THE URINARY EXCRETION OF CALCIUM IN EXPERIMENTAL STAPHYLOCOCCAL PYELONEPHRITIS

By ALEXANDER KENNEDY, M.D. Department of Pathology, University of Liverpool

IT is now over twenty years since Flocks (1939, 1940) pointed out that many patients with urinary calculi have a hypercalciuria which is not attributable either to hyperparathyroidism or to bone disease. This phenomenon, which has been called “ idiopathic hypercalciuria,” is still unex- plained. Workers in Boston, Massachusetts, have advanced the hypothesis that idiopathic hyper- calciuria is a result of staphylococcal pyelonephritis (Albright and Reifenstein, 1948 ; AIbright et a/., 1953 u, h ; Henneman et a/., 1958). This hypothesis has been quoted many times but it has never been substantiated (Rosenberg, 1954 ; Cottet and Vittu, 1955 ; Jesserer, 1957 ; Modlin, 1957 ; Hodgkinson, 1961, Bhandarkar and Nordin, 1962 ; Krane, 1962 ; Royer et a/., 1962; Lichtwitz et a/., 1963). There does not seem to have been any attempt to test the hypo- thesis experimentally ; the present paper describes an investigation into the urinary calcium excretion of rabbits with staphylococcal pyelonephritis.

Materials and Methods.-Two series of experiments were performed using adult male rabbits of mixed breeds. Technical details of the general methods of study have been published elsewhere (Kennedy, 1965). In one series of experiments the animals were fed on diet SG1 (Oxoid Limited) and in the other series they were fed on “Coney Brand” pellets (British Extracting Company Limited). The calcium and vitamin D contents of diet CB are much higher than those of diet SG I . These diets will be referred to as SCI and CB respectively.

After a period of acclimatisation to the particular diet, groups of six rabbits were transferred to metabolism cages and their mean food consumption and daily urinary calcium output were measured over a five or a six-day period. Blood was collected from each rabbit for the estimation of levels of serum urea.

Following these baseline observations, four of the rabbits i n each group were given an intravenous injection of a suspension of the Oxford staphylococcus ( S O x lo6 organisms/ml.) ; the administration of a dose of 1 ml./kg. of body weight is a reliable procedure for the production of pyelonephritis in the rabbit (Navasquez, 1950). The remaining two animals in the group served as controls and were injected with an equivalent dose of saline. Following the injections the urinary calcium excretions of the rabbits were studied continuously for two weeks. Further five-day periods of study in the metabolism cages were conducted at intervals for up to six months after the original injections ; during each period the blood urea was estimated. This experiment was repeated in a total of three groups of rabbits on diet SGI each ofwhich was treated identically. The same experiment was performed on a second series consisting of four groups of rabbits on diet CB.

RESULTS

The animals injected with staphylococci lost their appetites for‘the first few days after the injection but their consumption of food improved during the second week. Pyuria appeared on the third or fourth day after the injection and continued for a week or ten days. No pyuria occurred in any of the controls.

Pathological Changes.-Most of the kidneys showed changes similar to those described by In animals which died in the first week or two of the experiment, yellowish

Many polymorphs were present in the Navasquez. inflammatory lesions were present in the renal cortex.

171

172 B R I T I S H J O U R N A L O F U R O L O G Y

interstitial tissue of the cortex and in the tubules of the medulla (Fig. I ) . Pus was present in the renal pelvis and in some cases there were abscesses in the pyramid. Most of the animals survived for three or six months and they were then killed. In these rabbits the renal cortices showed multiple scars, 5 or 6 mm. in diameter, which were slightly depressed below the

FIG. 1 Acute pyelonephritis nine days after the injection of staphylococci. Pus is

x 105. present in the tubules of the medulla. (H. & E.)

FIG. 2

staphylococci. (H. & E.) x42. Pyelonephritic scar in the renal cortex. Six months after the injection of

surrounding surface of the kidney. Histologically, the lesions consisted of areas of reparative fibrosis from which most of the tubules had disappeared (Fig. 2). A variable number of inflam- matory cells were still present both in the scars and beneath the pelvic mucosa.

None of these changes was seen in the control animals and it may be concluded that the rabbits suffered an attack of acute pyelonephritis which had a temporal relation to the injection of staphylococci and which had healed leaving renal scars. Rabbits do sometimes suffer from a spontaneous focal scarring but such spontaneous lesions are quite different from the large scars produced by staphylococci.

This attack of pyelonephritis did not impair renal function to a sufficient extent to raise the

U R I N A R Y E X C R E T I O N O F C A L C I I J M 173

level of urea in the serum. In the few cases in which subsequent histological examination failed to detect evidence of pyelonephritis the results of analysis of the urine were discarded as of no interest.

1400.

1200

2 IOOO-

s . z 8 0 0 -

I 2 2 6 0 0 -

z 3 400- z

w

2 2 0 0 .

