Renal structure and function
hypothyroidism and hyperparathyroidism
An experimental study in the pig
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BENGT FÂHRÆUS
med.lic.
UMEÄ 1974
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS No. 17 1974
From the Departments of Urology and Pathology, University of Umeå, Umeå, Sweden
RENAL STRUCTURE AND FUNCTION IN HYPOTHYROIDISM AND HYPERPARATHYROIDISM
An experimental study in the pig
By Bengt Fåhraeus
UMEÅ 1974
These investigations were supported by grants from the Swedish Medical Research Council (Projects No. B72 - 17X - 3499 - 01, B73 - 17X - 3499 - 02 and B74 - 1 7X - 3499 - 0 3 ), the Medical Research Council of the Swedish Life Offices and the Medical Faculty, University of Umeå.
The statistical calculations were performed by Fil. Kand. Anders Baud in a.nd Pil. Kand. Stig Wall
Translated by Ann-Mari Fåhrasus
This survey is based on the following papers which will be referred to in the text by their Roman numerals:
I F â h r æ u s , B. & Lorentzon, R.: Renal clearance studies in the pig. Surgical procedures, performance of investigations and physiological data. Urol. Research. Acc. for publication.
II Fâhr æ u s , B.: Renal function after thyroidectomy and para
thyroid hormone administration. An experimental study in the pig. Urol. Research. Acc. for publication.
III Boquist, L. & F â h r æ u s , B.: Renal morphology in pigs with experimental hyperparathyroidism. Light microscopic findings and some functional aspects. Virchows Archiv. Acc. for publication.
IV Boquist, L. & F â h r æ u s , B.: Renal morphology in pigs with experimental hyperparathyroidism. Ultrastructural findings.
Virchows Archiv. Acc. for publication.
INTRODUCTION
In 1880 Sandström discovered the parathyroid glands hut not until 1 9 0 4 was a parathyroid tumour with concomitant ostitis fibrosa cystica generalisata observed (Askanazy, 1 9 0 4 ). In 1925>with
M a n d l 's (19 2 6 ) extirpation of a parathyroid tumour with considerable postoperative improvement in a patient with ostitis fibrosa cystica generalisata, the association between this disease and parathyroid tumours was established.
In 1 9 0 5 MacCallum observed simultaneous occurrence of a parathyroid tumour and renal changes and his report was followed later by several descriptions of simultaneous alterations in the parathyroid glands and the kidneys, e.g. by Bergstrand (1921).
When, independently of each other, Hanson and Collip succeeded in producing a stable parathyroid extract (Hanson, 1924; Collip, 1925)?
it was possible to demonstrate experimentally that administration of the parathyroid hormone (PTH) caused a rise in serum calcium and increased excretion of calcium in the kidneys.
Albright et al. (1934) were the first to establish the association between hyperparathyroidism and renal complications. They described 3 different types of renal lesions, 1 : calcium phosphate stone formation, 2 : calcium deposits in the renal parenchyma and 3 : acute parathyroid poisoning with calcium deposition in the kidneys and other organs. They also emphasized that hyperparathyroidism should be suspected in patients with urinary calculi and that renal changes could occur without simultaneous bone alterations. An active search for hyperparathyroidism in patients with renal calculi led to an increasing number of diagnoses of this disease.
It was also established that hyperparathyroidism could be the
cause of duodenal ulcers and pancreatitis (Hellström & Ivemark, I9 6 2 ) and that some mental disorders could arise from hypercalcaemia (Petersen, 1 9 6 8 ).
By means of improved laboratory methods it is nowadays possible to discover hypercalcaemia even in patients who are free from symptoms (Purnell et al., 1971)- Cases of normocalcaemic hyperparathyroidism are also diagnosed increasingly frequently (Grimelius et al., 1973)*
The clinical picture of primary hyperparathyroidism is thus very varied but most diagnoses are probably still made because of urinary calculi (Johansson et al., 1972).
When Copp et al. (1 9 6 2 ) discovered calcitonin (CT), a hormone which lowers serum calcium, a further inducement was offered to research into hypercalcaemic disorders.
