Renal Handling of /&-Microglobulin in Experimental Renal Disease
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A. FREDRIKSSON Dept. of Surgery, University Hospital, Uppsala, Sweden
Fredriksson, A. Renal Handling of bz-Microglobulin in Experimental Renal Disease. Scand. J. din. Lab. Invest. 35.591600, 1975. Renal extraction and urinary excretion of *251-labelIedDo-microglobulin was studied in rats. The effect of ischaemic renal injury, experimental pyelonephritis, and unilateral nephrectomy was investigated. The tubular secretion of o-iodohippurate (OIN)was measured for comparison. The urinary excretion was calculated as the ratio between the clearance of protein and the glomerular filtration rate. The glomerular filtration rate was estimated as clearance of polyethylene glycol (PEG 1000). The renal arteriovenous concentration difference was lower for P?-microglobulin than for PEG 1000 in all the experimental groups. In unilateral renal disease the ,$*-microglobulin excretion of the intact kidneys was similar to that of the diseased kidneys. A significant difference was noted only after ischaemic renal injury. The same was found for OIH. After removal of the intact kidneys the excretion of /j+microglobulin increased about 10-fold in pyelonephritic animals and 2- to 30-fold in animals with ischaemic renal injury. One hour after unilateral nephrectomy in normal animals the ratio increased about 50 per cent. The tubular secretion of OIH did not change noticeably. It is concluded that the glomerular filtration is a main step in the intrarenal catabolism of bzmicroglobulin and that its urinary excretion is considerably influenced by a reduction in the functioning kidney mass. Key-words: pz-microglobulin; proteinuria; renal disease, experimental; renal function A. Fredriksson, M.D.,Dept. of Surgery, Universiry H o s P i l d , 750 14 UPPsofa 14, Sweden
Proteinuria has long been used as a qualitative icdication of renal disease. During recent years it has been well established that theurinary excretion of plasma proteics is different in diseases primarily affecting the glomeruli or the tubuli (for review, see Refs. 3 and 35). There is currently interest in using quantitative estimations of various proteins for evaluation of proximal renal tubular dysfunction, since such an approach may be more discriminatory than electrophoretic analyses or qualitative estimations of the total urinary protein (22). $,-microglobulin, a low molecular weight protein present in biological fluids (2). may be the protein whose excretion is most sensitive to tubular dysfunction (16, 21, 29). The serum concentration of P,-microglobulin appears
to be a function mainly of the glomerular filtration rate (6, 11,16,41). Under physiological conditions only small amounts of B,-microglobulin are excreted into the urine, but in tubular disease greatly elevated levels of urinary $,-microglobulin are observed (29). The purpose of this investigation wa; to study in rats the renal handling of P,-microglobulin in experimentally induced renal disease. Special attention was focused on the effect of reducing the number of functioning nephrons, since clinical observations have indicated that this might influence the urinary excretion of plasma proteins (7, 22, 42). The tubular secretion of o-iodohippurate was measured for comparison.
592
A . Fredriksson
MATERIAL AND METHODS
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Animals
Male albino rats (n = 27) of the Sprague-Dawley strain (Anticimex, Stockholm, Sweden) were used throughout. Their average weight was 250 g at the induction of kidney damage. The individual weight at the time of the clearance investigation varied between 370 and 500 g. The rats were given a standard diet (Ewos, Sodertalje, Sweden) and had free access to food and drinking water until the start of the experiments. Experimental groups Pyetonephritis was induced in the left kidney in two ways. The kidney was exposed by an abdominal incision during ether anaesthesia. In one group (Py I , n = 4)a method modified from that described by Shankel et al. (36) was used. The kidney was punctured 100 times with a needle that was repeatedly dipped in a culture of enterococci (lolo bacteria per ml). In a second group (Py 2, n = 3) 0.15 ml of the enterococcal culture was injected into the renal pelvis at the beginning of 30 niin of ureteral occlusion. In groups Py 1 and Py 2 the right kidney was left intact. In a third group (Py S,n = 4)the left kidney was treated in the same way as in Py 1, but simultaneously the right kidney was removed. The functional studies were performed 2 to 4 months after induction of pyelonephritis. At the start of the clearance experiments urine samples were obtained by needle aspiration from the exposed bladder. The urine samples were kept at +4 "C until cultures were set up on standard laboratory media after serial dilutions of the samples in phosphate-buffered saline. Quantitative cultures were also made of sections from the kidney, excised at the time of killing with a disinfected but nonsterile knife. The sections were immediately placed in sterile tubes at $4 "C.The number of bacteria per gram of kidney tissue was calculated from cultures on standard laboratory media. After completion of the clearance studies the animals were killed by intra-arterial air injection. The kidneys were excised, freed from the adiposz capsule, immediately weighed, sectioned lon-
gitudinally, and fixed in 10 per cent formaldehyde solution. Deparaffinized, hdematoxylin and eosinstained sections were studied by light microscopy. Ischaeniic renal injury was induced by occlusion of the left renal artery with a 0000 silk thread for 60 min during anaesthesia with intraperitoneally injected pentobarbital sodium (NembutalB, Abbott), 45 mg/kg body weight. The body temperature was held constant at 37 0.5 "Cwith a thermostat-controlled heating pad. In one group ( I 60, n = 4) the right kidney was left intact, but in another group (IS, n = 5) it was removed after 8 weeks. The functional studies were carried out 8 weeks after the induction of kidney damage in group I 60 and 4 weeks after removal of the right kidney in group IS. Morphological studie- were accomplished as described for the pyelonephritic kidneys. Normal single kidneys ( N S , n = 7) were studied after unilateral nephrectomy in normal rats. Clearance experiments for two periods were performed before nephrectomy, and the following periods were started 1 hr after the nephrectomy. Morphological studies showed no abnormalities.
+
Substances used for clearance studies
Glomerular filtration rate (GFR) was estimated as the clearance for polyethylene glycol (PEG lOOO), molecular weight 800-1000 (Kebo, Stockholm, Sweden) (5). For the evaluation of proximal tubular secretory capacity, ortho-iodohippurate sodium (OIH) (Hippuranm, Mallinckrodt, New York, USA) was used with the addition of ~ r t h o - [ ~ ~ ~ I ] h i p p u r a t e sodium (AB Atomenergi, Studsvik, Sweden). P,-microglobulin was isolated from urine of patients with tubular disease. The purification procedure was essentially that of Berggard & Bearn (2). The protein, which was homogenous by several chemical, physicochemical, and immunological criteria, was labelled with lZ5I (Amersham, England) by the iodine monochloride method of Mc Farlane (24). No preoxidation was carried out. The preparations used contained about 0.5 to 0.7 mol of iodine per mol of protein. The labelled P,-microglobulin was indistinguishable from its native counterpart on examination by crossed immunoelectrophoresis (17). In the
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~2-Microglobulirtin Experimental Renal Disease
593
present investigation, only material that gave the same fractional catabolic rate when screened in one animal before measurement as when directly injected was used.
trichloroacetic acid and in urine by the same procedure but after the addition of 0.1 ml 10 per cent serum albumin to avoid losses. The radioactivity was counted in a gamma spectrometer (Autowell).
Clearance experiments
Calculations
The animals were anaesthetized with 5-sec-butyl5-ethyl-2-thiobarbituric acid (Inactin@, Promonta, Hamburg), 120 mg/kg body weight, injected intraperitoneally. Tracheostomy was performed, and the body temperature was held constant at 37 0.5 "C by a thermostat-controlled heating pad. Polypropylene catheters (PP 10 or 50) were inserted into a jugular vein for infusions and into a femoral artery for blood sampling and blood pressure recording. Urine was collected under oil through polyethylene catheters (PE 10) inserted into the distal ureteral ends. A constant rate of infusion of 200 mg PEG 1O00, 225 mg o-iodohippurate sodium, 5 pC 1311-labelledo-iodoMppurate sodium, and 4 pg 1z51-labelledPz-microglobulin in 2.5 ml saline was given per hour. Blood samples (0.3 ml) were withdrawn in the middle of the urine collection periods from a femoral artery and the exposed renal veins. Renal vein samples were taken with a 20-gauge needle introduced through the inferior vena cava. In the NS group renal vein samples were taken only from the left kidney. At least three clearance periods were performed in each animal. The duration of the clearance periods was varied, to ascertain that an appropriate amount of urine was obtained ( 20.2 ml). The amount of urine was determined by weighing.