Normal Urinary Calcium Output .-The limits of normal excretion have been previously established for the rabbit in observations in this laboratory using the same diets which have been used in the present work (Kennedy, 1965). The normal ranges are shown in Figures 3 and

.

220

2 0 0

iao

I 140

z I 120 3 2 loo U

w 8 0 z ’ 6 0

f 5 40

2 0

u 2 0

I , , , , , , , , ‘ 0 2 0 40 60 80 100 I20 140 153 180

MEAN FOOD CONSUMPTION IN G/DAY MEAN FOOD CONSUMPTION IN G/DAY

FIG. 3 FIG. 4

Fig. 3.-The range of normal for the urinary calcium excretion of the rabbit at different intakes of diet SGI. The horizontal line in the middle of each rectangle represents the mean and the rectangles represent

two standard deviations on either side of the mean.

Fig. 4.-The range of normal for the urinary calcium excretion of the rabbit at different intakes of diet CB. The horizontal line in the middle of each rectangle represents the mean and the rectangles represent

two standard deviations on either side of the mean.

4 ; the range of calcium excretion is plotted against the mean food consumption. The mean calcium output for any range of food intake is represented by a horizontal line in the middle of each rectangle and each rectangle represents two standard deviations above and below the mean. Any point falling within the rectangles may be regarded as normal.

Pyelonephritic Rabbits on Diet SG1.-Ten rabbits were injected with staphylococci and nine survived long enough to develop pyelonephritis. There were six rabbits in the control group. The mean calcium’excretions of the animal are shown in Table I ; as the rabbit’s urinary calcium is dependent on its food consumption, the mean food intake is also recorded.

It will be seen that the calcium excretion of the pyelonephritic group did not rise significantly above the baseline excretion or above the excretion of the controls. On the only occasion (nine weeks) at which the pyelonephritic group excreted more than the controls the difference was only 3 mg./day which is not statistically significant (P>0.9). The means of the two groups are not truly comparable because the food consumptions differed, but if the figures for the pyelonephritic group are plotted on Figure 3 it will be seen that all the points lie within the normal range.

Out of thirty-eight individual measurements of the calcium excretions in the pyelonephritic