It was soon possible to establish that CT is produced in the thyroid (thyrocalcitonin) by the C-cells which originate from the ultimobranchial body and that it plays an important part in the regulation of the serum calcium level. Elimination of CT production causes increased sensitivity to administration of calcium or calcium-mobilizing substances (Sorensen, 1970; Bartlet, 1972). CT also affects the mineralization of bone and has some renal effects, such as increased excretion of calcium, magnesium, phosphates and sodium (Clark & Kenny, 1969; Pak et al.,1970; Russel & Fleisch, I9 6 8 ).
The effects of CT on the renal function measured by clearance determinations have not been studied but cannot be excluded, as Pak et al. (1970) found increases in the clearance of inulin (Cljn ) after the administration of CT to dogs. These changes, however, might have been caused by variations in the serum calcium level (Kaufman & BiScala, 197"0*
Thyroidectomy would probably eliminate CT production and thereby exclude the increased CT levels in blood during hypercalcaemia.
However, thyroidectomy would also eliminate the other thyroid hormones and this affects the renal function measured by clearance determinations. In clinical investigations C1T , the clearance of
I n ’
para-amino-hippuric acid (Cl^.^) and the transfer maximum of PAH P A H
(Tnip^) have been found to be lower than normal in hypothyroidism (Corcoran & Page, 1947; Luft & Sjögren, 1950; Hlad & Bricker, 1954;
Yount & Little, 1955). In hyperthyroidism, on the other hand, there is an increase in these clearance values (Corcoran & Page, 1947;
Hlad & Bricker, 1954). S imilar results were obtained from experiments with dogs (White et al., 1947).
Clinical studies on hyperparathyroidism have as a rule shown a decrease in Cl , Cl and Tm^^. (Edwall, 1958; Hellström &
Ivemark, 19^2; Ohlsson, 1970; and others). There is also a reduction in the maximum urine concentration capacity (Hellström, 1954). The renal morphology in hyperparathyroidism is well described by Anderson (1939)2 ”There is interstitial infiltration by chronic inflammatory cells, interstitial fibrosis and interstitial deposition of calcium, particularly in or adjacent to tubular basement membranes ... Frequently tubules are dilated and lined by flattened epithelium ... Glomeruli are partially or completely fibrosed but evidence of proliferative or exudative changes in glomeruli ... are absent. Vascular involvement is usually slight;
when present it appears to be an unrelated change. Many of the calcium deposits, both those within tubules ... and peritubular masses may give rise to obstruction of the tubule and an effect on other portions of the nephron ... It is obvious that widespread obstruction of tubules may severely damage the kidney and produce insufficiency” .
Similar changes have been established in experiments in dogs (Learner, 1929; Cantarow et al., 1938; Carone et al., I960;
Yano et al., 19^5; Berry, 1970), in rats (McJunkin et al., 1932;
Engfeldt et al., 1958) and in mice (Schneider et al., I960;
Caulfield & Schräg, 19 6 4 ).
These authors disagree as to where the changes first occur: in the proximal tubules (Cantarow et al., 1938; Engfeldt et al., 1958; Caulfield & Schräg, 19&4; Yano et al., 19^5 ; Berry, 19 7 0) or in the distai tubules (Carone et al., i9 6 0 ).
Neither is it established whether the calcifications are primary (Learner, 1929; Schneider et al., i9 6 0 ) or secondary to degenerative changes in the cells (McJunkin et al., 1932; Cantarow et al., 1938;
Carone et al., i9 6 0 ).
The animals in these experiments were as a rule given large doses of PTH which produced a rise in serum calcium to values "between 16 and 24 m g / 100 m l .
Experiments on the renal function measured by the clearance technique and determinations of the renal blood flow immediately after PTH administration have shown that the renal blood flow increases (Charbon, 1 9 6 8 ) as well as the glomerular filtration rate (GER) and the renal plasma flow (RPF) (Handler et al., 1951;
Widrow & Levinsky, 1 9 6 2 ). 24 hours after PTH administration, how
ever, there is generally a reduction in the clearance values (Epstein et al., 1959) and a decrease in these values was also found in experiments with PTH administration over a longer period of time (Engfeldt et al., I9 6 2 ).