Glomerular filtration rate was calculated by the formula GFR = 'ir *Cu/Cp(PEG), [I1 where 3 is the urine flow rate in ml/min, and Cu/Cp (PEG) is the ratio of the urine to plasma concentration of PEG 1OOO. The ratio between the clearance for p,-microglobulin and PEG lo00 was calculated as
Analytical mcthods
PEG 1000 was analysed tvrbidimetrically with a micromodification of the method used by HydCn (20). Protein and sulphate were precipitated with barium hydroxide and zinc sulphate. Exactly 5 min after the addition of trichloroacetic acid, turbidity was determined at 400 nm with a spectrophotometer. The coefficient of variation of the present method was 5.7 per cent (n = 15). For analysis of I 3 l I , 50 pl of plasma or urine was diluted in 1 ml of distilled water. Proteinbound lZ5l in plasma was recovered by the precipitation of the proteins with ice-cold 10 per cent 8
-
Scand. J. clin. Lab. Invert.
The amount of o-iodohippurate (OIH) excreted into the urine per ml of glomerula: filtrate was calculated as
0 . c (OIH) ~ i'.Cu/Cp (PEG)
(P2)
-
Cu (OIH) Cu/Cp (PEG) '
PI 1
(P2)
Cu and Cp are the urinary and plasma concentrations of P,-microglobulin, respectively, and Cu (OIH) is the urinary concentration of o-iodohippurate. The renal extraction of a substance (x) is calculated as E(x) = 1
-
renal vein concentration . arterial concentration
[41
If it is assumed that no reabsorption of p,-microglobulin occurs distally to the end-proximal tubular segment, the amount of (&-microglobulin excreted, Cu (p,) .V, equals Ctf . h f , where Ctf (9,) is the concentration of P,-microglobulin and Vtf the volume flow rate in the end-proximal tubular segment. Dividing Cu (p,) .'? = Ctf (p,) * vtf by GFR gives
(P,)
where Ctf (PEG) is the concentration of PEG in the end-proximal tubular fluid. Ctf/Cp (PEG) is a reflection of the fractional reabsorption of fluid in the proximal tubules.
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A. Fredriksson
RESULTS
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Eactrriological and morphological findings
Urine cultures showed more than lo5 bacteria per ml in about 60 per cent of the pyelonephritic animals. Renal tissue cultures shohed more than lo4 bacteria per g cf tissue both for right and left kidneys when urine cultures were positive. No other bacteria than enterococci were found. The left kidneys in the Py 1 group were contracted with a scarrcd surface.' Their weight was moderately but not significantly reduced compared with the right control kidneys (Table I), which grossly appeared to be normal. Light microscopy showed moderate chronic-subchronic interstitial nephritis in the left kidneys, but minimal changes were also found in some of the right kidneys. In Py 2 the left kidneys showed only a few superficial scars, and their weight was only slightly reduced. Light microscopic findings were essentially as in Py 1 rats. In Py S the kidneys were largely hypertrophied and irregularly shaped and had a greyish discoloration. Their weight had increased substantially compared with the kidneys in the other groups. Light microscopy showed somewhat more marked inflammatory changes
than in Py 1 rats and more tubular dilatation as well. The left kidneys subjected to ischaemia (group I 60) were contracted, and their weight was considerably reducsd compared with the intact right kidneys (Table I). In group IS the remaining kidney was hypertrophied, but with large individual variations. Light microscopy of the ischaemically injured kidneys showed necroses surrounded by mononuclear infiltration in both groups. The tubules were dilated throughout with flattened epithelial cells and the occurrence of some casts. The changes were most advanced in the inner medullary zone and corticomedullary junction. The glomeruli appeared normal, with the exception of those in the necrotic areas. The right kidneys in group I 60 only showed moderate hypertrophy. In all experimental groups except the IS group, the animals appeared to be in a good health and gained weight like the normal control rats. In the IS group the animals appeared anaemic and in a slightly poor condition, but all except one gained weight even after the unilateral nephrectomy. Systolic blood pressure during anaesthesia wqs within the normal range except for one animal in
Table I. Characteristics of the experimental groups* Body weight, Group (number of rats) Normal, NS (7) Before nephrectomy Right kidney Left kidney After nephrectomy Left kidney Ischaemic renal injury 60 min, I 60 (4) Right kidney Left kidney 60 min, IS (5) Pyelonephritis
PY 1 (4)
GFR/100 g body weight, ml/min
1.6910.08
0.2910.02 0.34*0.03
1.7210.06
0.2910.04
2.1330.08 1.0310.09 2.08+0.36
0.37h0.03 0.12+0.01 0.2110.07
1.9930.10 1 ,5310.09
0.21f0.02 0.1710.01
1.79&0.13 1.56t0.08 2.8810.12
0.1610.02 0.1310.02 0.32*0.03
433115
4011 9 416119 4901 9
Right kidney Left kidney
PY 2 (3)
477113
Right kidney Left kidney PY s (4)
* All values are mean
Kidney weight,
g
440116
1S.E.M.GFR=glomerular filtration rate.
Bz-Microglobulin in Experirnental Renal Diseuse
595
Table IT. Mean differences between left and right kidney in the excretion of P,-microglobulin and o-iodohippurate (OIH)?
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~~
~~~~~~~~
Clearance P,-microglobulin
Cu(0IH)
Group (number of rats)
GFR Mean (P=0.95)*
Mean (P=0.95)*
Normal, NS (7) Ischaemic renal injury 60 min, I 60 (4) Pyelonephritis PY 1 (4) PY 2 (3)
- 0.0014j0.0015
- 0.007&0.145
Cu/Cp(PEG)
0.0032*0.0022* *
- 0.197&0.195**
O.OOOljO.0038
0.02110.155 - 0.12010.327
- 0.0014*0.0064
t GFR=glomerular filtration rate ; Cu=urine concentration; Cp=plasma concentration;PEG-polyethylene glycol.
* Confidence limits. ** Significant difference (P=0.95). the IS group (190 mm Hg) and one in the Py 2 group (160 mm Hg). Some characteristics of the experimental groups are summarized in Table I. Tubular transfer of o-iodohippurate
Equation [3] related to the plasma concentration of o-iodohippurate, Cp (OIH), was used to assess the tubular secretory capacity (14). The data for the various experimental groups are summarized in Table 11. It can be seen in the Table that the mean differences between the left and the right kidney were small for the animals in the experimental groups. The only significant difference was for group I60 (confidence limits, P = 0.95). The animals in group IS were unilaterally nephrectomiyed, and the secretory capacity of OIH of the remaining kidney was therefore compared with values obtained in normal rats before and after unilateral nephrectomy. It can be seen in Fig. 1 that there was no easily detectable difference. It is therefore conceivable that the tubular secretory capacity, as measured by OIH excretion, did not respond noticeably to a reduction in the functioning kidney mass. Renal extraction o f P,-rnicroglobulin
Since glomerular filtration of P,-microglobulin appears to be of great importance in the elimination of the protein from plasma, it appeared to be of interest to meascre the renal extraction of the protein in the groups of animals with various types of renal disease.
It can be seen in Table 111 that the ratio of the renal arteriovenous differences for p,-microglobulin and PEG lo00 was about 0.70 for all groups except Py S. In the latter group the average value was only 0.26. It is of interest that all mean values were lower than unity, which indicates that PEG 1000 may pass the glomerular barrier more easily than P,-microglobulin.
.
'
0 . 0
a5-
0'
0.5 1.0 1.5 Cp(0l H) rnglml Fig. 1. Urinary excretion of o-iodohippurate (OIH)
per ml of glomerular filtrate as a function of plasma concentration. The points represent values for each clearance period. (0)= group NS before nephrectomy; (0) = group NS after nephrectomy; and (v) = group IS.
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A. Fredriksson
Table 111. Clearance and renal extraction of P,-microglobulin. Mean values for all clearance periods? Clearance &../GFR
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Group (number of rats) Normal, NS (7) Before nephrectomy After nephrectomy Ischaemic renal injury 60 min, I 60 (4) Right kidney Left kidney 60 min, I S t t (5)
n
Mean (P=0.95)*
S.D.
n
Mean (P=0.95)*
S.D.
