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Effect of fasting on lysosomes in kidney cortex of glomerulonephritic rats
Kidney International, Vol. 34 (1988), pp. 766—773
Effect of fasting on lysosomes in kidney cortex of glomerulonephritic rats HANS-JACOB HAGA, KNUT-JAN ANDERSEN, BJARNE M. IVERSEN, JARLE OFSTAD, MILOSLAV DOBROTA, and ROALD MATRE Medical Department A, University of Bergen, Haukeland Sykehus, N-5016, Bergen, Norway; Robens Institute of Industrial and Environmental Health and Safety, University of Surrey, Guildford, United Kingdom; and Broegelman Research Laboratory for Microbiology and Departement of Microbiology, The Gade Institute, University of Bergen, Norway
Effect of fasting on lysosomes in kidney cortex of glomerulonephrltlc rats. The effect of food restriction (FR) on the kidney cortex lysosomes prepared by rate and isopycnic zonal centrifugation was studied in rats with passive Heymann glomerulonephritis (PHN). FR reduced the renal
progression of functional deterioration mainly in patients with tubulointerstitial disease, and that diet has only a marginally beneficial effect in patients with chronic glomerulonephritis [8].
mass by 41%, but the capacity for handling of labelled endocytosed proteins by the lysosomes was not different from fed PHN rats. While
ischemic induced tubular necrosis has also been demonstrated
The dramatic protective effect of dietary protein prior to
PHN with heavy proteinuria increased the recovery of lysosomal in rat models [9]. enzymes in the large lysosomes located in the proximal tubule, no We have previously demonstrated significant effects on the changes were observed in FR-PHN rats in spite of significant proteinuria. The density of the small lysosomes was significantly shifted! lysosomes in the rat kidney cortical tubules induced by acute reduced (from 1,200 and 1,235 g/mI to 1,185 and 1,225 g/ml, respectively) in both fed and FR-PHN rats, suggesting that the handling of extra loads of protein may enhance the absorbtive function of small
lysosomes found in the lower part of the nephron. FR reduced the mechanical fragility of lysosomes in the kidney cortex of PHN-rats. The highly increased urinary excretion of lysosomal enzymes in fed PHN
rats was not observed in FR-PHN rats. As a conclusion, FR reduces both the fragility of lysosomes and the proportion of digestive enzymes in fragile lysosomes. These lysosomal enzymes may be of pathogenic importance in PHN causing cell damage when liberated from disrupted lysosomes.
passive Heymann glomerulonephritis (PHN) [10, 11]. PHN induced a significant increase in the cortical activity of lysosomal enzymes mainly in the large lysosomes or 'protein droplets' in the proximal tubule [10]; PHN also induced significant changes in various other populations of small lysosomes found in the cortical nephron, probably reflecting an increased reabsorption of protein caused by proteinuria. Lysosomal en-
zymes are considered to be of pathogenic importance in the development of glomerulonephritis [12, 13], and increase in
urinary lysosomal enzyme activity has been widely accepted as indicative of kidney disease or kidney damage [14—16]. We have previously demonstrated increased urinary activity of several Food restriction, which involves either low protein diet or lysosomal enzymes in rats with acute passive Heymann glomeraccess to food on alternate days, has been shown to extend the ulonephritis (PHN) [10, 11]. Since food restriction reduces the life span of normal rats [1], and to retard the progression of urinary excretion of protein in glomerulonephritic rats [3—6], the nephntis in rat models and man [2—4]. It has been postulated favorable effect of food restriction on the development of
that the effect of food restriction may be caused by altered
glomerular flow reducing the progression of glomeruloscierosis [5, 6], or may be directly attributable to restriction of protein [3, 4] or dietary phosphorous [7]. However, the exact mechanisms by which food restriction influences the course of nephritis are still unknown. Most of the studies concerning the protective effect of foodl protein restriction have been devoted to hemodynamic changes
glomerulonephritis may be due to lysosomal changes in the nephron because of a reduced protein load. The particular aim
of this study was therefore to examine the effect of food restriction on the lysosomes in the kidney cortical tubules of PHN rats having a high urinary excretion of protein. Methods
Preparation of acute Heymann glomerulonephritis
in the glomeruli resulting in reduced urinary excretion of
Antiserum to Fx1A was raised in rabbits as previously
protein and retarded progression of glomerulosclerosis, but a few recent studies have demonstrated that protein restriction described [17]. Rabbit IgG was prepared/purified by ion-exmay also have significant effects on the tubules. It has, for change chromatography on DEAE- cellulose (Whatman DE-52) instance, been demonstrated that protein restriction retards the equilibrated with 0.015 M phosphate buffer pH 7.6, followed by a gel filtration on a Sephadex G-200 Column equilibrated with 0.015 M phosphate buffered saline pH 7.2 (PBS) [18]. IgG prepared from normal rabbits served as control. Male Wistar rats (weight 130 to 170 g) used in the experiments
