Scand J Urol Nephrol 17: 373-376, 1983

RENAL FUNCTION AND la-HYDROXYVITAMIN D3 IN RABBITS WITH

NORMAL RENAL FUNCTION AND RENAL INSUFFICIENCY

Erling Tvedegaard

From Medical Depurtrnent P , Division of Nephrology und Depurrmenr of Experimental Puihology, Rigshospirulet, Copenhagen, Denmurk

(Submitted for publication December 9, 1982)

Absrrucr. The effect of la-hydroxyvitamin D, ( laOHD3) on the renal clearance of creatinine was investigated in groups of rabbits with stable renal insufficiency and nor- mal controls. Urine was collected in the two groups for two one-week periods during placebo treatment. Then, laOHD3, 0.02 pg/kg BW/day, was adminstered orally and urine was coilected during the next two weeks. Weekly blood samples were obtained. The treatment had no sig- nificant effect on the serum concentrations of calcium and phosphate in either group. Creatinine clearance decreased significantly in both groups during the IaOHD, treatment @<0.05), mainly due to a decreased urinary excretion of creatinine. It is suggested that changes in the renal tubular handling of creatinine caused by laOHD3 may be the explanation of the observed effect.

Treatment of uraemic osteodystrophy with hydrox- ylated derivatives of vitamin D3, la-hydroxychole- calciferol ( laOHD3) and 1.25-dihydroxycholecaIci- ferol ( 1 .25(OH),D3), has been established as effec- tive and valuable in many patients (Madsen, 1979). Recently, reports of a nephrotoxic effect of these drugs have appeared concerning both predialytic uraemic patients (Tougaard, Skensen, Brgichner Mortensen, Christensen, Rgidbro & S@rensen, 1976; Christiansen, Rgdbro, Christensen, Hartnack & Transbgil, 1976; Nielsen, Rgjmer, Melsen, Chris- tensen & Hansen, 1980) and patients with normal renal function (Jensen, Christiansen & Transbgil, 1982), as the drugs are also used in the treatment of senile osteoporosis. Other investigators have not been able to detect this effect (Madsen, 1979; Compston, Dutt, Mitchell, Nunan & Woodhead, 1981; Naik, Cundy, Robinson, Russel & Kanis, 1981), but the methods used have not been identical and the question remains unsettled.

In a previous study on rabbits no effect of laOHD3 on the renal function deterioration rate

24-838263

was found measured by the plasma clearance of "Cr-EDTA (Tvedegaard & Ladefoged, 1982). In the present study, two weeks treatment with laOHD3 significantly decreased the calculated renal creatinine clearance, mainly by decreasing the urinary excretion of creatinine. The question is therefore raised whether creatinine clearance is a reliable measure of changes in renal function during treatment with laOHD3.

MATERIAL AND METHODS Male rabbits of the White Danish country strain were used at the age of three months, weighing 3-3.5 kg. In ten rabbits chronic renal failure (CRF) was induced by cauter- ization of 1/2-2/3 of the parenchyma of the left kidney with subsequent contralateral nephrectomy three weeks later (Kamstrup, Tvedegaard & Stender, 1980). Six con- trol rabbits were sham operated twice. Phenobarbital in- travenously was used for anaesthesia.

After two weeks during which the rabbits still received standard pellets, a diet containing 1.0% Ca, 0.9% P and 0.2% Mg with addition of 2000 I U of vitamin D3 per kg, was administered as pellets. The animals were housed in individual metabolic cages and received 100 g of the diet per day. Access to tap water was free. After 4 weeks on this regimen the body weights were stable. The rabbits were now given 0.3 ml of water orally by a syringe once daily as placebo treatment. Urine and faeces were collect- ed quantitatively and the consumption of food registered during two one-week periods. Venous blood were ob- tained at the start and after one and two weeks. By the same method laOHD3 was then administered, 0.02 pg per kg body weight per day, as 0.10 ml/kg/day of a solution containing 0.20 pg/ml. Collection of urine and faeces was continued for two one-week periods, food consumption was registered and venous blood was drawn after one and two weeks on this treatment.

