- Email: [email protected]
The effect of adrenalectomy on the proteinuria of spontaneously hypertensive rats and normotensive controls
Exp. Pathol. 30, 233- 242 (1986) VEB Gustav Fischer Verlag J ena Department of Pathology, University }Iedical School, Debrecen, Hungary
The effect of adrenalectomy on the proteinuria of spontaneously hypertensive rats and normotensive controls By M. SZOKOL, M. B. SOLTES Z, A. NAGY, Z. LENGYEL and Sz. GOMBA With 7 figures Address for correspondence: Dr. M. B. SOLTESZ, Department of Pathology, University Medical School, P.O.B. 23, H - 4012 Debrecen, Hungary Ke y w or ds: proteinuria; spontaneously hypertensive rats; adrenalectomy ; glucocorticoid treatment; mineralocorticoid treatment; DO CA; dexamethasone
Summary Urinary proteins were studied by quantitative and electrophoretic methods in 6-monthold spontaneously hypertensiv e rats and normotensive controls. Protein analysis was carried out before and after adrenalectomy and during gluco- or mineralocorticoid treatment. Urinary protein excretion was significantly diminished after adrenalectomy both in the hypertensive and control gr oups. The original level of protein excretion was restored only by glycocorticoid treatment . Normal or pathologic electrophoretic pattern of urinary proteins was not influenced by the experimental procedure. Moderately non-selective glomerular proteinuria persisted in the spo ntaneously hypertensive r at s r eferring t o a definitely damaged glomerular barrier. One protein fraction of about 130,000 dalton molecular weight disappeared from the urine of hypertensive animals after adrenalectomy and reappeared after glu cocorticoid treatment only. This fraction probably represents the dimeric form of albumin. Quantitative changes of urinary protein excretion can be explained by haemodynamic factors.
I ntroduction
It is well-known that proteinuria evoked by the injection of different pressure substances (renin, angiot ensin II, norepinephrine etc.) will be lowered to a normal level after bilat eral adrenalectomy (ADDIS et al. 1950; MASSON et al. 1952 ; PESSWA et al. 1972). Contradi ctory data are existing in the literature how the different adrenocortical hormone prepamtes elevate urinary protein excretion in adrenalectomized rats (DEODHAR et al. 1964 ; PESSINA and PEART 1972). Mechanisms by which pressure subst ances exert proteinuria are also not clear. Structural and hemodynamic alterations in the glomeruli are assumed to be responsible for the effect of adrenalectomy and pressure substances (BAUMAN 1979, 1980). Experim ental models used for the study of proteinuri a evoked by pressure substances and modified by adrenalectomy were differ ent concerning the initial state of the glomerular barrier: Permeability of the glomeruli was originally normal in most of the investigations (DEODHAR et al. 1964; PESSINA and PEART 1972; PESSINA et al. 1972; BAUMAN 1979). But new inform ation was obtained if aminonucleoside nephro sis and pathologic proteinuria preceded the injection of pressure substances (BAUMAN 1980). Th e effect of adrenalectomy was not investigated in this model. In our experiment 6-month-old spontaneously hypertensive rats were chosen to study the role of adrenal glands and adrenocortical hormones in the regulation of quantity and composition of urinary proteins. The glomerular barrier was definitely damaged in these animals accompanied by a constantly pathologic pr oteinuria.
233
....
~
00
....<:>
<:0 ,-..
~
::r ~
"'d ~
'?
~
t:
~ ~
SHR WKR SHR WKR
Food intake (gfday)
0.9
0.8
± 0.4
± 0.6
22.2 + 19.9 20.6 21.1 ±
14
SHR WKR
40.8 36.4
± 3.5 ± 5.0 47.1 ± 3.1 36.6 ± 6.9
After adrenalectomy
0.7***
DOCA
± 4.4
+ 2.8
46.9 41.3
Dexamethasone
± 7.2
68.6 38.8
+ 6.2***
±
±
11.3 + 1.0*** 0.9*** 10.9 14.6 + 0.6*** 1.0*** 12.1
± 0.6*** 0.7*** ± 0.8***
-3.6 + -4.6 -2.8 + - 3.4
±
154 + 9*** 79 f 3*** 153 + 10* 2*** 86
After adrenalectomy
After adrenalectomy with hormonal treatment
*** p < 0.001 as compared to the values after adrenalectomy.
11
15 11
SHR WKR
n
Table 1a. Saline intake (gfday)
p < 0.05 } ++ P < 0.01 as compared to the values after adrenalectomy +++ p < 0.001
+
0.2
± 0.5 0.2 ± 0.3
0.5 + 0.7 0.5 + 1.2
±
±
4 181 102 ± 2 177 + 5 101 3
Before adrenalectomy
as compared to the preoperative values
15 11 14 11
SHR WKR SHR WKR
Body weight gain (gfday)
* p < 0.05 *** P < 0.001
15 11 14 11
SHR WKR SHR WKR
Blood pressure (mmHg)
15 11 14 11
n
±
1.0+++
± 0.6+++ 0.6+++ ± 0.9+
± 0.7+++
+ 1.0++ ± 1.0 + 0.4+++
18.6 + 15.9 19.6 + 15.9
-0.8 -0.7 2.4 3.8
±
153 + 7 104 f 2 176 + 9+ 100 2+
After adrenalectomy with hormonal treatment
DOCA
Dexamethasone
DOCA
Dexamethasone
DOCA
Dexamethasone
Table 1. Blood pressure, body weight gain and food intake of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKR) before and after adrenalectomy and during hormonal treatment. Values are means SEM
g;
~
Il'>-
C$l
..s
I-' <0
---
~
~
t:r'
~
"'d
':'
~
t
11
15 11 14
15 11 14 11
15 11 14 11
4.23 + 0.44 1.95 ± 0.38 ±
+ 3.82
20.85 0.85 12.87 0.99
0.88 + 0.11 0.35 ± 0.08 ±
5.70 + 0.63 0.40 0.06 3.17 + 0.75 0.09 0.48
± 0.19 + 3.87 ± 0.21
± ± 0.82** 0.09*
1.17***
7.23+++
± 0.21 ++ 0.22 ± 0.06
35.42 + 1.28 0.92 + 0.23
±
0.12*'1< 0.02**
± 0.07*
±
0.44 + 0.11 0.23 0.08
7.50 + 1.02+ ++ 0.08+++ 0.50
±
4.5 + 0.6 3.0 1.7++ 3.0 + 0.5+++ 1.7± 0.3+++
After adrenalectomy with hormonal treatment
0.24***
± 0.02**
±
± ±
5.8 ± 0.6** 5.0 ± 0.7*** 5.4 + 0.5* 5.1 0.7***
After adrenalectomy
3.4 + 0.3 2.1 0.4 3.7 + 0.4 0.2 1.9
Before adrenalectomy
< 0.05 } as compared to the preoperative values P < 0.01 p < 0.001 P < 0.01 } as compared to the values after adrenalectomy p < 0.001
p
SHR WKR SHR WKR
Protein excretion (mg/6 h)
*
SHR WKR SHR WKR
Protein concentration (mg/ml)
** *** ++ +++
SHR WKR SHR WKR
Urine volume (ml)
n
DOCA
Dexamethasone
DOCA