30 16
0.01l+0.001
0.003 0.005
15 10
0.65k0.21 0.57&0.09
0.38 0.12
15 14
0.010*0.001 0.013f0.003 0.034f0.015
0.002
13 12
0.67h0.09 0.73h0.17
0.15 0.29
4
0.017IfI0.003
0.004
*+
0.305f0.090 0.348k0.079 0.020h0.004
0.010 0.011 0.052 0.046 0.003
13 14
0.007~0.001 0.008&0.001
0.002 0.002
12 12
0.77h0.14 0.73f0.15
0.22 0.24
10 10 11
0.01Of 0.001 0.008f0.001 0.079f0.016
0.001 0.002 0.024
7 6 10
0.6950.37 0.79h0.30 0.26&0.15
0.33 0.29 0.16
5 3 3
5
Pyelonephritis PY 1 (4) Right kidney Left kidney PY 2 (3) Right kidney Left kidney PY s (4)
E (P,P))/E(PEG)
0.040*0.015
t n=number of clearance periods. tt Values for individual animals are given, because of large interindividual variation. * Confidence limits. ** No extraction values were recorded for group IS because of technical failure. Urinary excretion of P,-microglobulin
The urinary excretion of P,-microglobulin is expressed as the ratio between the clearance of P,-microglobulin and GFR (eqn. [2]). The mean differences between values for right and left kidney were generally small (Table If). They were significant only for group I 60 (confidence limits, P = 0.95). The mean values for each experimental group are presented in Table I11 and the individual values for each clearance period are summarized in Fig. 2. Groups I 60, Py 1 , and Py 2 did not differ from group NS before nephrectomy. It should be noted, however, that after unilateral nephrectomy in group NS, the ratio of the P,-microglobulin clearance to GFR was increased by about 50 per cent. The animals in group IS showed a 2- to 30-fold increase in the ratio compared with the values obtained for the left kidneys of animals in group I 60.The urinary excretion of P,-microglobulin in
most animals of group IS is also higher than for group NS, irrespective of whether the comparison is made before or after unilateral nephrectomy in the latter group. The urinary excretion of P,-microglobulin is considerably elevated in the Py S group. A 10-fold increase in the excretion is, thus, noted in comparison with the amounts excreted both by right and left kidneys in the Py 1 and Py 2 groups. The Py S group excreted on the average about five times as much P,-microglobulin as the unilaterally nephrectomized animals in group NS. It should be noted that the G F R was about 60 per cent in the left kidneys of rats in groups I 60 and Py 1, when compared with the remaining kidneys in groups IS and Py S, respectively. DISCUSSION Chronic interstitial nephritis is commonly seen in clinical practice. It probably involves some proximal tubular dysfunction (19, 33, 34). A slight
P2-Microglobulin in Experimental Renal Disease
available, but the human protein was used in the present investigation. Most probably this does not introduce any significant nonphysiological reactions since (3,-microglobulin from dogs (38) and rabbits (4) are structurally and antigenically very similar to the human protein. Furthermore, studies of the metabolism of endogenous retinol-binding protein (25) and amylase (13) corroborate results obtained with human P,-rnicroglobulin (6, 16). Thus it appears reasonable to conclude that the exogenous P,-microglobulin is metabolized similarly to its endogenous counterpart. The urinary excretion of P,-microglobulin was estimated as the ratio of the (3,-microglobulin clearance to the GFR. Theexcretion from the right I and left kidneys in the normal rats (group NS) was NS I60 If Pyl PyZ PyS before after r I r l r l very similar, thus lending evidence to the view that Fig. 2. The ratio of clearance of P2-microglobulin the sampling of blood from the left renal vein did to glomerular filtration rate (GFR) for the experi- not substantially affect the protein excretion. A mental group. Log scale is used. The points represent values from each clearance period. In pitfall in the present studies may be that trauma is group NS, (0) = right and left kidneys before known to increase the (3,-microglobulin excretion and ( 0 ) = left kidneys after removal of the right (43). This would mean that the ab.rohrteamount of kidney. In the other groups, (0)= kidneys without excreted protein may be too high, but since the induced damage, and (0) = kidneys with induced data are analysed in terms of comparativcJvalues, damage. r = right kidneys; 1 = left kidneys. no seriously distorted information should be proteinuria of the mixed glomerular-tubular type obtained. It should be noted that renal extraction data in is commonly noted (32). The pyelonephritis induced in this study is morphologically similar to general may be unreliable. This is especially so because the usual difference between arterial and the chronic interstitial nephritis found in man. In the ischaemically injured kidneys, marked venous concentrations are only about 10 to 25 per generalized damage was found in both proximal cent. The experimental error may, thus, be magand distal tubules. This model is of value in study- nified 3- to 4-fold in estimations of the renal ing the effect of ischaemic tubular lesions in extraction value. The present determinations of comparison with chemically induced injuries. It is P,-microglobulin could be performed with a high also related to clinical situations -for example, degree of precision, since the protein was radiorenal embolism, kidney damage in shock, and actively labelled, but owing to its rapid catabolism ischaemically damaged kidney grafts. The lesion a substantial accumulation of free iodine occurs. is probably of an irreversible type, since structural Even very small amounts of free iodine trapped in alterations and a decreased G F R are apparent the protein precipitates may thus influence the after 11-15days(15)aswellasafter2or3months. renal extraction values. The PEG lo00 analyses Such observations are in accordance with pre- are subject to a coefficient of variation of 5.7 per vious studies (12, 28). cent, which of course also has bearing on the The renal handling of P,-microglobulin was extraction data. examined in the various experimentally induced Despite these limitations it appears reasonable kidney lesions. This protein was chosen because to conclude that the ratio E ( P 2 p ) to E (PEG) is several previous studies have documented that its below 1.0 for all experimental groups (Table 111). elimination is greatly dependent on a normal renal This indicates that the glomerular filtration of pzphysiology (6,7, 1 I , 16,22,29, 31,41,42). The rat microglobulin is a main step in its intrarenal cathomologue of (3,microglobulin is as yet not abolism.Ravnskov et al. (31) have measured the
.
'i
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Scand J Clin Lab Invest Downloaded from informahealthcare.com by University of Auckland on 11/09/14 For personal use only.
598
A. Fredriksson
renal extraction of P,-microglobulin in humans and suggested that P,-microglobulin may be eliminated not only by glomerular filtration but also by peritubular uptake. From the present data no mechanism other than glomerular filtration has to be invoked. In fact, ultrastructural studies support the hypothesis that the essential renal uptake of P,-microglobulin occurs in proximal tubular cells from the brush-border side (9,23,27). For insulin a renal extraction exceedin$glomerular filtration is observed (8, 30), but insulin is a hormone readily taken up from the blood by most cells. The low ratio of E ((3& to E (PEG) observed for group Py S is significantly different from the ratios found in the other experimental groups. At present, no readily acceptable explanation for this finding can be put forth. After unilateral nephrectomy in normal rats a moderate increase in P,-microglobulin excretion was observed in this study. Increased urinary excretion thus may be a sign of progressive reduction of functioning renal tissue. In rats an increased excretion of total protein and albumin is found after unilateral nephrectomy, normalized after 1 month (39). In unilateral renal disease, no or only small differences in P,-microglobulin excretion were found between the diseased and intact kidneys. If, however, the intact kidneys were removed, a greatly increased excretion of P,-microglohulin in relation to G F R was found for the various types of injured kidneys. The adaptation to a reduction of the functioning kidney mass thus seems to considerably influence the urinary excretion of P,-microglobulin in normal as well as in disease conditions. This is in accordance with the inverse correlation between excretion of low molecular weight proteins and GFR found in clinical studies (22,42). The tubular transfer of o-iodohippurate (OIH) appears to react to about the same degree as the (3,-microglobulin excretion in animals with one diseased and one intact kidney. OIH did not, however, in this study react noticeably to a reduction in the amount of functioning kidney mass, in contrast to the urinary P,-microglobulin excretion. Similar results were observed in an earlier study (1 5).
There are several factors that may have a bearing on the augmented (3,-rnicroglobulin excretion in response to reduction in functioning kidney mass. The following may be of importance: (i) overload of the tubular reabsorptive capacity, (ii) competition between various proteins for tubular reabsorption, (iii) the existence of factors in uraemic plasma inhibiting reabsorption, and (iv) variation in the fractional reabsorption of salt and water in the proximal tubules and tubular flow rates. An increased GFR per nephron (37) and a possible increase in the permeability to proteins in renal insufficiency may overload the tubular reabsorptive capacity. To give a proportionslly increased protein excretion, however, these changes must be proportional to the reduction inGFR. In some experimental groups (notably Py S and IS) the plasma concentration of endogenous P2microglobulin, as well as other low molecular weight proteins, might be increased above a possible threshold value for protein reabsorption. On the other hand, changes in the urinary excretion of amylase, similar to those noted for p,-microglobulin, have been found without any concomitant alterations in its plasma concentration (13). The possibility remains, however, that the tubular reabsorptive mechanism is nondiscriniinatory with regard to individual proteins (18). Plasma from uraemic patients has been demonstrated to reduce proximal tubular function (40) and might also influence protein reabsorption. Since protein reabsorption in the distal tubules probably is small (I, 35) renal excretion may be expressed as the result of end-proximal concentration and fractional reabsorption of water in proximal tubules (see Calculations, eqn. [5]). In micropuncture studies (10,26,39)very different absolute concentrations of proteins have been found, but a constant concentration along the proximal tubules has been observed. This would mean that protein reabsorption in proximal tubules is influenced by protein concentration and fluid reabsorption. In that case the reduced fractional reabsorption found in renal insufficiency (37) could produce increased protein excretion. The change in time of contact with tubular cells, due to variations of fractional fluid reabsorption and tubular flow rate, may also be important. In conclusion, this investigation provides ev-
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Bz-Microglobulin in Experimental Renal Disease
idenca that glomerular filtration is a main step in the intrarenal elimination of P,-microglobulin. The ratio P,-microglobulin clearance t o GFR is not or only slightly changed in unilateral renal disease, even with marked tubular damage. When the total kidney mass is reductd, this ratio is considerably increased, probably du- more t o functional adaptation than t o the kidney damags per se.