Received for publication January 28, 1988 and in revised form July 25, 1988
were fed on Brood Stock Feed R3. This diet is composed of
© 1988 by the International Society of Nephrology
21.0% crude protein, 19.0% pepsin digested protein, 6% crude 766
Haga et a!: Kidney lysosomes in fasted PHN rats
767
fat, 52.0% carbohydrates, 1.2% calcium, 1.0%. phosphorous, of the eight homogenates thus obtained were kept for further 0.09% sodium and 0.7% potassium. Forty-two percent of the assays of enzyme activities and protein before the homogenates total fatty acids consisted of linoleic acid (C18:2) and 8.2% of were pooled and further separated into the nuclear 'N', mitolinolenic acid (C18:3). Feed consumption was 10 to 15 glrat/day chondrial-lysosomal 'ML', microsomal 'Mic' and cytosolic and metabolizable energy was 3.10 Meal/kg. In this experiment supernatant 'Sup' fractions by differential pelleting [20, 21]. we studied three groups of rats with eight rats in each group: The 'ML' fraction was further subfractionated by rate and control rats, passive Heymann glomerulonephritic (PHN) rats isopycnic centrifugation techniques as previously described in and food restricted/fasted (FR) PHN rats. PHN was induced by detail by Andersen et al [20]. a single intravenous injection of 7.3 mg IgG with an anti-Fx1A titer of 1:2048. Control rats received 7.3 mg normal rabbit IgG Chemical and enzyme assay in homogenates and subcellular without detectable antibodies against Fx1A. Antibodies against fractions glomerular basement membrane were not detectable in either of Protein (mg per ml) was measured by the protein dye method the IgG preparations [171. described by Bradford [22] using bovine serum albumin as a The effect of food restriction was studied by feeding PHN standard. DNA was measured as described by Boer [23] and rats every alternate day from induction of glomerulonephritis to was expressed as g. The lysosomal enzymes, N-acetyl-/3-Dsacrifice 14 days later, and all rats were allowed to drink tap glucosaminidase (NAG), acid /3-galactosidase, acid /3-glycerowater ad libitum during the days without food. The glomerulo- phosphatase, acid RNA'ase and cathepsin D were assayed as nephritic model was not found to cause infiltration of white previously described [20]. To minimize lysosomal autolysis blood cells in the renal parenchyma [17]. during incubation, latency measurements were performed for only 10 minutes at 37°C. Latency measurements were perImmunofluorescence technique formed as previously described [24]. Cathepsin D units are Kidney specimens from rats in the experimental groups were expressed as A280/min/fraction and acid ribonuclease frozen in liquid nitrogen. Sections 4 m thick were cut on a (RNA'ase) as zi A2/min/fraction. The other enzyme activities cryostat and placed on microscopic slides. Air dried sections are expressed as pmol/min/fraction. were incubated for 34 minutes in a humid atmosphere with Labelling of '251-lysozyme and '°9Cd-thionein was performed twofold dilutions of FITC-labelled swine anti-rabbit IgG. The as previously described [20]. The labelled lysozyme (18 Ci per sections were then washed in PBS for 30 minutes, mounted in animal) and labelled Cd-thionein (5 pCi per animal) were glycerol-PBS and examined in a Leitz Orthoplan microscope injected intravenously into each rat (N = 8) 30 minutes before with a HBO-200 mercury lamp. sacrifice in two different experiments. Fluorescine-isothiocyanate (FITC) labelled swine IgG was Serum albumin was estimated fluorometrically using rat purchased from DAKO Immunoglobulin A/S, Copenhagen, albumin as standard [25]. Creatinine in serum was measured by Denmark. an automated version of the picrate method [17], and expressed as tmol/liter. Collection and preparation of urine Urine was collected on ice at the height of proteinuria 14 days Statistical methods following injection of IgG [17]. The rats were housed in metaStudents t-test for non-paired and paired observations was bolic cages where they were without admission to food but had used for statistical evaluation. All P values were considered free access to tap water during the 24-hour collection period. significant when smaller than 0.05. All urine were centrifuged (2000 rev/mm for 5 mm) and diafiltered in an Amicon UF 12 cell (Amicon Corp., Danvers, Results Massachusetts, USA) at 50 psi using an Um-lO filter [11] to Glomerulonephritis remove enzyme inhibitors of low molecular weight. Glomerulonephritis was confirmed by direct fluorescence Chemical and enzyme assay in urine staining of cryosections from kidneys (Fig. 1) 14 days after Urinary protein was measured by the biuret method [19] induction of passive Heymann glomerulonephritis (PHN). Food