Dexamethasone
DOCA
Dexamethasone
Table 2. Urine volume. protein concentration and 6 h protein excretion of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKR) before and after adrenalectomy and during hormonal treatment. Values are means ± SEM
Materials and Alethods Forty male 24-28 weeks old spontaneously hypertensive rats (Okamoto strain) of 280-330 g body weight, and 30 normotensive controls (Wistar-Kyoto strain) of the same age and sex were placed in individual cages. The animals were offered a commercial food (LA TI) and demineralized water ad libitum. Body weight, water and food intake were measured daily. Systolic blood pressure was registered by tail plethysmography in a light aether-narcosis every other day (BYROM and WILSON 1938). Three different experimental periods were performed each lasting 5 days. During the first period intact rats were observed. Bilateral adrenalectomy was carried out on the 5th day. One per cent saline as drinking fluid was offered to all the animals after the operation till the end of experiment. 11 spontaneously hypertensive and 8 normotensive rats died during the first 2-3 postoperative days without any remarkable autopsy findings probably because of disturbances in water and electrolyte metabolism. Hormonal treatment was performed during the third experimental period in the following way: Dexamethasone (Oradexon-Organon) was given subcutaneously every day in a dose of 0.5 mg to 15 spontaneously hypertensive and 11 normotensive rats. Desoxicorticosterone acetate (Decosteron-Kobanyai Gy6gyszergyar) was injected intramuscularly into 14 spontaneously hypertensive and 11 normotensive animals. The dose (0.2 mg/day) was twice as high as the recommended one for substitution therapy (BARNES and ELTHERINGTON 1973). Two urine samples were collected in each experimental period from each animal between 8 a.m. and 2. p.m. Blood samples were obtained from the cut tail at the end of each period. Rats were sacrificed by bleeding. Heart weights were registered and kidneys fixed in 10 per cent buffered formaldehyde solution for light microscopy. Protein concentration of the urine samples was measured by means of Folin reagent (HARTREE 1972) using bovine plasma albumin as the standard. Urinary and plasma proteins were separated by sodium dodecyl sulphate polyacrylamide gel (SDS-PAAG) disc electrophoresis (WEBER and OSBORNE 1969). The protein samples were treated only with SDS (BOESKEN et al. 1973) but in some cases mercaptoethanol was also added to the system. Gels removed from the tubes were stained by Coomassie briIIant blue R 250. Periodic acid Schiff reaction (ZAHARIUS and ZELL 1969) and alcian blue staining (WARDI et aI. 1972) were also performed. Molecular weight of the different protein bands was determined from a plot of the electrophoretic mobility against the logarithm of the known molecular weights of standard proteins (Serva). Results were recorded by scanning the gels with a Kipp-Zonen Micrograph 805 (red filter). Paraffin sections of the kidneys were stained with hemalum eosin, periodic acid Schiff reaction, MOVAT (1961) silver impregnation for the basement membranes, and LENDRUM'S (1962) method. Statistical analysis was carried out by Student's t-test (paired data). Values in the text, tables and figures are means ± S.E.M.
Results The blood pressure sank after adrenalectomy both in the hypertensive and normotensive rats. A return to the original level was evoked by gluco- or mineralocorticoid substitution in the normotensive group. A similar effect was registered in the spontaneously hypertensive rats after mineralocorticoid (DOCA) treatment only (table 1). Hormonal treatment induced some increase in food intake but it remained below the preoperative level (table 1). Body weight and food intake decreased after adrenalectomy (table 1). Saline was taken up in a large quantity by the normotensive and hypertensive animals during the postoperative days (table 1a). Desoxicorticosterone acetate brought about a rapid body weight gain by its sodium and water retaining effects. On the contrary in all the animals treated by dexamethasone a progressive loss of body weight was registered (table 1). The 6 hours urine volume increased, urinary protein concentration and the quantity of the excreted protein decreased after adrenalectomy. The urine volume decreased during the hormonal substitution in all the experimental groups (table 2). Protein concentration and 6 hours' excretion was elevated above the preoperative levels by dexamethasone treatment. Desoxicorticosterone acetate failed to produce this effect (table 2). The electrophoretic pattern of the urinary proteins proved to be pathologic in the spontaneously hypertensive rats already in the preoperative period: the bands qf albumin and transferrin widened and protein fractions above 100,000 dalton molecular weight appeared in the urine. This picture corresponded to the moderately non-selective type of glomerular proteinuria (BoEsKEN et al. 1975) (fig. 1). Urine samples of the normotensive animals con-
236
Exp. Pathol. 30 (1986) 4
180 &; ,
(\2
0
r
0,"
0.6
0,8
lRM
N
,
Q,2
0,"
A
MW
002- 180
t-~)
B
Ie
lRM
H
TA 1
2
Fig. 1. Electrophoretic pictures with densitometric curves of urinary proteins of a normotensive control (N) and a spontaneously hypertensive rat (H) before adrenalectomy. In contrast to the normotensive animal the bands of albumin (A) and transferrin (T) widened and proteins above 100,000 dalton molecular weight appeared in the urine. OD: optical density, MW: molecular weight, RM: relative mobility. Fig. 2. Microprotein fractions in the urine of a normotensive Wistar-Kyoto rat before (A) and after (B) the operation and during dexamethasone treatment (C). No alterations can be seen. Abbreviations see fig. 1.