ACKNOWLEDGEMENTS
I am grateful t o Dr. Per A. Peterson for supply of P,-microglobulin and t o Professors Stig Hyden and Ivar Sperber for P E G analyses. The investigation was supported by grants from the Medical Faculty, University of Uppsala, and from the Swedish Medical Research Ccuncil, project 3552-01. REFERENCES 1. Aukland, K. Stop flow analysis of renal protein excretion in the dog. Scand. J . din. Lab. Invest. 12, 300, 1960. 2. Berggird, J. & Bearn, A. G. Isolation and properties of a low molecular weiglit B2-globulin occurring in human biological fluids. J . biol. Chein. 243, 4095, 1968. 3. Berggird, I. Plasma proteins in normal human urine. p. 7 in Manuel, Y., Revillard, J. P. & Betuel, H. (eds.) Proteins in Normal and Pathological Urine. Karger, Basel, 1970. 4. Berggird, I. Personal communication. 5. Berglund, F., Engberg, A., Persson, E. & UI. fendahl, H. Renal clearances of labelled inulin and a polyethylene glycol (PEG 1000) in the rat. Acta physiol. scand. 76, 458, 1969. 6. Bernier, G. M. & Conrad, M. E. Catabolism of human /"2-microglobulin by the rat kidney. Anier. J . Physiol. 217, 1359, 1969. 7. Bernier, G. M., Cohen, R. J. & Conrad, M. E Microglobulinemia in renal failure. Nature (Lond.) 218, 598, 1968. 8. Chamberlain, M. J. & Stimmler, L. The renal handling of insulin. J. d i n . Invest. 46, 911. 1967. 9. Christensen, E. I. & Maunsbach, A. B. Intralysosomal digestion of lysozyme in renal proximal tubule cells. Kidney Int. 6, 396, 1974. 10. Dirks, J. H., Clapp, J. R. & Berliner, R. W. The protein concentration in the proximal tubule of the dog. J . din. Invest. 43, 916, 1964 11. Evrin, P.-E. & WibeII, L. The serum levels and urinary excretion of ~2-microglobulin in apparently healthy subjects. Scand. J . clin. Lab. Invest. 29, 69, 1972.
599
12. Fox, M. Progressive renal fibrosis following acute tubular necrosis: An experimental study. I . Urol. 97, 196, 1967. 13.Fredriksson, A. & Jacobson, G. Influence of renal function on the urinary excretion of amylase. An experimental study in the rat. S c a d 1. clin. Lab. Invest. 33, 313, 1974. 14. Fredriksson, A,, Engberg, A., Persson, E. & Ulfendahl, H. Tubular transfer of glucose and o-iodo-hippurate in normal rat kidneys. Scand. J. Urol. Nephrol. 9, Suppl. 26, 27, 1975. 15. Fredriksson, A., Engberg, A., Persson, E. & Hamberg, H. Tubular transfer of glucose and o-iodo-hippurate in experimental renal disease. Scand. J . Urol. Nephrol. 9, Suppl. 26, 45, 1975. 16. Fredriksson, A. & Peterson, P. A. The effect of renal dysfunction on B2-microglobulin metabolism. Scand. J. Urol. Nephrol. 9, Suppl. 26, 61, 1975. 17. Ganrot, P. 0. Crossed immunoelectrophoresis Scand. J . clin. Lab. Invest. 29, Suppl. 124, 39: 1972. 18. Hardwicke, J. & Squire, J. R. The relationship between plasma albumin concentration and protein excretion in patients with proteinuria. Clin. Sci. 14, 509, 1955. 19. Hoang Tuan, Hagemon, I., Briedigkeit, H. SC Dutz, W. Experimental pyelonephritis. Route of infection, course and immunological aspects. Int. Urol. Nephrol. 4, 285, 1972. 20. Hydin, S. A turbidimetric method for the determination of higher polyethylene glycols in biological materials. Kungl. Lantbrukshiigskolans Ann. 22, 139, 1955. 21. Johansson, B. G. & Ravnskov, U. Analyses of proteins in urine. Aids and limitations. Scand. J . din. Lab. Invest. 29, Suppl. 126, 25, 1972. 22. Johansson, B. G. & Ravnskov, U. The serum level and urinary excretion of ae-microglobulin, ,&-microglobulin and lysozyme in renal disease. Scand. J . Urol. Nephrol. 6, 249, 1972. 23. Maunsbach, A. B. & Christensen, E. I. Lysosoma1 accumulation and digestion of Bn-microglobulin in rat kidney tubules. IRCS 2, 1737, 1974. 24. Mc Farlane. A. S. Efficient trace-labelling of proteins with iodine. Nature (Lond.) 182, 53. 1958. 25. Ostberg, L., Fredriksson, A., Rask, L., Peterson, P. A. & Larsson, L. Renal handling of the vitamin A-transporting protein complex. Unpublished observations. 26. Oken, D. E., Cotes, S. C. & Mende, C. W. Micropuncture study of tubular transport of albumin in rats with aminonucleoside nephrosis. Kidney Int. 1, 3, 1972. 27. Ottosen, P. D. & Maunsbach. A. B. Transport of peroxidase in flounder kidney tubules studied by electron microscope histochemistry. Kidney Int. 3, 315, 1973.
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28. Parsons, F. M., Raper, F. P., Markland, C. & Fox, M. Cadaveric renal transplantation. Brit. med. J . 1, 930, 1963. 29. Peterson, P. A., Evrin, P.-E. & Berggbrd, I. Differentiation of glomerular, tubular and normal proteinuria: Determinations of urinary excretion of /&-microglobulin, albumin and total protein. J . clin. Invest. 48, 1189, 1969. 30. Rabkin, R., Norman, M. S., Steiner, S. & Colwell, J. A. Effect of renal disease on renal uptake and excretion of insulin in man. N . Engl. J . Med. 282, 182, 1970. 31. Ravnskov, U., Johansson, B. G. & Gothlin, J. Renal extraction of &-microglobulin. Scand. J . clin. Lab. Invest. 30, 71, 1972. 32. Revillard, J. P., Manuel, Y., Francois, R. & Traeger, J. Renal diseases associated with tubular proteinuria. pp. 209-219 in Manuel, Y., Revillard, J. D. & Betuel, H. (eds.) Proteins in Normal and Pathological Urine. S . Karger, Basel, 1970. 33. Schainuck, L. I., Striker, G. E. & Cutler, R. E. The functional significance of interstitial pathology in chronic renal disease. Clin. Res. 16, 168,1968. 34. Schirmeister, J. Pathophysiologie der Pyelonephritis in L o w H. & Kienitz, M. (HIS.) Die Pyelonephritis. Georg Thieme Verlag, Stuttgart, 1966. 35. Schultze, H. E. & Heremans, J. F. p. 670 in Molecular Biology of Human Proteins, Vol. 1 . Elsevier, New York, 1966. Received 2 November 1973 Accepted 21 April 1975
36. Shankel, S. W., Robson, A. M. & Bricker, N. S. On the mechanism of the splay in the glucose titration curve in advanced experimenta1 renal disease in the rat. J . clin. Invest. 46, 164, 1967. 37. Seldin, D. W., Carter, N. W. & Rector, F. C., Jr. Consequences of renal failure and their management. in Straws, M. B. & Welt, L. G. (eds.) Diseases of the Kidney. Little, Brown and Co., Boston, 1971. 38. Smithes, 0. & Poulik, M. D. Dog homologue of human 02-microglobulins. Proc. nat. Acad. Sci. (Wash.) 69, 2914, 1972. 39.Van Liew, J. B., Stolte, H. & Boylan, J. W. Micropuncture studies of proximal tubular protein reabsorption in normal and hypertrophied rat kidney. Fed. Proc. 26, 375, 1967. 40. White, A. G. Uremic serum inhibition of renal paraaminohippurate transport. Proc. SOC. exp. Biol. (N.Y.) 127, 309, 1966. 41. Wibell, L., Evrin, P.-E. & Berggbrd, I. Serum P2-microglobulin in renal disease. Nephron 10, 320,1973. 42. Wibell, L. & Evrin, P.-E. Urinary gp-microglobulin in patients with renal disease - a study during augmented diuresis. Acta Univ. Upsal. 183, 1974. 43. Wide, L. & Thoren, L. Increased urinary clearance for albumin, /j’p-microglobulin, insulin and luteinizing hormone following surgical or accidental trauma. Scand. J. clin. Lab. Invest. 30, 275, 1972.