Anulysis Ionized calcium was measured on fresh serum by an automatic analyzer, Orion Biomedical, Model SS-20

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374 E . Tvedegaard

Table I. Body weight, serum concenlrations and creatinine clearances (mean k SD) in normal and CRF rabbits during two weeks on placebo and two weeks treatment with laOHD3, 0.02 pglkg BWlday

CRF rabbits (n=7) Normal rabbits (n=6)

Placebo laOHD3 Placebo laOHD3

Body weight,

Creatinine, mmoH Urea, mmoVl Total calcium, mmoUl Ionized calcium, mmoH Phosphate, mmoVl Magnesium, mmoM Creatinine clearance, l/week

kg 3.20+0.20**

0.159k0.019** 10.0k 1.5** 3.69f0.16** I .67+0.05 1.69k0.35 0.80t0.06 57.5t 10.7**

3.21 fO.21

0.174f0.021 11.52 1.4 3.69f0.21 1.63k0.07 1.92k0.38 0.82k0.08 48.2t 11.4*

3.52k0.22

0.103f0.017 5.720.6

3.43t0.13 1.67fO.W 1.50f0.04 0.82f0.05 97.8f 10.7

3.53t0.22

0.101f0.016 5.8f0.6

3.39k0.10 1.69f0.06 1.51 20.09 0.82f0.07 88.3f12.9*

* Significant change within the group during laOHD3 treatment, p<0.05. ** Significantly different from normal rabbits during placebo p<O.Ol.

(Madsen & glgaard, 1977). Serum obtained from the ve- nous blood samples and samples from the urine collec- tions were stored at -20°C until further analysis. The concentrations of creatinine, urea, phosphate, calcium and magnesium were determined by an Automatic Clini- cal Analyzer (ACA, Wilmington, Mass. USA). Creatinine clearance was calculated for the urine collection periods using the mean of the serum creatinine concentrations at the start and end of each period.

Statistics The groups were compared by Student’s t-test and changes within the groups were evaluated by the r-test for paired comparison.

RESULTS

Two CRF rabbits died of enterocolitis before the start of the placebo treatment and one CRF rabbit was excluded because of a continuous large weight loss during the whole experimental period.

In Table I the mean serum concentrations, the body weights and creatinine clearances are shown. The serum values during the placebo period are based on the means of the three blood samples obtained at the start and after one and two weeks. The values during l a O H D 3 treatment are based on the means of the two blood samples drawn after one and two weeks of treatment. The clearance values are calculated from the means of two one- week periods during placebo and two during laOHD3 treatment.

The mean body weight of rabbits with normal renal function was slightly but significantly higher than in the CRF group (Table I). For all rabbits included in the study the consumption of food was

constant and the body weight was stable through- out the study with individual variations less than 2%. The method for induction of CRF resulted in a moderate but significant reduction of renal func- tion, as evidenced by the increased serum concen- tration of creatinine and decreased creatinine clear- ance (p<O.Ol, Table I).

The mean serum concentration of total calcium was significantly higher in the CRF rabbits com- pared to the sham operated controls (p<0.05). No difference was seen in ionized calcium levels. Treatment with laOHD3 caused no significant changes in these two parameters within any of the two groups. The mean serum concentration of phosphate in the CRF group did not differ signifi- cantly from the level in normal rabbits. Treatment with laOHD3 caused no significant change of se- rum phosphate neither in the CRF rabbits nor in the normal group. Serum magnesium was identical in the two groups and no effect of laOHD3 was regis- tered (Table I).

The mean urinary excretion of calcium in the CRF group increased by 15% during laOHD3 treatment, magnesium increased by 27 % and phos- phate by 15%. In the normal rabbits the increase was 15 % for calcium, 21 % for magnesium and 20% for phosphate. All these changes were statistically significant @<O.Oi).

Of the measures of renal function, the mean se- rum concentration of urea was not significantly affected by treatment with laOHD3 neither in the normal nor in the CRF rabbits. In Fig. 1 the individ- ual values for each rabbit are shown for serum creatinine concentrations, urinary excretions of

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la-hydroxyvitamin D3 and renal function 375

CRF Controls - + , - +

I

I----

.05<P<.lO ' P>.30

CRF Controls CRF Controls - + I - + - + I - +

I I

I I I

~

P<.O1

120.