tained only microproteins and albumin in a small quantity (fig. 1). The general electro phoretic pattern did not change either in the normotensive or hypertensive groups after adrenalectomy and during the hormonal treatment (fig. 2, 3, 4). Moderately non-selective proteinuria persisted in the hypertensive animals. But one fraction with 130,000 dalton molecular weight disappeared after adrenalectomy. Dexamethasone treatment induced its reappearance (fig. 3). Desoxicorticosterone acetate failed to produce the same effect (fig. 4). Electrophoretic analysis of plasma proteins detected a fraction of 130,000 dalton molecular weight before and after adrenalectomy and during the hormonal treatment (fig. 5). When the electrophoretic separation was carried out from freshly collected urine samples (omitting the few days storage at -20°C) the protein fraction of 130,000 dalton molecular weight was not detected. Mercaptoethanol added to the system containing frozen urine samples led to a Exp. Pathol. 30 (1986) 4
237
A
c
o
0.2
0,4
0,11
011
B .j
Fig. 3. Urinary proteins of a hypertensive rat before (A) and after (B) adrenalectomy and during dexamethasone treatment (0). Protein fraction with 130,000 dalton molecular weight (arrow) disappeared after the operation and reappeared under hormonal influence. Abbreviations see fig. 1. Fig. 4. Protein fractions in the urine of a hypertensive rat before (A) and after (B) adrenalectomy and during desoxicorticosterone acetate treatment (0). Protein of 130,000 dalton molecular weight (arrow) which disappeared in the adrenalectomized animal did not return while given mineralocorticoid. Abbreviations see fig. 1.
similar result. This protein fraction proved to be negative with alcian blue staining and periodic acid Schiff reaction. In the histologic picture alterations of glomeruli referred to a serious damage of their barrier: thickening of the basement membrane (fig. 6), hyalino sis and fibrinoid necrosis of the glomerular loops were equally noticed (fig. 7).
Discussion Considering the influence of adrenal glands and adrenocortical hormones on the quantity and composition of urinary proteins in normal and pathological states, some problems are not definitely settled. Contradictory results were registered about the effect of different hormone preparations on urinary protein excretion after bilateral adrenalectomy. Protein-
238
Exp. Pathol. 30 (1986) 4
o
1\2
0,4
o
o,~
1RM
u Fig. 5. Electrophoretic analysis of serum (S) and urinary proteins (U) from a hypertensive rat after adrenalectomy. A fraction of 130,000 dalton molecular weight (arrow) could be found among the serum proteins but it did not appear in the urine. Abbreviations see fig. 1.
uria induced by angiotensin II was quantitatively restored by cortisone or DOCA (DEODHAR et al. 1964). Some authors found that only corticosterone was effective (PESSINA and PEAR 1972). Using hormonal substances with a strong glucocorticoid (dexamethasone) and mineralocorticoid (aldosterone) effect, BAUMAN (1979) observed an elevated urinary protein excretion only after dexamethasone treatment. Our results are in accordance with the latter observation. The effect of adrenalectomy, substitution treatment and their interaction with proteinuria induced by pressure substances are still not clear. They probably influence urinary protein excretion through the modification of renal circulation. Renal plasma flow (RPF) and glomerular filtration rate (GFR) are diminished after adrenalectomy (HARRISON' and DARROW 1939; TALBOTT et al. 1942; LOCKETT 1949). Glucocorticoids, first of all the widely studied cortisone increase the quantity of urinary proteins by the elevation of GFR and RPF. Mineralocorticoids (e.g. DOCA, aldosterone) have no direct haemodynamic effect. They tend to normalize salt and water balance resulting in a moderate elevation of GFR and RPF (LUFT and SJOGREN 1949; GAUDINO and LEWITT 1949; GARROD et al. 1955). Pressure hormones increase the filtration fraction, which seems to be essential for their proteinuric effect (PESSlNA and PEART 1972; BAUMAN 1981). Another explanation was also Exp. Pathol. 30 (1986) 4
239
Fig. 6. Glomerulus of a spontaneously hypertensive rat. The basement membrane is almost diffusely thickened. The mesangial matrix also reveals black staining. Movat's silver impregnation, X 128.
Fig. 7. Glomerulus of a spontaneously hypertensive rat with extensive hyalinosis. Fibrinoid necrosis colouring in orange red can be seen in some glomerular loops (arrows). Lendrum's staining, X 128. Green filter.
240
Exp. Pathol. 30 (1986) 4
suggested by BAUMAN (1979) for the angiotensin-induced proteinuria. He assumed that permeability of the glomerular barrier will be enhanced if electrostatically negative groups of its polyanions are neutralized by angiotensin II, a compound with cationic feature (SRAER et al. 1977). Later studies of BAUMAN (1981) proved unequivocally that quantitative changes of proteinuria evoked by angiotensin infusion in aminonucleoside nephrosis are strongly parallel with the alterations of the filtration fraction. Proteinuria of spontaneously hypertensive rats has not been analysed in connection with the adrenal glands and adrenocortical hormones. In our experimental model the glomerular barrier was already definitely damaged in the 4-month-old hypertensive rats in contrast to the transiently enhanced glomerular permeability induced by pressure hormones (e.g. angiotensin II, norepinephrine). Therefore diminished protein excretion after adrenalectomy was not a consequence of an "improvement" in the glomerular permeability: electrophoretic analysis demonstrated the persistence of moderately non-selective glomerular proteinuria. Quantitative alterations of the urinary protein after adrenalectomy and hormonal substitution are probably in connection with haemodynamic factors. The applied dose of DOCA was effective as proved by body weight gain, increased food intake and decreased urine volume. The quantity of the excreted proteins was not influenced by DOCA treatment. This hormone has no direct effect on the glomerular circulation (GARROD et al. 1955). The exact nature of the protein fraction that had disappeared from the urine after adrenalectomy, was not determined. Spontaneously hypertensive rats have a special protein fraction in the serum (serum post albumin or alpha1-globulin) with a 130,000 dalton molecular weight. The absence of positive PAS-reaction and alcianophilia opposes the identity of urinary protein with serum post albumin (KYOGOKU et al. 1972). The 130,000 dalton molecular weight suggests that this fraction may be the dimeric form of albumin. Further different kinds of indirect evidence also support this idea:
1. This fraction disappeared from the urine sample after mercaptoethanol treatment. 2. Dimerisation proceeds during the storage of samples below 0 DC temperature (DOMAN et al. 1980; BOESKEN 1976) but not in freshly collected urine. 3. Glucocorticoids (hydrocortisone) support dimerisation of albumin, e.g. in the urine of human nephrotic cases (DOMAN et al. 1980). Apart from these kinds of evidence further studies are needed to determine the exact nature of the discussed protein fraction by immunochemical and biochemical methods.