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A. FREDRIKSSON Dept. of Surgery, University Hospital, Uppsala, Sweden
Fredriksson, A. Renal Handling of bz-Microglobulin in Experimental Renal Disease. Scand. J. din. Lab. Invest. 35.591600, 1975. Renal extraction and urinary excretion of *251-labelIedDo-microglobulin was studied in rats. The effect of ischaemic renal injury, experimental pyelonephritis, and unilateral nephrectomy was investigated. The tubular secretion of o-iodohippurate (OIN)was measured for comparison. The urinary excretion was calculated as the ratio between the clearance of protein and the glomerular filtration rate. The glomerular filtration rate was estimated as clearance of polyethylene glycol (PEG 1000). The renal arteriovenous concentration difference was lower for P?-microglobulin than for PEG 1000 in all the experimental groups. In unilateral renal disease the ,$*-microglobulin excretion of the intact kidneys was similar to that of the diseased kidneys. A significant difference was noted only after ischaemic renal injury. The same was found for OIH. After removal of the intact kidneys the excretion of /j+microglobulin increased about 10-fold in pyelonephritic animals and 2- to 30-fold in animals with ischaemic renal injury. One hour after unilateral nephrectomy in normal animals the ratio increased about 50 per cent. The tubular secretion of OIH did not change noticeably. It is concluded that the glomerular filtration is a main step in the intrarenal catabolism of bzmicroglobulin and that its urinary excretion is considerably influenced by a reduction in the functioning kidney mass. Key-words: pz-microglobulin; proteinuria; renal disease, experimental; renal function A. Fredriksson, M.D.,Dept. of Surgery, Universiry H o s P i l d , 750 14 UPPsofa 14, Sweden
Proteinuria has long been used as a qualitative icdication of renal disease. During recent years it has been well established that theurinary excretion of plasma proteics is different in diseases primarily affecting the glomeruli or the tubuli (for review, see Refs. 3 and 35). There is currently interest in using quantitative estimations of various proteins for evaluation of proximal renal tubular dysfunction, since such an approach may be more discriminatory than electrophoretic analyses or qualitative estimations of the total urinary protein (22). $,-microglobulin, a low molecular weight protein present in biological fluids (2). may be the protein whose excretion is most sensitive to tubular dysfunction (16, 21, 29). The serum concentration of P,-microglobulin appears
to be a function mainly of the glomerular filtration rate (6, 11,16,41). Under physiological conditions only small amounts of B,-microglobulin are excreted into the urine, but in tubular disease greatly elevated levels of urinary $,-microglobulin are observed (29). The purpose of this investigation wa; to study in rats the renal handling of P,-microglobulin in experimentally induced renal disease. Special attention was focused on the effect of reducing the number of functioning nephrons, since clinical observations have indicated that this might influence the urinary excretion of plasma proteins (7, 22, 42). The tubular secretion of o-iodohippurate was measured for comparison.
592
A . Fredriksson
MATERIAL AND METHODS
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Animals
Male albino rats (n = 27) of the Sprague-Dawley strain (Anticimex, Stockholm, Sweden) were used throughout. Their average weight was 250 g at the induction of kidney damage. The individual weight at the time of the clearance investigation varied between 370 and 500 g. The rats were given a standard diet (Ewos, Sodertalje, Sweden) and had free access to food and drinking water until the start of the experiments. Experimental groups Pyetonephritis was induced in the left kidney in two ways. The kidney was exposed by an abdominal incision during ether anaesthesia. In one group (Py I , n = 4)a method modified from that described by Shankel et al. (36) was used. The kidney was punctured 100 times with a needle that was repeatedly dipped in a culture of enterococci (lolo bacteria per ml). In a second group (Py 2, n = 3) 0.15 ml of the enterococcal culture was injected into the renal pelvis at the beginning of 30 niin of ureteral occlusion. In groups Py 1 and Py 2 the right kidney was left intact. In a third group (Py S,n = 4)the left kidney was treated in the same way as in Py 1, but simultaneously the right kidney was removed. The functional studies were performed 2 to 4 months after induction of pyelonephritis. At the start of the clearance experiments urine samples were obtained by needle aspiration from the exposed bladder. The urine samples were kept at +4 "C until cultures were set up on standard laboratory media after serial dilutions of the samples in phosphate-buffered saline. Quantitative cultures were also made of sections from the kidney, excised at the time of killing with a disinfected but nonsterile knife. The sections were immediately placed in sterile tubes at $4 "C.The number of bacteria per gram of kidney tissue was calculated from cultures on standard laboratory media. After completion of the clearance studies the animals were killed by intra-arterial air injection. The kidneys were excised, freed from the adiposz capsule, immediately weighed, sectioned lon-
gitudinally, and fixed in 10 per cent formaldehyde solution. Deparaffinized, hdematoxylin and eosinstained sections were studied by light microscopy. Ischaeniic renal injury was induced by occlusion of the left renal artery with a 0000 silk thread for 60 min during anaesthesia with intraperitoneally injected pentobarbital sodium (NembutalB, Abbott), 45 mg/kg body weight. The body temperature was held constant at 37 0.5 "Cwith a thermostat-controlled heating pad. In one group ( I 60, n = 4) the right kidney was left intact, but in another group (IS, n = 5) it was removed after 8 weeks. The functional studies were carried out 8 weeks after the induction of kidney damage in group I 60 and 4 weeks after removal of the right kidney in group IS. Morphological studie- were accomplished as described for the pyelonephritic kidneys. Normal single kidneys ( N S , n = 7) were studied after unilateral nephrectomy in normal rats. Clearance experiments for two periods were performed before nephrectomy, and the following periods were started 1 hr after the nephrectomy. Morphological studies showed no abnormalities.
+
Substances used for clearance studies
Glomerular filtration rate (GFR) was estimated as the clearance for polyethylene glycol (PEG lOOO), molecular weight 800-1000 (Kebo, Stockholm, Sweden) (5). For the evaluation of proximal tubular secretory capacity, ortho-iodohippurate sodium (OIH) (Hippuranm, Mallinckrodt, New York, USA) was used with the addition of ~ r t h o - [ ~ ~ ~ I ] h i p p u r a t e sodium (AB Atomenergi, Studsvik, Sweden). P,-microglobulin was isolated from urine of patients with tubular disease. The purification procedure was essentially that of Berggard & Bearn (2). The protein, which was homogenous by several chemical, physicochemical, and immunological criteria, was labelled with lZ5I (Amersham, England) by the iodine monochloride method of Mc Farlane (24). No preoxidation was carried out. The preparations used contained about 0.5 to 0.7 mol of iodine per mol of protein. The labelled P,-microglobulin was indistinguishable from its native counterpart on examination by crossed immunoelectrophoresis (17). In the
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~2-Microglobulirtin Experimental Renal Disease
593
present investigation, only material that gave the same fractional catabolic rate when screened in one animal before measurement as when directly injected was used.
trichloroacetic acid and in urine by the same procedure but after the addition of 0.1 ml 10 per cent serum albumin to avoid losses. The radioactivity was counted in a gamma spectrometer (Autowell).
Clearance experiments
Calculations
The animals were anaesthetized with 5-sec-butyl5-ethyl-2-thiobarbituric acid (Inactin@, Promonta, Hamburg), 120 mg/kg body weight, injected intraperitoneally. Tracheostomy was performed, and the body temperature was held constant at 37 0.5 "C by a thermostat-controlled heating pad. Polypropylene catheters (PP 10 or 50) were inserted into a jugular vein for infusions and into a femoral artery for blood sampling and blood pressure recording. Urine was collected under oil through polyethylene catheters (PE 10) inserted into the distal ureteral ends. A constant rate of infusion of 200 mg PEG 1O00, 225 mg o-iodohippurate sodium, 5 pC 1311-labelledo-iodoMppurate sodium, and 4 pg 1z51-labelledPz-microglobulin in 2.5 ml saline was given per hour. Blood samples (0.3 ml) were withdrawn in the middle of the urine collection periods from a femoral artery and the exposed renal veins. Renal vein samples were taken with a 20-gauge needle introduced through the inferior vena cava. In the NS group renal vein samples were taken only from the left kidney. At least three clearance periods were performed in each animal. The duration of the clearance periods was varied, to ascertain that an appropriate amount of urine was obtained ( 20.2 ml). The amount of urine was determined by weighing.