AIM OF THE PRESENT STUDY
The aim of the present investigation was to study:
1) the effects on renal function and structure of experimental hyperparathyroidism, with moderately increased serum calcium values, in the absence of CT production.
2) the effects on renal function and structure of thyroidectomy, which is necessary for the elimination of CT production.
CHOTCE OF LABORATORY ANIMALS
The pig was chosen as laboratory animal for the following reasons:
In pigs the thyroid and the parathyroid glands are completely separated (Vermeulen, 1917; Littledike, 19^7);
By thyroidectomy elimination of all CT production is obtained in the pig, even in hypercalcaemia (Arnaud et al., 1970).
There are specific radioimmuno-assays for determination of PTH and CT in the pig.
MATERIAL AND METHODS
Laboratory animals: In all, 4 0 female pigs of the Swedish Land race, Swedish Yorkshire race and crosses between these two races were used. At the beginning of the experiments they weighed between I6 . 5 and 28 kg and were 10 to 14 weeks old. They were kept in large
cages and were given commercial pig food with an energy content of 3.2 Meal and with 1200 I U of vitamin D^, 8 . 4 g of calcium salts and 6.0 g of phosphates per daily ration. The pigs had free access to drinking water.
The pigs were divided into 3 groups: one control group primarily consisting of 12 pigs (SHAM), one group of animals subjected to thyroidectomy, primarily comprising 8 pigs (TX) and one group of 11 -thyroidectomized pigs which received PTH injections (TX + P T H ) .
Anaesthesia: The animals were anaesthetized with halothane-oxygen- nitrous-oxide on a semi-open system. Por the induction of the general anaesthesia the same anaesthetics or thiopentone sodium (Penthotal sodium, Abbott) were used.
Surgical procedures: Central venous catheters were implanted in the superior vena cava by way of the external or the internal jugular vein (Christison & Curtin, I9 6 9 ).
Cystostomy, using a Pezzer or Couvelaire catheter, was carried out combined with ligature and division of the urethra.
Thyroidectomy or a sham operation was performed through a mid-ventral incision (Romack et al., 1 9 6 4 ).
Kidney biopsies were done in 33 pigs, each on 2 different occasions through a transversal incision below the last rib.
Experimental procedures: The first clearance investigation was performed 2-3 days after the implantation of venous catheters and the cystostomy and was immediately followed by thyroidectomy or a sham operation. The TX + PTH group received the first PTH injection
on the second day (i.e. 44 hours) after the thyroidectomy. PTH was injected subcutaneously 3 times a day, 8 a.m., 3 p.m. and 10 p.m.
The total daily dose was 25 ÏÏSP units/kg. On the 5th day after the thyroidectomy the second clearance investigation was carried out, followed by a left kidney biopsy, and 3 days later the third, followed by a right kidney biopsy.
Laboratory methods: Serum calcium and serum magnesium were
determined using an atomic absorption spectrophotometer (Unicam, SP 90 B ) . The calcium ion activity was determined by means of an ion
specific flow-through electrode (Orion Research Corp.). The other serum electrolytes were determined using a multichannel auto analyzer (Technicon auto analyzer SMA 6 /6 0 ).
Clearance studies: The following clearance studies were performed:
clearance of 51Cr complexed with ethylene-diamine-tetra-acetic acid ( 51Cr-EDTA) calculated both from the activity in plasma (Cl 51Cr-EDTA-p) and from the activity in whole blood after correction for the haematocrit (Cl ^ C r - E D T A - w . b .), clearance of
12 e) 12 e)
I-labelled hippuran (Cl I-hippuran), clearance of para- amino-hippuric acid (Cl ), the tubular transfer maximum of
Jr A h
para-amino-hippuric acid (Tnip^) , clearance of inulin (Clp^) and the tubular reabsorption of phosphates (TRP).
