2). The serum level of calcium, phosphorus, 25-OH-I), and 1 ,25-(OH)2D in another end-stage renal failure patient not treated with 1-OH-I)3 was 6.4 mg/dl, 9.2 mg/dl, 21.5 ng/ml, and 19.5 pg/mi, respectively. The l-hydroxylase activity of this patient was 13.6 pg/mg tissue/20 mm. Discussion

Recently, Brown and DeLuca reported [16] that l0-oxo-l9nor-25-OH-D3 exactly coelutes with 1 ,25-(OH)2D3 on normal Group A The stature and body weight were within normal range (data

not shown). Serum level of calcium, inorganic phosphorus, 25-OH-D and 1,25-(014)20 ranged from 8.6 to 9.8 mg/dl (age matched control: 8.6 to 9.8 mg/dl), 4.0 to 5.5 mg/dl (age matched control: 3.9 to 5.5 mg/dl), 12.3 to 30.1 ng/ml (normal children: 8.0 to 36.1 ng/ml [14]), and 29.2 to 80.0 pg/mI (normal children: 19.9 to 80.4 pg/mI [14]) were within the normal range, suggesting no evidence of impaired mineral metabolism. Normal GFR suggests that there had been no impairment of renal function in these patients (Table 2). Histological study by light microscopy showed the tubules were normal in these cases (Table 3). Their renal 1-hydroxylase activity measured 83.2 37.7 pg/mg tissue/20 mm (Fig. 2).

Group B Normal GFR suggests that there was no impairment of renal function. Serum concentration of calcium and phosphorus, and Tmp/GFR were also in the normal range. On the other hand, the ratio of urinary calcium/creatinine (0.18 0.07 vs. 0.07 0.03, Pc 0.01) and the serum level of 25-OH-D (10.5 2.2 ng/ml vs. 18.9 6.1 ng/ml, P < 0.05) were significantly different from those of group A (Table 2). Histological study by light micros-

phase HPLC using the same system used in this study. The presence of this compound in the sample could interfere with the enzyme assay by binding to the receptor for 1 ,25-(OH)2D3 [16]. However, Okabe et al reported [17] that the 10-oxo-19-

nor-25-OH-D3 does not bind specifically to the receptor for 1,25-(OH)2D3. Brown and DeLuca [161 also indicated that lO-oxo-l9-nor-25-OH-D is not formed in significant amounts in

incubations of intact mitochondria. Also, using this assay system neither 10-oxo-19-nor-25-OH-D3 nor 1,25-(OH)2D3 were

detected in preparations from thyroparathyroidectomized rats [9]. Thus, it is quite unlikely that lO-oxo-l9-nor-25-OH-D3 is formed or detected by our assay system. The l-hydroxylase activity in patients with asymptomatic proteinuria and/or hematuria (group A) was 83.2 37.7 pg/mg

tissue/20 mm. Since these patients did not show impaired mineral metabolism or pathological change in renal tubules where the 1-hydroxylase is found, we assume that these results

represent normal values of renal 1-hydroxylase activity in children. In addition, the l-hydroxylase activity from normal five-month-old rats is 53.1

21 pg/mg tissue/20 mm [91; our

results from the patients of group A are also in accord with these values. It has been suggested that glucocorticoid-induced alterations

in calcium metabolism are responsible for the decrease in copy showed that the tubules were normal (Table 3). The intestinal calcium absorption produced by glucocorticoid ex1-hydroxylase activity did not differ from that of group A (Fig. cess. However, the reported results are contradictory. Initial 2).

Group C The mean OFR was significantly lower (65.1 6.1 vs. 105.6 14.5 ml/min/l.73 m2, P C 0.05) than those of group A. Serum

studies showed a glucocorticoid-induced decrease in both serum 25-OH-D [18] and l,25-(OH)2D [191, but more recent work has shown that 25-OH-I) is normal [20] and that 1 ,25-(OH)2D is

elevated [21] or unchanged [221. Using a primary monolayer

culture of mouse kidney epithelial cells, Fukase et al [231

715

Satomura et a!: 25-OH-D3-1-hydroxylase activity in renal disease

Table 2. Laboratory findingsa GFR ml/minll.73 m2 Blood Calcium mg/dl Phosphorus mg/dl 25-OH-D ng/ml 1,25-(OH)2D pg/mI

Tmp/GFR mg/dl 100 GF Urine Calcium/creatinine

105.6

9.2 4.7 18.9 66.5 4.03

0.07

Group C 65.1 6.1"

Group B

Group A

110.8

20.3

0.4

9.1

0.5 6.1

4.3

0.8 1.0 2.2"

14.5

10.5

8.1

1.4 1.5'

12.8

0.7 1.0 2.8

21.9

6.3

4.1 0.43"

41.4 183b ND

0.08

ND

9.0 4.6

0.45

53.8 4.00

0.71

77.9 3.45

0.03

0.18

0.07"

0.08

19.5

12.3

Group D NDd 8.7

Abbreviations are: GFR, glomerular filtration rate; Tmp/GFR, renal threshold of phosphorus; ND, not determined. Values are the mean SD. "Significantly different (P <0.05) from the value in group A. C Significantly different (P < 0.01) from the value in group A. d The GFR of case no. 24 showed 3.5 ml/minll .73 m2. Table 3. Histological findings Group A Minor glomerular abnormalities with normal tubules Focal GN with normal tubules Diffuse mesangial proliferative GN with normal tubules Membranous GN with normal tubules Group B Minor glomerular abnormalities with normal tubules Diffuse mesangial proliferative GN with normal tubules Group C Diffuse mesangial proliferative GN with global sclerosis (50— 57%) and focal tubular atrophy Group D End stage kidney with extensive tubular atrophy