Glomerular filtration rate was calculated by the formula GFR = 'ir *Cu/Cp(PEG), [I1 where 3 is the urine flow rate in ml/min, and Cu/Cp (PEG) is the ratio of the urine to plasma concentration of PEG 1OOO. The ratio between the clearance for p,-microglobulin and PEG lo00 was calculated as
Analytical mcthods
PEG 1000 was analysed tvrbidimetrically with a micromodification of the method used by HydCn (20). Protein and sulphate were precipitated with barium hydroxide and zinc sulphate. Exactly 5 min after the addition of trichloroacetic acid, turbidity was determined at 400 nm with a spectrophotometer. The coefficient of variation of the present method was 5.7 per cent (n = 15). For analysis of I 3 l I , 50 pl of plasma or urine was diluted in 1 ml of distilled water. Proteinbound lZ5l in plasma was recovered by the precipitation of the proteins with ice-cold 10 per cent 8
-
Scand. J. clin. Lab. Invert.
The amount of o-iodohippurate (OIH) excreted into the urine per ml of glomerula: filtrate was calculated as
0 . c (OIH) ~ i'.Cu/Cp (PEG)
(P2)
-
Cu (OIH) Cu/Cp (PEG) '
PI 1
(P2)
Cu and Cp are the urinary and plasma concentrations of P,-microglobulin, respectively, and Cu (OIH) is the urinary concentration of o-iodohippurate. The renal extraction of a substance (x) is calculated as E(x) = 1
-
renal vein concentration . arterial concentration
[41
If it is assumed that no reabsorption of p,-microglobulin occurs distally to the end-proximal tubular segment, the amount of (&-microglobulin excreted, Cu (p,) .V, equals Ctf . h f , where Ctf (9,) is the concentration of P,-microglobulin and Vtf the volume flow rate in the end-proximal tubular segment. Dividing Cu (p,) .'? = Ctf (p,) * vtf by GFR gives
(P,)
where Ctf (PEG) is the concentration of PEG in the end-proximal tubular fluid. Ctf/Cp (PEG) is a reflection of the fractional reabsorption of fluid in the proximal tubules.
594
A. Fredriksson
RESULTS
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Eactrriological and morphological findings
Urine cultures showed more than lo5 bacteria per ml in about 60 per cent of the pyelonephritic animals. Renal tissue cultures shohed more than lo4 bacteria per g cf tissue both for right and left kidneys when urine cultures were positive. No other bacteria than enterococci were found. The left kidneys in the Py 1 group were contracted with a scarrcd surface.' Their weight was moderately but not significantly reduced compared with the right control kidneys (Table I), which grossly appeared to be normal. Light microscopy showed moderate chronic-subchronic interstitial nephritis in the left kidneys, but minimal changes were also found in some of the right kidneys. In Py 2 the left kidneys showed only a few superficial scars, and their weight was only slightly reduced. Light microscopic findings were essentially as in Py 1 rats. In Py S the kidneys were largely hypertrophied and irregularly shaped and had a greyish discoloration. Their weight had increased substantially compared with the kidneys in the other groups. Light microscopy showed somewhat more marked inflammatory changes
than in Py 1 rats and more tubular dilatation as well. The left kidneys subjected to ischaemia (group I 60) were contracted, and their weight was considerably reducsd compared with the intact right kidneys (Table I). In group IS the remaining kidney was hypertrophied, but with large individual variations. Light microscopy of the ischaemically injured kidneys showed necroses surrounded by mononuclear infiltration in both groups. The tubules were dilated throughout with flattened epithelial cells and the occurrence of some casts. The changes were most advanced in the inner medullary zone and corticomedullary junction. The glomeruli appeared normal, with the exception of those in the necrotic areas. The right kidneys in group I 60 only showed moderate hypertrophy. In all experimental groups except the IS group, the animals appeared to be in a good health and gained weight like the normal control rats. In the IS group the animals appeared anaemic and in a slightly poor condition, but all except one gained weight even after the unilateral nephrectomy. Systolic blood pressure during anaesthesia wqs within the normal range except for one animal in
Table I. Characteristics of the experimental groups* Body weight, Group (number of rats) Normal, NS (7) Before nephrectomy Right kidney Left kidney After nephrectomy Left kidney Ischaemic renal injury 60 min, I 60 (4) Right kidney Left kidney 60 min, IS (5) Pyelonephritis
PY 1 (4)
GFR/100 g body weight, ml/min
1.6910.08
0.2910.02 0.34*0.03
1.7210.06
0.2910.04
2.1330.08 1.0310.09 2.08+0.36
0.37h0.03 0.12+0.01 0.2110.07
1.9930.10 1 ,5310.09
0.21f0.02 0.1710.01
1.79&0.13 1.56t0.08 2.8810.12
0.1610.02 0.1310.02 0.32*0.03
433115
4011 9 416119 4901 9
Right kidney Left kidney
PY 2 (3)
477113
Right kidney Left kidney PY s (4)
* All values are mean
Kidney weight,
g
440116
1S.E.M.GFR=glomerular filtration rate.
Bz-Microglobulin in Experirnental Renal Diseuse
595
Table IT. Mean differences between left and right kidney in the excretion of P,-microglobulin and o-iodohippurate (OIH)?
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~~
~~~~~~~~
Clearance P,-microglobulin
Cu(0IH)
Group (number of rats)
GFR Mean (P=0.95)*
Mean (P=0.95)*
Normal, NS (7) Ischaemic renal injury 60 min, I 60 (4) Pyelonephritis PY 1 (4) PY 2 (3)
- 0.0014j0.0015
- 0.007&0.145
Cu/Cp(PEG)
0.0032*0.0022* *
- 0.197&0.195**
O.OOOljO.0038
0.02110.155 - 0.12010.327
- 0.0014*0.0064
t GFR=glomerular filtration rate ; Cu=urine concentration; Cp=plasma concentration;PEG-polyethylene glycol.
* Confidence limits. ** Significant difference (P=0.95). the IS group (190 mm Hg) and one in the Py 2 group (160 mm Hg). Some characteristics of the experimental groups are summarized in Table I. Tubular transfer of o-iodohippurate
Equation [3] related to the plasma concentration of o-iodohippurate, Cp (OIH), was used to assess the tubular secretory capacity (14). The data for the various experimental groups are summarized in Table 11. It can be seen in the Table that the mean differences between the left and the right kidney were small for the animals in the experimental groups. The only significant difference was for group I60 (confidence limits, P = 0.95). The animals in group IS were unilaterally nephrectomiyed, and the secretory capacity of OIH of the remaining kidney was therefore compared with values obtained in normal rats before and after unilateral nephrectomy. It can be seen in Fig. 1 that there was no easily detectable difference. It is therefore conceivable that the tubular secretory capacity, as measured by OIH excretion, did not respond noticeably to a reduction in the functioning kidney mass. Renal extraction o f P,-rnicroglobulin
Since glomerular filtration of P,-microglobulin appears to be of great importance in the elimination of the protein from plasma, it appeared to be of interest to meascre the renal extraction of the protein in the groups of animals with various types of renal disease.
It can be seen in Table 111 that the ratio of the renal arteriovenous differences for p,-microglobulin and PEG lo00 was about 0.70 for all groups except Py S. In the latter group the average value was only 0.26. It is of interest that all mean values were lower than unity, which indicates that PEG 1000 may pass the glomerular barrier more easily than P,-microglobulin.
.
'
0 . 0
a5-
0'
0.5 1.0 1.5 Cp(0l H) rnglml Fig. 1. Urinary excretion of o-iodohippurate (OIH)
per ml of glomerular filtrate as a function of plasma concentration. The points represent values for each clearance period. (0)= group NS before nephrectomy; (0) = group NS after nephrectomy; and (v) = group IS.
596
A. Fredriksson
Table 111. Clearance and renal extraction of P,-microglobulin. Mean values for all clearance periods? Clearance &../GFR
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Group (number of rats) Normal, NS (7) Before nephrectomy After nephrectomy Ischaemic renal injury 60 min, I 60 (4) Right kidney Left kidney 60 min, I S t t (5)
n
Mean (P=0.95)*
S.D.
n
Mean (P=0.95)*
S.D.