A complete clearance investigation consisted of 4 clearance periods of 30 min each. Cl 51Cr-EDTA was determined in all clearance periods, and Cl 1 25I-hippuran in 2 and Ti^p^H in 2. Cl^.
was determined only in 6 pigs to establish the correlation between ClIn and Cl 51Cr-EDTA.
Blood samples for the clearance determinations were taken in the middle of each period.
At the beginning and end of each clearance period the urinary bladder-was irrigated with 0.9 % saline and emptied by means of air inflation and abdominal compression.
Inulin was determined according to Heyrovsky (1956).
PAH was determined according to Brun (1951).
The isotope concentrations in the blood, plasma and urine were determined in 2 ml samples with a well-crystal scintillation detector (Landis & G-ühr) to a statistical precision of - 1 A microhaematocrit centrifuge (No. 490, International Equipment Company) was used for the haematocrit determination.
Tm.pAjj was calculated from the formula
UPAH • 7 “ 0,83 • 01 51Cr-EDTA-p . PpAH
where U = urine concentration, V = volume of urine in ml/min and
P = plasma concentration. 0.83 is a constant given "by Reubi (i9 6 0 ) to correct for the PAH bound to protein.
TRP was determined in 11 pigs. TRP was calculated from the formula
P^, . Cl 51Cr-ELTA-p Ph
where Ph = phosphates.
Morphological studies: Renal biopsies were performed in 25 pigs on two different occasions. Specimens were taken for light and electron microscopic investigations.
Light microscopic procedures: The specimens were fixed in 10 $ formalin. The following light microscopic stains were used:
van Gieson's stain,hematoxylin-eosin, periodic acid -Schiff (PAS), von Kossa's stain, Ladewig's stain, Laidlaw's stain and Sudan Black B.
Electron microscopic procedures: The specimens were fixed in 2.5 i<>
glutaraldehyde in 0.34 M Veronal acetate buffer adjusted to pH 7.4»
followed by postfixation in 1 ^ osmium tetroxide in the same buffer.
After fixation the specimens were rinsed, dehydrated and embedded in Epon 812. The sections were cut on an Ultrotome III and thick sections stained with toluidine blue were used for light microscopic identification of suitable areas for the thin sections. After staining with uranyl acetate and lead citrate the sections were examined in a Siemens Elmiscope 101.
Statistical methods: Standard statistical methods were used for the calculation of mean values, linear regressions and correlation coefficients (Pearson's product moment correlation coefficient).
In paper II the material was analyzed using a two-factor analysis of variance design. P-tests were applied to judge whether the changes were significant. If significant changes were found, further analyses were made using Scheffé's method for multiple comparisons.
RESULTS
RENAL CLEARANCE STUDIES IN THE PIG (i).
The values for the serum electrolytes agree well with the values for miniature pigs of the same age reported earlier (McClellan et al., 1 9 6 6 ; Marshall et al., 1972).
The following mean values were obtained from the clearance studies in normal pigs:
n X S.D. S.E.]
21In ml/min/kg 4 4 . 1 5
Cl 51Cr-EDTA-p ml/min/kg 18 3.82 O. 4 2 0.10
Cl 51Cr-EDTA-w.b. ml/min/kg 17 3.89 O.4 I 0.10 Cl 1 25I-hippuran ml/min/kg 18 1 7 . 4 4 .I6 0.98
C1PAH ml/min/kg 18 21 . 4 6.95 1 . 6 4
t"Va h mg/min/kg 16 4 . 3 1 0.73 0.18
In 4 pigs with normal serum creatinine values but in which the maximum urine concentration capacity was investigated the day before the clearance studies, the following clearance values were recorded:
Cl ^ C r - E D T A - p 2.71 ml/min/kg 21 I6 . 3 ml/min/kg RAJd
Cl ^ C r - E D T A - w . b . 2.74 ml/min/kg TmPAH 4.26 mg/min/kg Cl 1 25I-hippuran 13*6 ml/min/kg
The correlations between 2 1 ^ and Cl 51Cr-EDTA are shown in Pigs.