Acknowledgements The authors are indebted to J. VIRAGH and I. VERSENYI for their technical help and Dr. P. HAJDU (Inst. Social Medicine, Univ. Med. Sch. Debrecen) for help in the statistical evaluation.
Literature ADDIS, T. J., J. MAMMORSTON, H. C. GOODMAN, A. L. SELLERS and M. SMITH, Effect of adrenalectomy on spontaneous and induced proteinuria in the rat. Proc. Soc. expo BioI. Med. 74,43-46 (1950). BARNES, C. D., and L. G. ELTHERINGTON, Drug dosage in laboratory animals. Univ. California Press, Berkeley, Los Angeles, London 1973, p. 278. BAUMAN, J. W., Corticoid effects of angiotensin- and norepinephrine-induced proteinuria in rats. Amer. J. PhysioI. 237, F133-F137 (1979). - On the mechanism of angiotensin-induced proteinuria. 1. Studies in aminonucleoside nephrotic rats and in saralasin blockade. Nephron 27, 47-50 (1981). BOESKEN, W. H., K. KOPF and P. SCHOLLMEYER, Differentiation of proteinuria diseases by discelectrophoretic molecular weight analysis of urinary proteins. Clin. Nephrol. 1, 311-318 (1973). - Discelectrophoretic molecular weight analysis of urinary proteins. Contr. NephroI. 1, 143-155 (1975).
]6
Exp. Pathol. 30 (1986) 4
241
BOESKEN, W. H., and E. NOLLER, Serum and urinary protein analysis by SDS-PAA electrophoresis combined with immunoprecipitation. Dimer albumin in the nephrotic syndrome. In: XXIII. Colloquium on protein and biological fluids (H. PETERS, ed.). Pergamon Press, Oxford 1976, pp. 437-440. BYROM, F. B., and C. WILSON, A plethysmographic method for measuring systolic blood pressure in the intact rat. J. Physiol. 93, 301-304 (1938). DEODHAR, S. D., F. E. CUPPAGE and E. GABLEMAN, Studies on the mechanism of experimental proteinuria induced by renin. J. Exp. Med. 120, 677-697 (1964). DOMAN, J., J. Fox and J. HARD WICKE, Conditions for albumin dimerisation in nephrotic urines. Dependence on an ultrafilter able factor. Clin. Chim. Acta 108, 201-210 (1980). GARROD, 0., S. A. DAVIES and G. CAHILL, The action of cortisone and desoxycorticosterone acetate on glomerular filtrate rate and sodium and water exchange in the adrenalectomized dog. J. Clin. Invest. 34, 761-776 (1955). GAUDINO, M., and M. F. LEWITT, Inulin space as a measure of extracellular fluid. Amer. J. Physiol. Hi7, 387-393 (1949). HARRISON, H. E., and D. C. DARROW, Renal function in experimental adrenal insufficiency. Amer. J. Physiol. 125, 631-643 (1939). HARTREE, E. F., Determination of protein. A modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48, 122-127 (1972). KYOGOKU, 1\£., H. HAEBARA, A. OOSIMA, Y. YAMORY, S. IKEHARA, T. OHTA, T. OKUDA and K. OKAMOTO, Further studies on the cardiovascular system in the spontaneously hypertensive rat. In: Spontaneous hypertension. Its pathogenesis and complications (K. OKAMOTO, ed.). Igaku Shoin Ltd, Tokyo, Springer, Berlin, Heidelberg, New York 1972, pp. 155-159. LEND RUM, A. C., D. S. FRASER, W. SLIDDERS and R. HENDERSON, Studies on the character and staining of fibrin. J. Clin. Path. Hi9, 401-413 (1962). LOCKETT, M. F., The influence of suprarenal deficiency and sympathectomy on creatinine clearance in conscious dogs. J. Physiol. 109, 250-257 (1949). LUFT, R., and B. S. SJOGREN, The effect of desoxycorticosterone acetate and sodium chloride on blood pressure and renal function. Acta Endocrinol. (Copenhagen) 3, 56-70 (1949). MASSON, G. M. C., A. C. CORCORAN and I. H. PAGE, Renal and vascular lesions elicited by renin in rats with desoxycorticosterone hypertension. Arch. Pathol. 53, 217-225 (1952). - F. DELGRECO, A. C. CORCORAN and I. H. PAGE, Factors influencing renin diuresis and proteinuria. Proc. Soc. expo BioI. Med. 83, 631-636 (1953). MOVAT, H. Z., Silver impregnation methods for electron microscopy. Amer. J. Clin. Path. 35, 528-537 (1961). PESCE, A. J., I. BOREISHA and V. E. POLLAK, Rapid differentiation of glomerular and tubular proteinuria by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Clin. Chim. Acta 40, 27-34 (1972). PESSINA, A. C., and W. S. PEART, Renin induced proteinuria and the effects of adrenalectomy. I. Haemodynamic changes in relation to function. Proc. Roy. Soc. London B 180, 43-60 (1972). - B. HULME and W. S. PEART, Renin induced proteinuria and the effect of adrenalectomy. II. Morphology in relation to function. Proc. Roy. Soc. London B 180, 61-71 (1972). SRAER, J., L. BAUD, J. P. COSYNS, P. VERRDUST, M. P. NIVEZ and R. ARDAILLOU, High affinity binding of 125J angiotensin II to rat glomerular basement membranes. J. CIin. Invest. 59, 69-81 (1977). TALBOTT, J. H., L. J. PECORA, R. S. MELVILLE and W. V. CONSOLAZIO, Renal function in patients with Addison's disease and in patients with adrenal insufficiency secondary to pituitary panhypofunction. J. CIin. Invest. 21, 107-119 (1942). WARDI, A. H., and W. S. ALLEN, Alcian blue staining of glycoproteins. Anal. Biochem. 48,621-623 (1972). WEBER, K., and M. OSBORNE, The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. J. BioI. Chem. 244, 4406-4412 (1969). ZAHARJUS, R. M., and T. E. ZELL, Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30, 148-151 (1969). (Received January 14, 1985)
242
Exp. Pathol. 30 (1986) 4
The effect of adrenalectomy on the proteinuria of spontaneously hypertensive rats and normotensive controls By M. SZOKOL, M. B. SOLTES Z, A. NAGY, Z. LENGYEL and Sz. GOMBA With 7 figures Address for correspondence: Dr. M. B. SOLTESZ, Department of Pathology, University Medical School, P.O.B. 23, H - 4012 Debrecen, Hungary Ke y w or ds: proteinuria; spontaneously hypertensive rats; adrenalectomy ; glucocorticoid treatment; mineralocorticoid treatment; DO CA; dexamethasone
Summary Urinary proteins were studied by quantitative and electrophoretic methods in 6-monthold spontaneously hypertensiv e rats and normotensive controls. Protein analysis was carried out before and after adrenalectomy and during gluco- or mineralocorticoid treatment. Urinary protein excretion was significantly diminished after adrenalectomy both in the hypertensive and control gr oups. The original level of protein excretion was restored only by glycocorticoid treatment . Normal or pathologic electrophoretic pattern of urinary proteins was not influenced by the experimental procedure. Moderately non-selective glomerular proteinuria persisted in the spo ntaneously hypertensive r at s r eferring t o a definitely damaged glomerular barrier. One protein fraction of about 130,000 dalton molecular weight disappeared from the urine of hypertensive animals after adrenalectomy and reappeared after glu cocorticoid treatment only. This fraction probably represents the dimeric form of albumin. Quantitative changes of urinary protein excretion can be explained by haemodynamic factors.