I74 B R I T I S H J O U R N A L O F U R O L O G Y

125

~~~

TABLE 1

The Mean Urinary Calcium Excretion (mg./day) and Mean Food Consumption (g./day) of the Rabbits on Diet SGI

134

Duration of Experiment ~ Baseline

Pyelonephritic rabbits- I I--- _ _ ~ p p

Urinary calcium i 76 excretion, nig./day

nig./day

Control rabbits-

Food consumption, I 131

One 1 TWO SIX Week 1 Weeks 1 Weeks

_ - p ~ l - - - - ~ p p - -

59 79 i 65

81 ~ 102 95

I

Nine Weeks

~ p - ~

77

I05

~~ -

Thirteen Weeks

78

91

Urinary calciuiii . 121 91 ~ 101 , 76 excretion, mg./day I

Foodconsumption, i 150 ~ 129 I 137 I04 I nig./day ~

I I

~ ~~

i ~

I - ~~~~

I .~

TABLE I I

The Mean Urinary Calcium Excretion (mg./day) and Mean Food Consumption (g./day) of the Rabbits on Diet CB

Duration of Experiment

Pyelonephritic rabbits- Urinary calcitim excretion,

Food consumption, mg./day

mg./day

Baseline

668

I27

Control rabbits- Urinary calcium excretion, 71 1

mg./day

mg./day Food consumption, I18

One 1 Two Week Weeks

I

Three ~ Six Weeks 1 Weeks

I 508 539

74 1 98

74 ~ 88 I

I18 I12

. - ~ ~

Thirteen Weeks

568

97

64 I

129

Nine- teen

Weeks

594

104

616

- 1 ~

Twenty- I

foul Weeks

631 1 113

530

110 , 114

group only two fell outside the normal range. In both these cases the rabbits concerned excreted an excessive amount of calcium on a single occasion and in one of them the upper limit of normal was exceeded by only 3 mg./day. At the end of the experiment all the pyelonephritic rabbits had normal urinary calcium excretions.

Pyelonephritic Rabbits on Diet CB.-Eighteen rabbits were injected with staphylococci and nine survived and developed pyelonephritis ; the control group consisted of six rabbits. The results obtained from these animals are shown in Table I1 ; the calcium excretions are enormous by human standards but are quite normal for rabbits on this diet. The pyelonephritic group excreted less calcium than the controls except during the observation period at six months, but the rise above the controls at this stage was not statistically significant (P>0.4). All the figures lie within the normal range shown in Figure 4.

U R I N A R Y E X C R E T I O N O F C A L C I U M I75

When the individual excretions of the pyelonephritic rabbits were compared with the normal only two out of a total of forty-eight figures were outside the normil range. In both the cases the high excretion occurred on one occasion only, and after three months all the rabbits hid normal calcium excretions.

In the early part of the experiment the pyelonephritic group showed a reduction in their urinary calcium excretion but this was related to the reduction in their food consumption which occurred during the acute phase of the illness. In no case did the urinary cilcium excretion fall below normal limits.

D I SCUSSl ON

These results indicate quite clearly that, in the rabbit, staphylococcal pyelonephritis does not result in an increase in the renal elimination of calcium. Furthermore, hyptrcalciuria did not occur during the early period of pyuria ; a finding which is not in accord with the reports by the Boston workers of hypercalciuria occurring during acute staphylococcal pyelonephritis in human patients (Albright and Reifenstein, 1948).

It might be argued that the length of the experiments was inadequate to all the animals to develop hypercalciuria. However, the structural changes in the kidneys appeared to be complete by three months and there was no recognisable further development in the kidneys examined at six months. Thus, any changes in the urinary calcium excretion should have app:ared by three months.

I t might perhaps be objected that in the pyelonephritis experiments the kidneys were so severely damaged that hypercalciuria was impossible. Firstly, the damage was not severe because the animals did not become urzmic. Secondly, those on diet CB were still capable of excreting 500 to 600 mg. of calcium per day. The renal pathology of the animals on diet SGI was the same as the pathology of those on diet CB, so that it seems reasonable to infer that those on diet SGI could have excreted 500 to 600 mg. of calcium per day. Even an excretion of half this amount by animals on diet SG I would have demonstrated the presence of hypercalciuria.

A further possible problem is the choice of the organism which was used to produce pyelonephritis. Albright and his colleagues (1953 a, h) reported the presence of Staph. albus in the urine of their patients with long-standing hypercalciuria and they suggested that this hypercalciuria was the result of staphylococcal pyelonephritis. In support of this suggestion they quoted two cases in which hypercalciuria occurred during acute staphylococcal pyelonephritis. Presumably the organism in these acute cases was Staph. aureus and not Staph. albus, but unfortunately they did not specify the type of staphylococcus responsible.

The present work has been conducted using Staph. aureuS only but it is clear that pyelo- nephritis due to this organism does not cause hypercalciuria in the rabbit. This conclusion applies only to the rabbit and there is the possibility that the effect of staphylocoxal pyelo- nephritis on the renal handling of calcium is subject to species differences. Nevertheless, such differences are, at present, purely speculative

SUMMARY

Albright suggested that " idiopathic hypercalciuria " may be due to staphylococcal pyelonephritis. In order to test this hypothesis experimental pyelonephritis was induced in rabbits by the intravenous injection of staphylococci and their urinary calcium excretion was studied for periods of up to six months afterwards. The pyelonephritic rabbits did not excrete more calcium in their urine than normal and they did not excrete more calcium than a group of control rabbits. It is concluded that staphylococcal pyelonephritis does not cause hyper- calciuria in the rabbit.

176 B R I T I S H J O U R N A L OF U R O L O G Y

I am indebted to Professor H. L. Sheehan for his advice at all stages of this work and for his criticism of the manuscript. I also wish to thank Mr F. Beckwith and his staff for preparing the histological sections, Mr J. Cowie for the supply of the staphylococcal suspensions and Mr A. C. Hemes for the photography.

REFERENCES

ALBRIGHT, F., HENNEMAN, P., BENEDICT, P. H., and FORBES, A. P. (1953 a) . J. clin. Endocr.. 13, 860. ~ (1953 b). Proc. Roy. SOC. Med., 46, 1077. ALBRICHT, F., and REIFENSTEIN, E. c. (1948). ‘‘ The Parathyroid Glands and Metabolic Bone

BHANDARKAR, S. D., and NORDIN, B. E. C. (1962). Brit. med. J . , 1, 145. COTTET, J., and VITTU, C. (1955). Presse med., 63, 878. FLOCKS, R. H. (1939). J. Amer. med. Ass., 113, 1466.

Disease.” (Baltimore : Williams and Wilkins.)

~ (1940). HENNEMAN, P., BENEDICT, P. H., FORBES, A. P., and DUDLEY, H. R. (1958).

J. U I O ~ . , 43, 214. New E/ig/. J. Med.,

259, 802. HODGKINSON, A. (1961). Proc. Ass. din. Biochem., 1, 52. JESSERER, H. (1957). Dtsch. med. Wschr., 82, 943. KENNEDY, A. (1965). J. comp. Path., 75, 69. KRANE, S. M. (1962). New Engl. J. Med., 267, 875.

nied., 71, 165. LICHTWITZ, A., DE SEZE, S., HIocO, D., MIRAVET, L., LANHAM, C., and PARLIER, R. (1963). Presse

MODLIN, M. (1957). S. Afr. med. J. , 31, 1010. NAVASQUEZ, S. DE (1950). J. Path. Bart., 62,429. ROSENBERG, M. L. (1954). Calif. Med., 81, 382. ROYER, P., MATHIEU, H., GERBEAUX, S., FREDERICH, A,. RODRIGUEZ-SORIANO, J., DARTOIS. A. M.,

and CUISINIER, P. (1962). Sem. H6p. Paris, 38, 767.