1 and 2.
m l / m i n C l j n
100
y = 1 . 0 7 5 X 5 0
5 0 100 m l / m i n C1 C r - E D T A - p
Fig. 1 . Correlation between simultaneous clearances of 51Cr-EDTA-p and inulin in 6 pigs.
ml /mi
100
1 0 0 m l / m i n 5 0
Fig. 2 . Correlation between simultaneous clearances of 51Cr-EDTA-w.b. and inulin in 6 pigs.
The correlation between Cl 1 25^I-hippuran and ClpAH is shown in
Fig. 3.
Cl PAH m l / mi n 7 00
5 00
300
100
500 m l/ mi n 100 300
-100
Fig. 5 . Correlation between simultaneous clearances of 1 25I-hippuran and PAH in 26 pigs.
The tubular reabsorption of phosphates (TR?) was > 80io (80.1 - 96.5) in the 11 cases in which it was calculated.
RENAL FUNCTION AFTER THYROIDECTOMY AND PARATHYROID HORMONE ADMINISTRATION (il).
In 16 out of 32 pigs the clearance investigations were completed.
Of these, 5 pigs belonged to the SHAM group, 6 to the TX group and 5 to the TX + PTH group.
In the other 16 pigs the clearance studies were interrupted due to technical complications, e.g. urinary leakage.
Serum electrolytes. No changes in sodium, potassium, magnesium,
Chlorides, bicarbonate or serum proteins were observed in any of the 3 groups. In the SHAM and TX groups no changes in creatinine, urea-N, calcium or phosphates were noted. In the TX + PTH group a significant (p'CO.O'l) increase in the serum calcium was found as well as a significant ( p < 0 . 0 l ) decrease in serum phosphates. A significant ( p < O . O l ) increase in serum creatinine and an increase in urea-N and the calcium ion activity (p> O.O5 ) were also noted.
Clearance studies. In the SHAM group no changes in the clearance values or TEP were observed. In the TX group there was a significant (p< O.OI) decrease in Cl ^ C r - E D T A - p and in TiUp^ and a not
significant (p> O.O5 ) decrease in Cl ^ ^ I-hip pur an and Clp^g*
TEP was unchanged (> 80%). In the TX + PTH group there were
significant ( p < 0 . 0 l ) decreases in all clearance values. TEP w a s < 80%.
(Pigs. 4 and 5)
m l ( m g ) / m i n / k g
PAH
D a y s
Cl 2 Cl 3
CM
\
\ m l ( m g ) / m i n / k g
\
\
\
\
\
\
\
\
\
'C l 1ZbI - h ip p u r a n
~ C l 51C r-E D T A
C 12 CI3 D a y s
Pig. 4 . Changes in clearance values in the TX + PTH and TX groups.
T X + P T H TX
100- 100
50.
50..
Cl 3 C M C12 Cl 3
eu C 12
Eig. 3 » Changes in clearance values in the TX + PTH and TX groups.
The values are expressed in per cent of initial values.
RENAL MORPHOLOGY IN PIGS WITH EXPERIMENTAL HYPERPARATHYROIDISM.
Light microscopic findings and some functional .aspects, (ill).
In the animals of the SHAM group no pathological renal alterations were found.
In the TX group, at the examinations both 5 and 8 days after the thyroidectomy, a slight to moderate dilatation of some tubules, mainly the proximal ones, was seen. Occasional hyaline casts and some desquamated epithelial cells could be noted in the tubular lumina. No calcifications or any conspicuous inflammatory cell infiltrates were found.
In the TX + PTH group the following changes were observed at the first examination 78 hours after the first PTH injection: Most glomeruli showed an ordinary cellularity and a normal appearance of the epithelial and endothelial cells. Deposits of calcium were infrequently observed in the capsular epithelium. There were no calcifications in Bowman's space. In the tubules dilatation affecting all parts of the nephron was a prominent feature. The tubular
epithelium was usually normal. Degenerative changes of varying severity were found, mainly in cells with calcareous inclusions.
Some epithelial cells were necrotic. There were also signs of epithelial regeneration with occurrence of mitotic figures.