30 16
0.01l+0.001
0.003 0.005
15 10
0.65k0.21 0.57&0.09
0.38 0.12
15 14
0.010*0.001 0.013f0.003 0.034f0.015
0.002
13 12
0.67h0.09 0.73h0.17
0.15 0.29
4
0.017IfI0.003
0.004
*+
0.305f0.090 0.348k0.079 0.020h0.004
0.010 0.011 0.052 0.046 0.003
13 14
0.007~0.001 0.008&0.001
0.002 0.002
12 12
0.77h0.14 0.73f0.15
0.22 0.24
10 10 11
0.01Of 0.001 0.008f0.001 0.079f0.016
0.001 0.002 0.024
7 6 10
0.6950.37 0.79h0.30 0.26&0.15
0.33 0.29 0.16
5 3 3
5
Pyelonephritis PY 1 (4) Right kidney Left kidney PY 2 (3) Right kidney Left kidney PY s (4)
E (P,P))/E(PEG)
0.040*0.015
t n=number of clearance periods. tt Values for individual animals are given, because of large interindividual variation. * Confidence limits. ** No extraction values were recorded for group IS because of technical failure. Urinary excretion of P,-microglobulin
The urinary excretion of P,-microglobulin is expressed as the ratio between the clearance of P,-microglobulin and GFR (eqn. [2]). The mean differences between values for right and left kidney were generally small (Table If). They were significant only for group I 60 (confidence limits, P = 0.95). The mean values for each experimental group are presented in Table I11 and the individual values for each clearance period are summarized in Fig. 2. Groups I 60, Py 1 , and Py 2 did not differ from group NS before nephrectomy. It should be noted, however, that after unilateral nephrectomy in group NS, the ratio of the P,-microglobulin clearance to GFR was increased by about 50 per cent. The animals in group IS showed a 2- to 30-fold increase in the ratio compared with the values obtained for the left kidneys of animals in group I 60.The urinary excretion of P,-microglobulin in
most animals of group IS is also higher than for group NS, irrespective of whether the comparison is made before or after unilateral nephrectomy in the latter group. The urinary excretion of P,-microglobulin is considerably elevated in the Py S group. A 10-fold increase in the excretion is, thus, noted in comparison with the amounts excreted both by right and left kidneys in the Py 1 and Py 2 groups. The Py S group excreted on the average about five times as much P,-microglobulin as the unilaterally nephrectomized animals in group NS. It should be noted that the G F R was about 60 per cent in the left kidneys of rats in groups I 60 and Py 1, when compared with the remaining kidneys in groups IS and Py S, respectively. DISCUSSION Chronic interstitial nephritis is commonly seen in clinical practice. It probably involves some proximal tubular dysfunction (19, 33, 34). A slight
P2-Microglobulin in Experimental Renal Disease
available, but the human protein was used in the present investigation. Most probably this does not introduce any significant nonphysiological reactions since (3,-microglobulin from dogs (38) and rabbits (4) are structurally and antigenically very similar to the human protein. Furthermore, studies of the metabolism of endogenous retinol-binding protein (25) and amylase (13) corroborate results obtained with human P,-rnicroglobulin (6, 16). Thus it appears reasonable to conclude that the exogenous P,-microglobulin is metabolized similarly to its endogenous counterpart. The urinary excretion of P,-microglobulin was estimated as the ratio of the (3,-microglobulin clearance to the GFR. Theexcretion from the right I and left kidneys in the normal rats (group NS) was NS I60 If Pyl PyZ PyS before after r I r l r l very similar, thus lending evidence to the view that Fig. 2. The ratio of clearance of P2-microglobulin the sampling of blood from the left renal vein did to glomerular filtration rate (GFR) for the experi- not substantially affect the protein excretion. A mental group. Log scale is used. The points represent values from each clearance period. In pitfall in the present studies may be that trauma is group NS, (0) = right and left kidneys before known to increase the (3,-microglobulin excretion and ( 0 ) = left kidneys after removal of the right (43). This would mean that the ab.rohrteamount of kidney. In the other groups, (0)= kidneys without excreted protein may be too high, but since the induced damage, and (0) = kidneys with induced data are analysed in terms of comparativcJvalues, damage. r = right kidneys; 1 = left kidneys. no seriously distorted information should be proteinuria of the mixed glomerular-tubular type obtained. It should be noted that renal extraction data in is commonly noted (32). The pyelonephritis induced in this study is morphologically similar to general may be unreliable. This is especially so because the usual difference between arterial and the chronic interstitial nephritis found in man. In the ischaemically injured kidneys, marked venous concentrations are only about 10 to 25 per generalized damage was found in both proximal cent. The experimental error may, thus, be magand distal tubules. This model is of value in study- nified 3- to 4-fold in estimations of the renal ing the effect of ischaemic tubular lesions in extraction value. The present determinations of comparison with chemically induced injuries. It is P,-microglobulin could be performed with a high also related to clinical situations -for example, degree of precision, since the protein was radiorenal embolism, kidney damage in shock, and actively labelled, but owing to its rapid catabolism ischaemically damaged kidney grafts. The lesion a substantial accumulation of free iodine occurs. is probably of an irreversible type, since structural Even very small amounts of free iodine trapped in alterations and a decreased G F R are apparent the protein precipitates may thus influence the after 11-15days(15)aswellasafter2or3months. renal extraction values. The PEG lo00 analyses Such observations are in accordance with pre- are subject to a coefficient of variation of 5.7 per vious studies (12, 28). cent, which of course also has bearing on the The renal handling of P,-microglobulin was extraction data. examined in the various experimentally induced Despite these limitations it appears reasonable kidney lesions. This protein was chosen because to conclude that the ratio E ( P 2 p ) to E (PEG) is several previous studies have documented that its below 1.0 for all experimental groups (Table 111). elimination is greatly dependent on a normal renal This indicates that the glomerular filtration of pzphysiology (6,7, 1 I , 16,22,29, 31,41,42). The rat microglobulin is a main step in its intrarenal cathomologue of (3,microglobulin is as yet not abolism.Ravnskov et al. (31) have measured the
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'i
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renal extraction of P,-microglobulin in humans and suggested that P,-microglobulin may be eliminated not only by glomerular filtration but also by peritubular uptake. From the present data no mechanism other than glomerular filtration has to be invoked. In fact, ultrastructural studies support the hypothesis that the essential renal uptake of P,-microglobulin occurs in proximal tubular cells from the brush-border side (9,23,27). For insulin a renal extraction exceedin$glomerular filtration is observed (8, 30), but insulin is a hormone readily taken up from the blood by most cells. The low ratio of E ((3& to E (PEG) observed for group Py S is significantly different from the ratios found in the other experimental groups. At present, no readily acceptable explanation for this finding can be put forth. After unilateral nephrectomy in normal rats a moderate increase in P,-microglobulin excretion was observed in this study. Increased urinary excretion thus may be a sign of progressive reduction of functioning renal tissue. In rats an increased excretion of total protein and albumin is found after unilateral nephrectomy, normalized after 1 month (39). In unilateral renal disease, no or only small differences in P,-microglobulin excretion were found between the diseased and intact kidneys. If, however, the intact kidneys were removed, a greatly increased excretion of P,-microglohulin in relation to G F R was found for the various types of injured kidneys. The adaptation to a reduction of the functioning kidney mass thus seems to considerably influence the urinary excretion of P,-microglobulin in normal as well as in disease conditions. This is in accordance with the inverse correlation between excretion of low molecular weight proteins and GFR found in clinical studies (22,42). The tubular transfer of o-iodohippurate (OIH) appears to react to about the same degree as the (3,-microglobulin excretion in animals with one diseased and one intact kidney. OIH did not, however, in this study react noticeably to a reduction in the amount of functioning kidney mass, in contrast to the urinary P,-microglobulin excretion. Similar results were observed in an earlier study (1 5).
There are several factors that may have a bearing on the augmented (3,-rnicroglobulin excretion in response to reduction in functioning kidney mass. The following may be of importance: (i) overload of the tubular reabsorptive capacity, (ii) competition between various proteins for tubular reabsorption, (iii) the existence of factors in uraemic plasma inhibiting reabsorption, and (iv) variation in the fractional reabsorption of salt and water in the proximal tubules and tubular flow rates. An increased GFR per nephron (37) and a possible increase in the permeability to proteins in renal insufficiency may overload the tubular reabsorptive capacity. To give a proportionslly increased protein excretion, however, these changes must be proportional to the reduction inGFR. In some experimental groups (notably Py S and IS) the plasma concentration of endogenous P2microglobulin, as well as other low molecular weight proteins, might be increased above a possible threshold value for protein reabsorption. On the other hand, changes in the urinary excretion of amylase, similar to those noted for p,-microglobulin, have been found without any concomitant alterations in its plasma concentration (13). The possibility remains, however, that the tubular reabsorptive mechanism is nondiscriniinatory with regard to individual proteins (18). Plasma from uraemic patients has been demonstrated to reduce proximal tubular function (40) and might also influence protein reabsorption. Since protein reabsorption in the distal tubules probably is small (I, 35) renal excretion may be expressed as the result of end-proximal concentration and fractional reabsorption of water in proximal tubules (see Calculations, eqn. [5]). In micropuncture studies (10,26,39)very different absolute concentrations of proteins have been found, but a constant concentration along the proximal tubules has been observed. This would mean that protein reabsorption in proximal tubules is influenced by protein concentration and fluid reabsorption. In that case the reduced fractional reabsorption found in renal insufficiency (37) could produce increased protein excretion. The change in time of contact with tubular cells, due to variations of fractional fluid reabsorption and tubular flow rate, may also be important. In conclusion, this investigation provides ev-
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Bz-Microglobulin in Experimental Renal Disease
idenca that glomerular filtration is a main step in the intrarenal elimination of P,-microglobulin. The ratio P,-microglobulin clearance t o GFR is not or only slightly changed in unilateral renal disease, even with marked tubular damage. When the total kidney mass is reductd, this ratio is considerably increased, probably du- more t o functional adaptation than t o the kidney damags per se.