80 -

60 -

40-

20-

C I

creatinine and creatinine clearances. During laOHD3 treatment no significant change of serum creatnine were registered within any of the two groups. The urinary excretion of creatinine in the CRF rabbits did not change significantly whereas a significant decrease was found in the normal group during laOHD3 treatment compared to placebo (p~0.01). The calculated creatinine clearance de- creased significantly both in the CRF group (p< 0.02) and in normal rabbits (pcO.05).

DISCUSSION

In the present study creatinine clearance in both normal and CRF rabbits decreased when treatment with laOHD3 was instituted and the decrease was due mainly to a decrease in the urinary excretion of creatinine.

A decrease in creatinine clearance during treat- ment with hydroxylated derivatives of vitamin D3 has been found in several clinical studies both in patients with normal renal function and with renal insufficiency (Tougaard et al., 1976, Christensen et al., 1978, Nielsen et al., 1980). The urinary excre- tion of creatinine is not stated in these reports but serum creatinine was found to increase. Other in- vestigators have not been able to demonstrate any changes in creatinine clearance during this kind of treatment (Madsen, 1979; Naik et al. 1981). Two studies have been published using the clearance of "Cr-EDTA as a measure of the glomerular filtra- tion rate (GFR) and in both reports no change in

P<.02 I P<.O5

Fig. I. Values of serum cre- atinine, urinary creatinine excretion, and creatinine clearance in two groups of rabbits with chronic renal failure (CRF) and normal renal function (controls), shown for each animal dur- ing placebo (-) and during laOHD3 treatment (+).

renal function caused by treatment with laOHD3 was registered (Compston et al., 1981; Tvedegaard et al., 1982).

These conflicting results might be due to the differences in the methods used to monitor renal function. Changes in creatinine clearance or serum creatinine do not neccessarily reflect changes in GFR. Indeed, this problem has been discussed in some reports (Compston et al., 1981; Jensen et a]., 1982). Creatinine clearance overestimates GFR, measured as renal clearance of inulin, due to a net tubular secretion of creatinine (Hagstam, Norden- felt, Svensson & Svensson, 1974). If treatment with laOHD3 caused an increase in tubular reabsorp- tion or a decrease in tubular secretion of creatinine, urinary creatinine excretion would initially de- crease. Assuming a constant endogenous produc- tion of creatinine and a constant GFR, the serum creatinine concentration would increase until uri- nary excretion had returned to normal and at this new steady-state the calculated creatinine clear- ance would remain decreased.

Another, perhaps less likely possibility is that 1 aOHD3 might decrease the formation of creati- nine from creatine phosphate in the muscle cells. That laOHD3 has an effect on muscle cell metabo- lism is supported by the rapid effect on the proxi- mal myopathy often seen in patients with severe uraemic osteodystrophy (Kanis, Earnshaw, Hen- derson, Heynen, Ledingham, Naik, Oliver, Russel, Smith, Wilkinson & Woods, 1977). This effect would cause a temporary decrease in the calculated

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376 E . Tvedegaard

creatinine clearance due to decreased urinary ex- cretion until serum creatinine had decreased to es- tablish a new steady-state. This possibility would thus explain only acute changes in creatinine clear- ance.

Both of the above mentioned possible mechan- isms might explain the changes in creatinine clear- ance found in the present study. The increase in serum creatinine in the CRF group might point to the first possibility as most probable but in the normal rabbits no tendency in serum creatinine concentrations were seen (Fig. 1). Until it has been proved that none of them are at work, creatinine clearance cannot be accepted as a reliable measure of GFR during these circumstances.