Calcium deposits were found in both normal and degenerated tubular epithelial cells. Calcifications were present in the lumina of both dilated and nondilated tubules. The calcium deposits were found in all parts of the nephron and collecting tubules but mostly in the proximal convoluted tubules. There was often a strong tendency to a patchy distribution of these changes.
Hyperaemia, as evidenced by dilated blood-filled vessels, was also noted.
At the examination 3 days later (i.e. I50 hours after the first PTH injection) the histopathologic changes were of the same character but even more advanced.
RENAL MORPHOLOGY IN PIGS WITH EXPERIMENTAL HYPE RPARATHYROIDISM.
Ultrastrueturai findings, (iv).
The electron microscopic examinations were mainly directed to the occurrence and morphological details of structures believed to represent calcium deposits.
In the SHAM group the renal parenchyma was normal and no calcifica
tions were seen.
In the TX group no conspicuous ultrastructural changes and no structures representing calcifications were observed.
In the animals of the TX + PTH group the light microscopic findings of degenerative changes in the tubular epithelium were verified.
In the tubular epithelial cells, mainly in those of the proximal convolution, electron dense structures of various kinds, inter
preted as calcium deposits, were found in the mitochondria and cytoplasmic vacuoles.
Tubular lumina with light microscopic evidence of calcifications were found to contain calcified remnants of degenerated tubular cells.
No marked degenerative changes were observed in epithelial cells without calcifications.
Only a few calcium deposits were found in the basement membranes of the tubular cells.
The light and electron microscopic findings indicated a development of large calcareous bodies from small electron dense needle-shaped and granular particles, primarily localized to mitochondria and cytoplasmic vacuoles in the tubular epithelium. The findings also denoted that the deposits in the tubular lumina had been discharged from the tubular epithelial cells, apparently due to cellular degeneration.
It seemed as if the calcium deposition preceded and also caused the degenerative changes.
DISCUSSION
Experiments on renal physiology have hitherto usually been performed in dogs in spite of the considerable anatomical and functional differences between the canine and the human kidney. However, the dog is not very suitable for studies on the interaction between PTH and CT as its thyroid and parathyroid glands are located close
to each other and 2 parathyroids are found in the thyroid.
Extirpation of either of these organs thus entails a great risk of damaging the other. Moreover there are no specific methods available so far for determinations of PTH and CT in the dog.
In the pig, however, the thyroid and the parathyroid glands are completely separated. There are only 2 parathyroids and they are located to the cranial part of the cervical portion of the thymus (Vermeulen, 1917; Littledike, 1967). Thus it is possible to extirpate either the thyroid or the parathyroid glands with no risk of
damaging the other organ.
In this study we wanted to eliminate increased CT production caused by hypercalcaemia. In the pig this can be achieved by thyroidectomy (Arnaud et al., 1970) and therefore this animal was chosen for the experiments.
There are specific methods for determinations of PTH and CT in the pig (Arnaud et al., 1970).
There are also anatomical and functional differences between the porcine and the human kidney, e.g. in the percentage of long looped nephrons, the relative medullary thickness (the medullary
thickness x 10/kidney size, where kidney size = the cube root of the product of the dimensions of the kidney) and the maximum urine concentration capacity (Schmidt-Nielsen & O'Dell, I9 6 I).
Man Pig Dog
Long looped nephrons % 14 3 100
Relative medullary thickness 3.0 1.6 4.3
Max. urine conc. capacity mOsm/kg 1 1 6 0 1080 2425
Only a few experiments on renal function have been carried out in pigs and there are great differences between the values for Cljn , Clp^jj and Tm^^. found at these investigations.
C1TIn C1PAH TmPAH
Dalgaard-Mikkelsen et al (1953) 3.65
Munsick et al (1958) 5 . 0 0 1 9 . 5 3 . 1 0
Suarez et al (1968) 3 .5 O
Gyrd-Hansen (19 6 8 ) 2 . 1 0 6.4 2 .3 O
Gyrd-Hansen (1970) 3 . 2 0 8.7
This investigation 4.11 21.4 4 . 3 1
The values are expressed in ml(mg)/min/kg.