I ntroduction
It is well-known that proteinuria evoked by the injection of different pressure substances (renin, angiot ensin II, norepinephrine etc.) will be lowered to a normal level after bilat eral adrenalectomy (ADDIS et al. 1950; MASSON et al. 1952 ; PESSWA et al. 1972). Contradi ctory data are existing in the literature how the different adrenocortical hormone prepamtes elevate urinary protein excretion in adrenalectomized rats (DEODHAR et al. 1964 ; PESSINA and PEART 1972). Mechanisms by which pressure subst ances exert proteinuria are also not clear. Structural and hemodynamic alterations in the glomeruli are assumed to be responsible for the effect of adrenalectomy and pressure substances (BAUMAN 1979, 1980). Experim ental models used for the study of proteinuri a evoked by pressure substances and modified by adrenalectomy were differ ent concerning the initial state of the glomerular barrier: Permeability of the glomeruli was originally normal in most of the investigations (DEODHAR et al. 1964; PESSINA and PEART 1972; PESSINA et al. 1972; BAUMAN 1979). But new inform ation was obtained if aminonucleoside nephro sis and pathologic proteinuria preceded the injection of pressure substances (BAUMAN 1980). Th e effect of adrenalectomy was not investigated in this model. In our experiment 6-month-old spontaneously hypertensive rats were chosen to study the role of adrenal glands and adrenocortical hormones in the regulation of quantity and composition of urinary proteins. The glomerular barrier was definitely damaged in these animals accompanied by a constantly pathologic pr oteinuria.
233
....
~
00
....<:>
<:0 ,-..
~
::r ~
"'d ~
'?
~
t:
~ ~
SHR WKR SHR WKR
Food intake (gfday)
0.9
0.8
± 0.4
± 0.6
22.2 + 19.9 20.6 21.1 ±
14
SHR WKR
40.8 36.4
± 3.5 ± 5.0 47.1 ± 3.1 36.6 ± 6.9
After adrenalectomy
0.7***
DOCA
± 4.4
+ 2.8
46.9 41.3
Dexamethasone
± 7.2
68.6 38.8
+ 6.2***
±
±
11.3 + 1.0*** 0.9*** 10.9 14.6 + 0.6*** 1.0*** 12.1
± 0.6*** 0.7*** ± 0.8***
-3.6 + -4.6 -2.8 + - 3.4
±
154 + 9*** 79 f 3*** 153 + 10* 2*** 86
After adrenalectomy
After adrenalectomy with hormonal treatment
*** p < 0.001 as compared to the values after adrenalectomy.
11
15 11
SHR WKR
n
Table 1a. Saline intake (gfday)
p < 0.05 } ++ P < 0.01 as compared to the values after adrenalectomy +++ p < 0.001
+
0.2
± 0.5 0.2 ± 0.3
0.5 + 0.7 0.5 + 1.2
±
±
4 181 102 ± 2 177 + 5 101 3
Before adrenalectomy
as compared to the preoperative values
15 11 14 11
SHR WKR SHR WKR
Body weight gain (gfday)
* p < 0.05 *** P < 0.001
15 11 14 11
SHR WKR SHR WKR
Blood pressure (mmHg)
15 11 14 11
n
±
1.0+++
± 0.6+++ 0.6+++ ± 0.9+
± 0.7+++
+ 1.0++ ± 1.0 + 0.4+++
18.6 + 15.9 19.6 + 15.9
-0.8 -0.7 2.4 3.8
±
153 + 7 104 f 2 176 + 9+ 100 2+
After adrenalectomy with hormonal treatment
DOCA
Dexamethasone
DOCA
Dexamethasone
DOCA
Dexamethasone
Table 1. Blood pressure, body weight gain and food intake of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKR) before and after adrenalectomy and during hormonal treatment. Values are means SEM
g;
~
Il'>-
C$l
..s
I-' <0
---
~
~
t:r'
~
"'d
':'
~
t
11
15 11 14
15 11 14 11
15 11 14 11
4.23 + 0.44 1.95 ± 0.38 ±
+ 3.82
20.85 0.85 12.87 0.99
0.88 + 0.11 0.35 ± 0.08 ±
5.70 + 0.63 0.40 0.06 3.17 + 0.75 0.09 0.48
± 0.19 + 3.87 ± 0.21
± ± 0.82** 0.09*
1.17***
7.23+++
± 0.21 ++ 0.22 ± 0.06
35.42 + 1.28 0.92 + 0.23
±
0.12*'1< 0.02**
± 0.07*
±
0.44 + 0.11 0.23 0.08
7.50 + 1.02+ ++ 0.08+++ 0.50
±
4.5 + 0.6 3.0 1.7++ 3.0 + 0.5+++ 1.7± 0.3+++
After adrenalectomy with hormonal treatment
0.24***
± 0.02**
±
± ±
5.8 ± 0.6** 5.0 ± 0.7*** 5.4 + 0.5* 5.1 0.7***
After adrenalectomy
3.4 + 0.3 2.1 0.4 3.7 + 0.4 0.2 1.9
Before adrenalectomy
< 0.05 } as compared to the preoperative values P < 0.01 p < 0.001 P < 0.01 } as compared to the values after adrenalectomy p < 0.001
p
SHR WKR SHR WKR
Protein excretion (mg/6 h)
*
SHR WKR SHR WKR
Protein concentration (mg/ml)
** *** ++ +++
SHR WKR SHR WKR
Urine volume (ml)
n
DOCA
Dexamethasone
DOCA
Dexamethasone
DOCA
Dexamethasone
Table 2. Urine volume. protein concentration and 6 h protein excretion of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKR) before and after adrenalectomy and during hormonal treatment. Values are means ± SEM