ACKNOWLEDGEMENTS
I am grateful t o Dr. Per A. Peterson for supply of P,-microglobulin and t o Professors Stig Hyden and Ivar Sperber for P E G analyses. The investigation was supported by grants from the Medical Faculty, University of Uppsala, and from the Swedish Medical Research Ccuncil, project 3552-01. REFERENCES 1. Aukland, K. Stop flow analysis of renal protein excretion in the dog. Scand. J . din. Lab. Invest. 12, 300, 1960. 2. Berggird, J. & Bearn, A. G. Isolation and properties of a low molecular weiglit B2-globulin occurring in human biological fluids. J . biol. Chein. 243, 4095, 1968. 3. Berggird, I. Plasma proteins in normal human urine. p. 7 in Manuel, Y., Revillard, J. P. & Betuel, H. (eds.) Proteins in Normal and Pathological Urine. Karger, Basel, 1970. 4. Berggird, I. Personal communication. 5. Berglund, F., Engberg, A., Persson, E. & UI. fendahl, H. Renal clearances of labelled inulin and a polyethylene glycol (PEG 1000) in the rat. Acta physiol. scand. 76, 458, 1969. 6. Bernier, G. M. & Conrad, M. E. Catabolism of human /"2-microglobulin by the rat kidney. Anier. J . Physiol. 217, 1359, 1969. 7. Bernier, G. M., Cohen, R. J. & Conrad, M. E Microglobulinemia in renal failure. Nature (Lond.) 218, 598, 1968. 8. Chamberlain, M. J. & Stimmler, L. The renal handling of insulin. J. d i n . Invest. 46, 911. 1967. 9. Christensen, E. I. & Maunsbach, A. B. Intralysosomal digestion of lysozyme in renal proximal tubule cells. Kidney Int. 6, 396, 1974. 10. Dirks, J. H., Clapp, J. R. & Berliner, R. W. The protein concentration in the proximal tubule of the dog. J . din. Invest. 43, 916, 1964 11. Evrin, P.-E. & WibeII, L. The serum levels and urinary excretion of ~2-microglobulin in apparently healthy subjects. Scand. J . clin. Lab. Invest. 29, 69, 1972.
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12. Fox, M. Progressive renal fibrosis following acute tubular necrosis: An experimental study. I . Urol. 97, 196, 1967. 13.Fredriksson, A. & Jacobson, G. Influence of renal function on the urinary excretion of amylase. An experimental study in the rat. S c a d 1. clin. Lab. Invest. 33, 313, 1974. 14. Fredriksson, A,, Engberg, A., Persson, E. & Ulfendahl, H. Tubular transfer of glucose and o-iodo-hippurate in normal rat kidneys. Scand. J. Urol. Nephrol. 9, Suppl. 26, 27, 1975. 15. Fredriksson, A., Engberg, A., Persson, E. & Hamberg, H. Tubular transfer of glucose and o-iodo-hippurate in experimental renal disease. Scand. J . Urol. Nephrol. 9, Suppl. 26, 45, 1975. 16. Fredriksson, A. & Peterson, P. A. The effect of renal dysfunction on B2-microglobulin metabolism. Scand. J. Urol. Nephrol. 9, Suppl. 26, 61, 1975. 17. Ganrot, P. 0. Crossed immunoelectrophoresis Scand. J . clin. Lab. Invest. 29, Suppl. 124, 39: 1972. 18. Hardwicke, J. & Squire, J. R. The relationship between plasma albumin concentration and protein excretion in patients with proteinuria. Clin. Sci. 14, 509, 1955. 19. Hoang Tuan, Hagemon, I., Briedigkeit, H. SC Dutz, W. Experimental pyelonephritis. Route of infection, course and immunological aspects. Int. Urol. Nephrol. 4, 285, 1972. 20. Hydin, S. A turbidimetric method for the determination of higher polyethylene glycols in biological materials. Kungl. Lantbrukshiigskolans Ann. 22, 139, 1955. 21. Johansson, B. G. & Ravnskov, U. Analyses of proteins in urine. Aids and limitations. Scand. J . din. Lab. Invest. 29, Suppl. 126, 25, 1972. 22. Johansson, B. G. & Ravnskov, U. The serum level and urinary excretion of ae-microglobulin, ,&-microglobulin and lysozyme in renal disease. Scand. J . Urol. Nephrol. 6, 249, 1972. 23. Maunsbach, A. B. & Christensen, E. I. Lysosoma1 accumulation and digestion of Bn-microglobulin in rat kidney tubules. IRCS 2, 1737, 1974. 24. Mc Farlane. A. S. Efficient trace-labelling of proteins with iodine. Nature (Lond.) 182, 53. 1958. 25. Ostberg, L., Fredriksson, A., Rask, L., Peterson, P. A. & Larsson, L. Renal handling of the vitamin A-transporting protein complex. Unpublished observations. 26. Oken, D. E., Cotes, S. C. & Mende, C. W. Micropuncture study of tubular transport of albumin in rats with aminonucleoside nephrosis. Kidney Int. 1, 3, 1972. 27. Ottosen, P. D. & Maunsbach. A. B. Transport of peroxidase in flounder kidney tubules studied by electron microscope histochemistry. Kidney Int. 3, 315, 1973.
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28. Parsons, F. M., Raper, F. P., Markland, C. & Fox, M. Cadaveric renal transplantation. Brit. med. J . 1, 930, 1963. 29. Peterson, P. A., Evrin, P.-E. & Berggbrd, I. Differentiation of glomerular, tubular and normal proteinuria: Determinations of urinary excretion of /&-microglobulin, albumin and total protein. J . clin. Invest. 48, 1189, 1969. 30. Rabkin, R., Norman, M. S., Steiner, S. & Colwell, J. A. Effect of renal disease on renal uptake and excretion of insulin in man. N . Engl. J . Med. 282, 182, 1970. 31. Ravnskov, U., Johansson, B. G. & Gothlin, J. Renal extraction of &-microglobulin. Scand. J . clin. Lab. Invest. 30, 71, 1972. 32. Revillard, J. P., Manuel, Y., Francois, R. & Traeger, J. Renal diseases associated with tubular proteinuria. pp. 209-219 in Manuel, Y., Revillard, J. D. & Betuel, H. (eds.) Proteins in Normal and Pathological Urine. S . Karger, Basel, 1970. 33. Schainuck, L. I., Striker, G. E. & Cutler, R. E. The functional significance of interstitial pathology in chronic renal disease. Clin. Res. 16, 168,1968. 34. Schirmeister, J. Pathophysiologie der Pyelonephritis in L o w H. & Kienitz, M. (HIS.) Die Pyelonephritis. Georg Thieme Verlag, Stuttgart, 1966. 35. Schultze, H. E. & Heremans, J. F. p. 670 in Molecular Biology of Human Proteins, Vol. 1 . Elsevier, New York, 1966. Received 2 November 1973 Accepted 21 April 1975
36. Shankel, S. W., Robson, A. M. & Bricker, N. S. On the mechanism of the splay in the glucose titration curve in advanced experimenta1 renal disease in the rat. J . clin. Invest. 46, 164, 1967. 37. Seldin, D. W., Carter, N. W. & Rector, F. C., Jr. Consequences of renal failure and their management. in Straws, M. B. & Welt, L. G. (eds.) Diseases of the Kidney. Little, Brown and Co., Boston, 1971. 38. Smithes, 0. & Poulik, M. D. Dog homologue of human 02-microglobulins. Proc. nat. Acad. Sci. (Wash.) 69, 2914, 1972. 39.Van Liew, J. B., Stolte, H. & Boylan, J. W. Micropuncture studies of proximal tubular protein reabsorption in normal and hypertrophied rat kidney. Fed. Proc. 26, 375, 1967. 40. White, A. G. Uremic serum inhibition of renal paraaminohippurate transport. Proc. SOC. exp. Biol. (N.Y.) 127, 309, 1966. 41. Wibell, L., Evrin, P.-E. & Berggbrd, I. Serum P2-microglobulin in renal disease. Nephron 10, 320,1973. 42. Wibell, L. & Evrin, P.-E. Urinary gp-microglobulin in patients with renal disease - a study during augmented diuresis. Acta Univ. Upsal. 183, 1974. 43. Wide, L. & Thoren, L. Increased urinary clearance for albumin, /j’p-microglobulin, insulin and luteinizing hormone following surgical or accidental trauma. Scand. J. clin. Lab. Invest. 30, 275, 1972.