The method used for the induction of CRF result- ed in only a moderate decrease in renal function. However, the significantly increased serum calcium concentration in the CRF rabbits indicates that de- spite this, the metabolism of the CRF rabbits was affected by this degree of renal insufficiency. In- creased serum calcium in rabbits with chronic renal failure has been reported previously but the mechanism is unknown (Tvedegaard, Nielsen & Kamstrup, 1982). Serum concentrations of calcium, phosphate and magnesium did not change signifi- cantly during the treatment with laORD3, but the significant increase in the urinary excretions proves that the dosage given was sufficient to influence mineral metabolism in both CRF and normal rab- bits.

The implications of the present study for clinical situations are limited, partly because it is an experi- mental study involving only a small number of ani- mals and partly because of the methodological con- siderations cited above. A comprehensive study in human subjects using several different techniques for GRF determinations simultaneously has been initiated to answer the questions raised by the pres- ent and previous studies in this field.

ACKNOWLEDGEMENTS This work was supported by the Michaelsen Foundation. The technical assistance of Doris Christoffersen and Kris- tian Sangill Christensen is gratefully acknowledged. Leo Pharmaceuticals, Ballerup, Copenhagen generously sup- plied the la-hydroxyvitamin D3.

REFERENCES Christiansen, C., Redbro, P., Christensen, M. S., Hart-

nack, B. & TransbZl, 1. 1978. Deterioration of renal function during treatment of chronic renal failure with 1.25-dihydroxycholecaIciferol. Lancet i i , 700.

Compston, J . E., Dutt, M. K., Mitchell, W. D., Nunan, T. 0. & Woodhead, J . S. 1981. The effect of l a - hydroxyvitamin D3 on renal function in normal sub- jects. Metab Bone Dis Re1 Res 2 , 297.

Hagstam, K. E., Nordenfelt, I., Svensson, L. & Svens- son, S. E. 1974. Comparison of different methods for determination of glomerular filtration rate in renal dis- ease. Scand J Clin Lab Invest 34, 31.

Jensen, G. F., Christiansen, C. & Transbel, I. 1982. 1.25(OH),D3 and renal function. Acra Med Scand 211, 51.

Kamstrup, O., Tvedegaard, R. & Stender, S. 1980. Effect of chronic uraemia on plasma lipids and the aortic accumulation of cholesterol in hypercholesterolemic rabbits. Nephron 26, 280.

Kanis, J. A., Earnshaw, M., Henderson, R. G., Heynen, G., Ledingham, J. G. G., Naik, R. B., Oliver, D. O., Russel, R. G. G., Smith, R., Wilkinson, R. H. & Woods, C. G. 1977. Correlations of clinical, biochemi- cal and skeletal responses to la-hydroxyvitamin D3 in renal bone disease. Clin Endocrinol 7 , Suppl, 45 s.

Madsen, S. 1979, Calcium and phosphate metabolism in chronic renal failure with special reference to the ef- fect of la-hydroxyvitamin D3. Acra Med Scand, Suppl. 638.

Madsen, S. & Olgaard, K. 1977. Evaluation of a new, automatic calcium ion analyzer. Clin Chem 23, 690.

Naik, R. B., Cundy, T., Robinson, B. H. B., Russel, R. G. G. & Kanis, J . A. 1981. Effects of vitamin D metabolites and analogues on renal function. Nephron 28, 17.

Nielsen, H. E., Remer, F. K., Melsen, F., Christensen, M. S. & Hansen, H. E. 1980. la-hydroxyvitamin D3 treatment of undialyzed patients with chronic renal failure. Effects on bone, mineral metabolism and kid- ney function. Clin Nephrol 13, 103.

Tougaard, L., Serensen, E., Brechner Mortensen, J., Christensen, M. S., Redbro, P. & Serensen, A. W. S. 1976. Controlled trial of la-hydroxycholecalciferol in chronic renal failure. Lancet, 1044.

Tvedegaard, E. & Ladefoged, 0. 1982. No effect of low- dose la-hydroxycholecalciferol treatment on the renal function deterioration rate in long-term uraemic rab- bits. Acra Pharmacol Toxic01 50, 385.

Tvedegaard, E., Nielsen, M. & Kamstrup, 0. 1982. Os- teosclerosis of the femoral head in long-term uraemic rabbits. Acra Pathol Microbiol Immunol Scand, Sect. A, 90, 235.

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