Materials and Alethods Forty male 24-28 weeks old spontaneously hypertensive rats (Okamoto strain) of 280-330 g body weight, and 30 normotensive controls (Wistar-Kyoto strain) of the same age and sex were placed in individual cages. The animals were offered a commercial food (LA TI) and demineralized water ad libitum. Body weight, water and food intake were measured daily. Systolic blood pressure was registered by tail plethysmography in a light aether-narcosis every other day (BYROM and WILSON 1938). Three different experimental periods were performed each lasting 5 days. During the first period intact rats were observed. Bilateral adrenalectomy was carried out on the 5th day. One per cent saline as drinking fluid was offered to all the animals after the operation till the end of experiment. 11 spontaneously hypertensive and 8 normotensive rats died during the first 2-3 postoperative days without any remarkable autopsy findings probably because of disturbances in water and electrolyte metabolism. Hormonal treatment was performed during the third experimental period in the following way: Dexamethasone (Oradexon-Organon) was given subcutaneously every day in a dose of 0.5 mg to 15 spontaneously hypertensive and 11 normotensive rats. Desoxicorticosterone acetate (Decosteron-Kobanyai Gy6gyszergyar) was injected intramuscularly into 14 spontaneously hypertensive and 11 normotensive animals. The dose (0.2 mg/day) was twice as high as the recommended one for substitution therapy (BARNES and ELTHERINGTON 1973). Two urine samples were collected in each experimental period from each animal between 8 a.m. and 2. p.m. Blood samples were obtained from the cut tail at the end of each period. Rats were sacrificed by bleeding. Heart weights were registered and kidneys fixed in 10 per cent buffered formaldehyde solution for light microscopy. Protein concentration of the urine samples was measured by means of Folin reagent (HARTREE 1972) using bovine plasma albumin as the standard. Urinary and plasma proteins were separated by sodium dodecyl sulphate polyacrylamide gel (SDS-PAAG) disc electrophoresis (WEBER and OSBORNE 1969). The protein samples were treated only with SDS (BOESKEN et al. 1973) but in some cases mercaptoethanol was also added to the system. Gels removed from the tubes were stained by Coomassie briIIant blue R 250. Periodic acid Schiff reaction (ZAHARIUS and ZELL 1969) and alcian blue staining (WARDI et aI. 1972) were also performed. Molecular weight of the different protein bands was determined from a plot of the electrophoretic mobility against the logarithm of the known molecular weights of standard proteins (Serva). Results were recorded by scanning the gels with a Kipp-Zonen Micrograph 805 (red filter). Paraffin sections of the kidneys were stained with hemalum eosin, periodic acid Schiff reaction, MOVAT (1961) silver impregnation for the basement membranes, and LENDRUM'S (1962) method. Statistical analysis was carried out by Student's t-test (paired data). Values in the text, tables and figures are means ± S.E.M.
Results The blood pressure sank after adrenalectomy both in the hypertensive and normotensive rats. A return to the original level was evoked by gluco- or mineralocorticoid substitution in the normotensive group. A similar effect was registered in the spontaneously hypertensive rats after mineralocorticoid (DOCA) treatment only (table 1). Hormonal treatment induced some increase in food intake but it remained below the preoperative level (table 1). Body weight and food intake decreased after adrenalectomy (table 1). Saline was taken up in a large quantity by the normotensive and hypertensive animals during the postoperative days (table 1a). Desoxicorticosterone acetate brought about a rapid body weight gain by its sodium and water retaining effects. On the contrary in all the animals treated by dexamethasone a progressive loss of body weight was registered (table 1). The 6 hours urine volume increased, urinary protein concentration and the quantity of the excreted protein decreased after adrenalectomy. The urine volume decreased during the hormonal substitution in all the experimental groups (table 2). Protein concentration and 6 hours' excretion was elevated above the preoperative levels by dexamethasone treatment. Desoxicorticosterone acetate failed to produce this effect (table 2). The electrophoretic pattern of the urinary proteins proved to be pathologic in the spontaneously hypertensive rats already in the preoperative period: the bands qf albumin and transferrin widened and protein fractions above 100,000 dalton molecular weight appeared in the urine. This picture corresponded to the moderately non-selective type of glomerular proteinuria (BoEsKEN et al. 1975) (fig. 1). Urine samples of the normotensive animals con-
236
Exp. Pathol. 30 (1986) 4
180 &; ,
(\2
0
r
0,"
0.6
0,8
lRM
N
,
Q,2
0,"
A
MW
002- 180
t-~)
B
Ie
lRM
H
TA 1
2
Fig. 1. Electrophoretic pictures with densitometric curves of urinary proteins of a normotensive control (N) and a spontaneously hypertensive rat (H) before adrenalectomy. In contrast to the normotensive animal the bands of albumin (A) and transferrin (T) widened and proteins above 100,000 dalton molecular weight appeared in the urine. OD: optical density, MW: molecular weight, RM: relative mobility. Fig. 2. Microprotein fractions in the urine of a normotensive Wistar-Kyoto rat before (A) and after (B) the operation and during dexamethasone treatment (C). No alterations can be seen. Abbreviations see fig. 1.