Since the results of previous investigations on porcine renal function disagree, which may be put down to differences in races, hydration or experimental techniques (Gyrd-Hansen, 1 9 7 0 ), a standardized investigation technique was worked out.
To make blood sampling and intravenous fluid administration possible, it was necessary to implant central venous catheters,and to ensure correct urine sampling cystostomy with ligature and division of the urethra had to be performed.
The animals were kept without food for 12 hours before the clearance investigations. To begin with the pigs were also deprived of water for studies on the maximum urine concentration capacity but this study had to be abandoned as 4 out of the 8 pigs subjected to this investigation became uraemic and all 8 had clearance values lower than those of the pigs with free access to drinking water.
To ensure high diuresis 0.2 - 0.3 ml/min/kg of isotonic saline was administered intravenously. The fluid volume was limited to 0.3 ml/min/kg as it has been established in studies in man that doses of this order probably do not affect the renal function measured by clearance determinations (Wesson, 1969).
It was necessary to perform the experiments in unsedated and un
restrained pigs as there is usually a reduction in the renal function under anaesthesia (Wesson, 1969)
In the present investigation Cl 51Cr-EDTA was chosen as the expression of GFR. Earlier studies have established that this clearance value is very close to that for inulin (Stacy & Thorburn, 1966; Lingårdh, 1972).
This fact was verified in the present study at simultaneous determinations of Cl-,, and Cl ^ C r - E D T A in 6 animals (i). The
In x y
correlations found agree well with those of studies published earlier.
EPF was determined by means of C k . ^ and Cl 1 25I-hippuran. It has been found earlier that Cl 1 25I-hippuran is distinctly lower than O l p ^
but also that there is a correlation between these clearance values (Maher & Elveback, 1970; Lingårdh, 1972).
The correlation found at the present study is consistent with that of earlier investigations (i).
In studies on the renal function in patients suffering from spontaneous hypothyroidism, low values for 01^n and 0 1 ^ ^ (or the clearance of diodrast (Cl^) ) were found (Corcoran & Page, 1947;
Luft & Sjögren,I9 5 O; Hlad & Bricker, 1954; Yount & Little, 1955).
It was also noted that there is a rapid return to normal values after administration of thyroxin (HIad & Bricker, 1954)« These findings were verified in experiments in dogs (White et al., 1947)«
However, there is no information about how quickly a reduction in the clearance values is obtained after thyroidectomy.
The results of the present study (il) imply that this reduction occurs very rapidly. In 2 animals in which clearance studies were performed 2 days after the thyroidectomy, a decrease in the clearance values to approx. the same level as that found after 5 days was noted. This decrease 5 days after the thyroidectomy was statistically significant ( p < 0 . 0 l ) for Cl ^ C r - E D T A and Tmp^. , but not for Clp^g (p^ 0.05). At investigations 8 days after the thyroidectomy a further reduction in the clearance values was observed. This decrease in the renal function was not associated with any marked morphological alterations in the kidneys (ill, IV).
Previous experiments on the renal function after PTH injections have shown that there is an initial increase in the renal blood flow (Charbon, 19^8) and a rise in Cljn and Clp^p- ( °r Cl^ ) values (Handler et al., 1951; Widrow & Levinsky, I9 6 2 ).
Morphological alterations occurred very rapidly (Carone et al., i9 6 0 ), in any case if the administered dose of PTH caused a marked in
crease in serum calcium ( 1 6 - 2 4 mg/100 ml ). 24 hours after the PTH injection to these animals a reduction was found in the renal function (Epstein et al., 1959). The morphological changes, i.e.
degenerative alterations in the cells and calcifications, occurred in all parts of the nephron and collecting tubules.
According to Carone et al.(l960) the most extensive changes should be found in the distal parts of the nephron and in the collecting tubules, but most other authors have stated that the alterations are most extensive in the proximal tubules (Cantarow et al., 1938;
Caulfield & Schräg, 19 6 4 ; Yano et al., 1965; Berry, 1970)
The findings of the present study ( III, IV ) 78 and I50 hours after the first PTH injection showed that the most pronounced changes are found in the proximal tubules.