NS NS

3 7 2 2 3

.E

ll

reported that physiological concentrations of hydrocortisone directly stimulate renal 1-hydroxylase activity. In the present study, the value of the 1-hydroxylase activity in the group treated with prednisolone did not differ from the group not treated with prednisolone, whereas the urinary excretion of calcium (the ratio of calcium/creatinine) was increased and Tmp/GFR remained unchanged. Thus, glucocorticoids at this level apparently do not affect the l-hydroxylase while nevertheless altering creatinine/calcium ratio in the urine. Considering the reduction of nephron mass in chronic renal disease, it could be anticipated that the 1-hydroxylase activity would fall. Recent studies have shown that serum l,25-(OH)2D concentrations fall with moderate and severe renal insufficiency

100

-I

Ii

50

GroupD GroupC GroupB GroupA Fig. 2. The renal 25-hydroxyvitamin D3-I-hydroxylase activity in pa-

tients with asymptomatic proteinuria and/or hematuria (group A), in

treated with prednisolone (group B), in patients with mild renal [24, 251. No information has been available, however, to patients insufficiency (group C), and in patients with severe renal insufficiency indicate at what level of renal insufficiency 1-hydroxylase (group D). Results are mean SD. Abbreviation for 25-hydroxyvitamin activity begins to fall. We have found that 1-hydroxylase D3- 1-hydroxylase is 1-hydroxylase.

activity is not reduced in mild renal insufficiency (GFR; 65.1

6.1 ml/min/l.73 m2) but that it is reduced in severe renal insufficiency. Recently, Portale et al [261 have reported that in children with moderate renal insufficiency (creatinine clear-

SE), a normal dietary intake of ance; 45.4 4, mean phosphorus is attended by a greatly increased FEPi, reflecting an increased rate of phosphorus excretion per nephron. It is possible that an increased intracellular concentration of phosphorus in the proximal tubules suppresses the activity of 1-hydroxylase even in the absence of an increased serum level of phosphorus. An increased serum level of parathyroid hormone in mild renal insufficiency may be expected to stimulate

1-hydroxylase activity. Therefore, the 1-hydroxylase activity might be unchanged as a result of two opposite factors.

The renal 1-hydroxylase activity is suppressed by 1,25(OH)2D3 and phosphorus [27]. The four patients in group D had been treated with l-OH-D3, which could be converted in vivo to

l,25-(OH)2D3. But the serum level of l,25-(OH)2D in these patients was significantly lower than that of group A (Table 2). Furthermore, the 1-hydroxylase activity in an end-stage renal failure patient not treated with 1-OH-D3 was also found suppressed. These data indicate that suppression of 1-hydroxylase

716

Satomura et a!: 25-OH-D3-i-hydroxy!ase activity in renal disease

activity in group D was not caused by treatment with 1-OH-D3. The serum level of phosphorus of the patients in group D was

significantly higher than that in group A (Table 2). In severe renal insufficiency, therefore, the 1-hydroxylase activity seems to be reduced by the combination of an increased serum level of

phosphorus and a markedly reduced nephron mass. These

phorus. J Biol Chem 66:375—400, 1925 12.

FOLIN 0, Wv H: A system of blood analysis. J

Biol Chem 38:81—

110, 1919

13. WALTON RJ, BIJvOET OL: Nomogram for derivation of renal threshold phosphate concentration. Lancet 2:309—310, 1975 14. YAMAOKA K, SEINO Y, I5HIDA M, IsHil T, SHIMOTSUJI T, TANAKA T, KUROSE H, MAT5UDA M, SATOMURA K, YABUUCHI H: Effect of

dibutyryl adenisine 3', 5'-monophosphate administration of plasma concentration of I ,25-dihydroxyvitamin D in pseudoparathyroidism Type 1. J Clin Endocrinol Metab 53:1096—1100, 1981

factors apparently are not compensated by an increased serum level of parathyroid hormone.

15. SEINO Y, YAMAOKA K, I5HIDA M, YABUUCHI H, ICHIKAWA M,

Acknowledgments This work was supported by a program project grant no, DK14881 from the National Institutes of Health and by the Harry Steenbock Fund of the Wisconsin Alumni Research Foundation. This study was

also supported in part by grants from the Ministry of Health and Welfare and the Ministry of Education of Japan.

IsHIGE H, Y05HIN0 H, AvioLl LV: Biochemical characterization of 1 ,25(OH)2D receptors in chick embryonal duodenal cytosol. Calczf Tissue mt 34:265—269, 1982 16. BROWN AJ, DELUcA HF: Production of lO-oxo-19-nor-25-hydroxyvitamin D3 by solubilized kidney mitochondria from chick kidney.

JBiol Chem

260:14132—14136, 1985

17. OKABE T, ISHIZUKA 5, FUJI5HIMA M, WATANARE J, TAKAKU F:

Reprint requests to Dr. Hector F. DeLuca, Department of Biochemistry, University of Wisconsin-Madison, 420 Henry Mall, Madison, Wisconsin 53706, USA.

Sarcoid granulomas metabolize 25-hydroxyvitamin D3 in vitro. Biochem Biophys Res Com 123:822—830, 18.

1984

KLEIN RG, ARNAUD SB, GALLAGHER JC, DELUCA HF, RIGGs BL:

Intestinal calcium absorption in exogenous hypereortisolism: Role of 25-hydroxyvitamin D and cortieosteroid dose. J Clin Invest 60:

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