tained only microproteins and albumin in a small quantity (fig. 1). The general electro phoretic pattern did not change either in the normotensive or hypertensive groups after adrenalectomy and during the hormonal treatment (fig. 2, 3, 4). Moderately non-selective proteinuria persisted in the hypertensive animals. But one fraction with 130,000 dalton molecular weight disappeared after adrenalectomy. Dexamethasone treatment induced its reappearance (fig. 3). Desoxicorticosterone acetate failed to produce the same effect (fig. 4). Electrophoretic analysis of plasma proteins detected a fraction of 130,000 dalton molecular weight before and after adrenalectomy and during the hormonal treatment (fig. 5). When the electrophoretic separation was carried out from freshly collected urine samples (omitting the few days storage at -20°C) the protein fraction of 130,000 dalton molecular weight was not detected. Mercaptoethanol added to the system containing frozen urine samples led to a Exp. Pathol. 30 (1986) 4
237
A
c
o
0.2
0,4
0,11
011
B .j
Fig. 3. Urinary proteins of a hypertensive rat before (A) and after (B) adrenalectomy and during dexamethasone treatment (0). Protein fraction with 130,000 dalton molecular weight (arrow) disappeared after the operation and reappeared under hormonal influence. Abbreviations see fig. 1. Fig. 4. Protein fractions in the urine of a hypertensive rat before (A) and after (B) adrenalectomy and during desoxicorticosterone acetate treatment (0). Protein of 130,000 dalton molecular weight (arrow) which disappeared in the adrenalectomized animal did not return while given mineralocorticoid. Abbreviations see fig. 1.
similar result. This protein fraction proved to be negative with alcian blue staining and periodic acid Schiff reaction. In the histologic picture alterations of glomeruli referred to a serious damage of their barrier: thickening of the basement membrane (fig. 6), hyalino sis and fibrinoid necrosis of the glomerular loops were equally noticed (fig. 7).
Discussion Considering the influence of adrenal glands and adrenocortical hormones on the quantity and composition of urinary proteins in normal and pathological states, some problems are not definitely settled. Contradictory results were registered about the effect of different hormone preparations on urinary protein excretion after bilateral adrenalectomy. Protein-
238
Exp. Pathol. 30 (1986) 4
o
1\2
0,4
o
o,~
1RM
u Fig. 5. Electrophoretic analysis of serum (S) and urinary proteins (U) from a hypertensive rat after adrenalectomy. A fraction of 130,000 dalton molecular weight (arrow) could be found among the serum proteins but it did not appear in the urine. Abbreviations see fig. 1.
uria induced by angiotensin II was quantitatively restored by cortisone or DOCA (DEODHAR et al. 1964). Some authors found that only corticosterone was effective (PESSINA and PEAR 1972). Using hormonal substances with a strong glucocorticoid (dexamethasone) and mineralocorticoid (aldosterone) effect, BAUMAN (1979) observed an elevated urinary protein excretion only after dexamethasone treatment. Our results are in accordance with the latter observation. The effect of adrenalectomy, substitution treatment and their interaction with proteinuria induced by pressure substances are still not clear. They probably influence urinary protein excretion through the modification of renal circulation. Renal plasma flow (RPF) and glomerular filtration rate (GFR) are diminished after adrenalectomy (HARRISON' and DARROW 1939; TALBOTT et al. 1942; LOCKETT 1949). Glucocorticoids, first of all the widely studied cortisone increase the quantity of urinary proteins by the elevation of GFR and RPF. Mineralocorticoids (e.g. DOCA, aldosterone) have no direct haemodynamic effect. They tend to normalize salt and water balance resulting in a moderate elevation of GFR and RPF (LUFT and SJOGREN 1949; GAUDINO and LEWITT 1949; GARROD et al. 1955). Pressure hormones increase the filtration fraction, which seems to be essential for their proteinuric effect (PESSlNA and PEART 1972; BAUMAN 1981). Another explanation was also Exp. Pathol. 30 (1986) 4
239
Fig. 6. Glomerulus of a spontaneously hypertensive rat. The basement membrane is almost diffusely thickened. The mesangial matrix also reveals black staining. Movat's silver impregnation, X 128.
Fig. 7. Glomerulus of a spontaneously hypertensive rat with extensive hyalinosis. Fibrinoid necrosis colouring in orange red can be seen in some glomerular loops (arrows). Lendrum's staining, X 128. Green filter.
240
Exp. Pathol. 30 (1986) 4
suggested by BAUMAN (1979) for the angiotensin-induced proteinuria. He assumed that permeability of the glomerular barrier will be enhanced if electrostatically negative groups of its polyanions are neutralized by angiotensin II, a compound with cationic feature (SRAER et al. 1977). Later studies of BAUMAN (1981) proved unequivocally that quantitative changes of proteinuria evoked by angiotensin infusion in aminonucleoside nephrosis are strongly parallel with the alterations of the filtration fraction. Proteinuria of spontaneously hypertensive rats has not been analysed in connection with the adrenal glands and adrenocortical hormones. In our experimental model the glomerular barrier was already definitely damaged in the 4-month-old hypertensive rats in contrast to the transiently enhanced glomerular permeability induced by pressure hormones (e.g. angiotensin II, norepinephrine). Therefore diminished protein excretion after adrenalectomy was not a consequence of an "improvement" in the glomerular permeability: electrophoretic analysis demonstrated the persistence of moderately non-selective glomerular proteinuria. Quantitative alterations of the urinary protein after adrenalectomy and hormonal substitution are probably in connection with haemodynamic factors. The applied dose of DOCA was effective as proved by body weight gain, increased food intake and decreased urine volume. The quantity of the excreted proteins was not influenced by DOCA treatment. This hormone has no direct effect on the glomerular circulation (GARROD et al. 1955). The exact nature of the protein fraction that had disappeared from the urine after adrenalectomy, was not determined. Spontaneously hypertensive rats have a special protein fraction in the serum (serum post albumin or alpha1-globulin) with a 130,000 dalton molecular weight. The absence of positive PAS-reaction and alcianophilia opposes the identity of urinary protein with serum post albumin (KYOGOKU et al. 1972). The 130,000 dalton molecular weight suggests that this fraction may be the dimeric form of albumin. Further different kinds of indirect evidence also support this idea:
1. This fraction disappeared from the urine sample after mercaptoethanol treatment. 2. Dimerisation proceeds during the storage of samples below 0 DC temperature (DOMAN et al. 1980; BOESKEN 1976) but not in freshly collected urine. 3. Glucocorticoids (hydrocortisone) support dimerisation of albumin, e.g. in the urine of human nephrotic cases (DOMAN et al. 1980). Apart from these kinds of evidence further studies are needed to determine the exact nature of the discussed protein fraction by immunochemical and biochemical methods.