We also found histological signs of hyperaemia (i.e. dilated and
"blood-filled vessels), which is in agreement with the increased renal "blood flow reported "by Charbon (1 9 6 8 ) and with Edwall 's ( 1958) findings of relative renal hyperaemia in patients who were to "be operated on because of hyperparathyroidism.
As PAH is secreted by the proximal tubular cells (Pitts, I9 6 8 ) it should be possible to estimate the extent of the cellular lesions from the reduction in Tmp ^ and values.
In the present study (il) we found a pronounced reduction in Cl-p^
and above all as early as 72 hours after the first PTH injection.
The findings of the present study (ill, IV) agree with the opinion of Learner (1929) and Schneider et al. (i9 6 0 ) that the calcifica
tions precede the degenerative changes in the tubular epithelium.
It also seems as if the cellular degeneration leads to a discharge of calcific material into the tubular lumina, sometimes followed by tubular obstruction.
The calcium deposition, which is related to the hypercalcaemia induced by PTH administration, thus appears to be of primary and great importance in the development of the structural lesions in the kidneys.
In the present study a development of rather large, sometimes concentric, calcareous bodies was noted. It is suggested tha'c these bodies are formed by coalescence of small electron dense particles, primarily localized to mitochondria and cytoplasmic vacuoles. It is probable that these bodies might later give rise to calcium deposits visible to the naked eye and subsequently to roentgenologically evident nephrocalcinosis.
The earliest calcium deposits were localized to mitochondria and also to cytoplasmic vacuoles, which is consistent with the fact that these organelles have a considerable capacity for uptake of divalent cat-ions (Lehninger, 1965).»
GENERAL SUMMARY AND CONCLUSIONS
A technique for clearance determinations in unsedated and u n restrained pigs has been described. This technique has been used to study the renal function after thyroidectomy and parathyroid hormone administration to thyroidectomized pigs.
Normal clearance values in Swedish domestic pigs have been established.
Thyroidectomy was followed by a rapid decrease in renal function without any marked concomitant morphological changes in the kidneys.
Parathyroid hormone administration to thyroidectomized pigs was followed by a rapid and pronounced reduction in renal function with concomitant morphological changes in the kidneys.
The most prominent morphological changes were calcium deposition, cellular degeneration, tubular dilatation, inflammation and
hyperaemia. The calcifications and degenerative changes were mainly, although not exclusively, localized to the proximal convoluted tubules.
The structural alterations suggested a development of large calcific bodies from small subunits, primarily localized to mitochondria and cytoplasmic vacuoles. The calcium deposition appeared to precede the degenerative changes.
There were also indications of a discharge of calcified material into tubular lumina as a result of the degenerative processes.
It has been established previously that parathyroid hormone increases the renal blood flow and the clearance values. Therefore the reduction found in these values must be due to the cellular lesions in the kidneys.
An attempt has been made to establish a correlation between the decrease in the clearance values and the extent of the cellular lesions, but as these lesions were extensive in all animals given parathyroid hormone, no such correlation could be established.
To demonstrate whether or not such a correlation exists, further studies, using a lower dosage of parathyroid hormone, are needed.
Further studies are also required to determine to what extent these cellular lesions are reversible.
In addition further studies are necessary to establish the effects of calcitonin on renal function and whether it modifies the effect of parathyroid hormone on renal function and structure.
ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to:
Professor Lennart Andersson for initiating this study, for the excellent working facilities he placed at my disposal and for all the support and constructive criticism he has given me throughout the investigation;
Professor Lennart Boquist for stimulating cooperation and constructive criticism;
Docent Gunnar Lingårdh for generous help and valuable advice;
Professor Sven Landgren for excellent working facilities;
Mrs Gudrun Andersson, Mrs Ulla-Britt Carlsson, Mrs Kristina Leppälaakso and Mrs Britt-Helene Sandgren for skilful technical assistance;
Mrs Solveig Isberg and Mrs Desiree Lundin for typing the manuscripts.
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