Acknowledgements The authors are indebted to J. VIRAGH and I. VERSENYI for their technical help and Dr. P. HAJDU (Inst. Social Medicine, Univ. Med. Sch. Debrecen) for help in the statistical evaluation.
Literature ADDIS, T. J., J. MAMMORSTON, H. C. GOODMAN, A. L. SELLERS and M. SMITH, Effect of adrenalectomy on spontaneous and induced proteinuria in the rat. Proc. Soc. expo BioI. Med. 74,43-46 (1950). BARNES, C. D., and L. G. ELTHERINGTON, Drug dosage in laboratory animals. Univ. California Press, Berkeley, Los Angeles, London 1973, p. 278. BAUMAN, J. W., Corticoid effects of angiotensin- and norepinephrine-induced proteinuria in rats. Amer. J. PhysioI. 237, F133-F137 (1979). - On the mechanism of angiotensin-induced proteinuria. 1. Studies in aminonucleoside nephrotic rats and in saralasin blockade. Nephron 27, 47-50 (1981). BOESKEN, W. H., K. KOPF and P. SCHOLLMEYER, Differentiation of proteinuria diseases by discelectrophoretic molecular weight analysis of urinary proteins. Clin. Nephrol. 1, 311-318 (1973). - Discelectrophoretic molecular weight analysis of urinary proteins. Contr. NephroI. 1, 143-155 (1975).
]6
Exp. Pathol. 30 (1986) 4
241
BOESKEN, W. H., and E. NOLLER, Serum and urinary protein analysis by SDS-PAA electrophoresis combined with immunoprecipitation. Dimer albumin in the nephrotic syndrome. In: XXIII. Colloquium on protein and biological fluids (H. PETERS, ed.). Pergamon Press, Oxford 1976, pp. 437-440. BYROM, F. B., and C. WILSON, A plethysmographic method for measuring systolic blood pressure in the intact rat. J. Physiol. 93, 301-304 (1938). DEODHAR, S. D., F. E. CUPPAGE and E. GABLEMAN, Studies on the mechanism of experimental proteinuria induced by renin. J. Exp. Med. 120, 677-697 (1964). DOMAN, J., J. Fox and J. HARD WICKE, Conditions for albumin dimerisation in nephrotic urines. Dependence on an ultrafilter able factor. Clin. Chim. Acta 108, 201-210 (1980). GARROD, 0., S. A. DAVIES and G. CAHILL, The action of cortisone and desoxycorticosterone acetate on glomerular filtrate rate and sodium and water exchange in the adrenalectomized dog. J. Clin. Invest. 34, 761-776 (1955). GAUDINO, M., and M. F. LEWITT, Inulin space as a measure of extracellular fluid. Amer. J. Physiol. Hi7, 387-393 (1949). HARRISON, H. E., and D. C. DARROW, Renal function in experimental adrenal insufficiency. Amer. J. Physiol. 125, 631-643 (1939). HARTREE, E. F., Determination of protein. A modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48, 122-127 (1972). KYOGOKU, 1\£., H. HAEBARA, A. OOSIMA, Y. YAMORY, S. IKEHARA, T. OHTA, T. OKUDA and K. OKAMOTO, Further studies on the cardiovascular system in the spontaneously hypertensive rat. In: Spontaneous hypertension. Its pathogenesis and complications (K. OKAMOTO, ed.). Igaku Shoin Ltd, Tokyo, Springer, Berlin, Heidelberg, New York 1972, pp. 155-159. LEND RUM, A. C., D. S. FRASER, W. SLIDDERS and R. HENDERSON, Studies on the character and staining of fibrin. J. Clin. Path. Hi9, 401-413 (1962). LOCKETT, M. F., The influence of suprarenal deficiency and sympathectomy on creatinine clearance in conscious dogs. J. Physiol. 109, 250-257 (1949). LUFT, R., and B. S. SJOGREN, The effect of desoxycorticosterone acetate and sodium chloride on blood pressure and renal function. Acta Endocrinol. (Copenhagen) 3, 56-70 (1949). MASSON, G. M. C., A. C. CORCORAN and I. H. PAGE, Renal and vascular lesions elicited by renin in rats with desoxycorticosterone hypertension. Arch. Pathol. 53, 217-225 (1952). - F. DELGRECO, A. C. CORCORAN and I. H. PAGE, Factors influencing renin diuresis and proteinuria. Proc. Soc. expo BioI. Med. 83, 631-636 (1953). MOVAT, H. Z., Silver impregnation methods for electron microscopy. Amer. J. Clin. Path. 35, 528-537 (1961). PESCE, A. J., I. BOREISHA and V. E. POLLAK, Rapid differentiation of glomerular and tubular proteinuria by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Clin. Chim. Acta 40, 27-34 (1972). PESSINA, A. C., and W. S. PEART, Renin induced proteinuria and the effects of adrenalectomy. I. Haemodynamic changes in relation to function. Proc. Roy. Soc. London B 180, 43-60 (1972). - B. HULME and W. S. PEART, Renin induced proteinuria and the effect of adrenalectomy. II. Morphology in relation to function. Proc. Roy. Soc. London B 180, 61-71 (1972). SRAER, J., L. BAUD, J. P. COSYNS, P. VERRDUST, M. P. NIVEZ and R. ARDAILLOU, High affinity binding of 125J angiotensin II to rat glomerular basement membranes. J. CIin. Invest. 59, 69-81 (1977). TALBOTT, J. H., L. J. PECORA, R. S. MELVILLE and W. V. CONSOLAZIO, Renal function in patients with Addison's disease and in patients with adrenal insufficiency secondary to pituitary panhypofunction. J. CIin. Invest. 21, 107-119 (1942). WARDI, A. H., and W. S. ALLEN, Alcian blue staining of glycoproteins. Anal. Biochem. 48,621-623 (1972). WEBER, K., and M. OSBORNE, The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. J. BioI. Chem. 244, 4406-4412 (1969). ZAHARJUS, R. M., and T. E. ZELL, Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30, 148-151 (1969). (Received January 14, 1985)
242
Exp. Pathol. 30 (1986) 4