using bovine serum albumin as standard. The acid hydrolases restriction had no effect on the fluoroscine staining in glomeruli N-acetyl-3-D-glucosaminidase, acid-3-galactosidase, acid phos- of PHN rats. No fluorescence was observed in kidneys of phatase and /3-glucosidase were assayed by a fluorometric assay control rats. As previously described [101 PHN induced highly [10], and enzyme activities were expressed as nmol/min per 24 increased urinary excretion of protein and urinary activity of all hours. three lysosomal enzymes studied (NAG, acid 3-galactosidase and acid phosphatase; Table 1) 2 weeks after induction. The Tissue homogenization and fractionation urinary excretion of protein was significantly lower in FR-PHN Male Wistar rats were starved 24 hours before sacrifice at day rats compared to fed PHN rats, and the urinary activity of NAG 14 after injection of IgG, and anesthetized with ether before the and acid /3-galactosidase was significantly lower in food rekidneys were removed after bleeding out the animal. Blood stricted compared to fed PHN rats two weeks after induction of samples were collected for determination of albumin and cre- PHN (Table 1). The partial correlation test demonstrated that in atinine. Individual pairs of renal cortices were homogenized in control rats (N = 38) no correlation was found between the 0.25 M sucrose with 5 m Tris-HC1 as previously described [20, urinary activity of NAG, acid /3-galactosidase, acid phospha21]. The homogenates were enriched in tubular material as the tase and urinary volume, while in PHN rats (N = 40) a slight glomeruli were retained on the coarse filter. Samples from each correlation was observed between the urinary activity of acid
Haga et a!: Kidney lysosomes in fasted PHN rats
768
Table 1. The excretion of protein and activity of 4 urinary enzymes in glomerulonephritic rats compared to control, and in fasted glomerulonephritic compared to fed glomerulonephritic rats
Control Activity and
amount
Urinary volume Urinary protein N-acetyl-fl-glucosaminidase Acid 13-galactosidase
Acid phosphatase /3-glucosidase
60.8
40,9
10.0 17.3 15.9
5.3 10.6
74.6 3.2 53.9 35.5
6.8 35.3 1.0
Fasted glomerulonephritic Activity and amount P valueb
Glomerulonephritic Activity and P valuea amount 52.4 341.5 53.8 77.2 368.4 3.4 54.0 24.6 232.3 2.2 230.9
29.9 119.7
24.2 30.3 124.8 1.9
NS P < 0.001 P < 0.01 P < 0.001 P < 0.05 NS NS
11.3
9.1
7.3 16.4
4.2
63.2 37.6 110.1
10.7 76.1
0.8 0.6
—P < 0.01 —P < 0.001 —P < 0.001
P < 0.001 NS NS —P < 0.05 P < 0.01
Serum creatinine 6.6 8.8 43.4 4.7 Serum albumin 2.0 6.3 —P < 0,002 34.6 4.0 190.3 23.2 23.4 P < 0.02 144.9 19.5 —P < 0.001 Total protein P < 0.05 1.6 0.3 0.8 1.3 0.3 —P < 0.05 Kidney weight 217.5 15.5 10.7 NS 180.1 17.9 —P < 0.001 Body weight Excretion of urinary protein is expressed as mg/24 hours, and enzyme activity as nmol/min per 24 hours. Serum albumin is expressed as mg/ml, serum creatinine as mol/liter, total protein in mg/pair of kidney cortices, and kidney (pair) and body weights in grams. All values represent mean of 8 experiments SD. a Significance versus control rats b Significance versus glomerulonephritic rats
body weight was significantly lower than for fed PHN rats at the
time of sacrifice 14 days after induction of PHN. The kidney weights and indeed the amount of kidney cortex protein of fasted/food restricted PHN rats were also significantly lower than for fed PHN and control rats (Table 1). While the amount of DNA in kidney cortex did not increase significantly in fed PHN rats compared to control, food restriction reduced the DNA content from 888.0 sg to 637.3 pg (—P < 0.005) in PHN rats. The lysosomal system
Activities of five lysosomal enzymes in cortical homogenate.
The total activities of all five lysosomal enzymes (but not
Fig. 1. Direct fluorescence staining in cryostat sections from kidney 14 days after induction of glomeru!onephritis. Granular deposits of rabbit anti-Fx1A IgG can be seen in glomeruli.
glucuronidase) increased in PHN rats compared to control two weeks after induction, but only the activities of NAG and acid /3-glycerophosphatase were significantly increased as ps eviously described (Table 2) [101. The total activities of all five enzymes studied were significantly lower in fasted/food restricted compared to fed PHN rats, and the activities of all the enzymes in fasted/food restricted PHN rats were even lower than in control rats (Table 2). The distribution profiles for protein in the classical 'N', 'ML',
'Mic' and 'Sup' fractions confirms our findings in previous
studies [20], and was almost identical in control and fed PHN rats. The recovery of protein was higher in the 'ML' fraction phosphatase and urine volume (r = 0.40; P < 0.05). The urinary from fasted/food restricted PHN rats (33.1%) compared to fed activity of /3-glucosidase did not increase in any of the experi- PHN rats (27.3%), however, with a concomitant lower recovery mental rats compared to control. in the 'N' and 'Sup' fractions. As previously demonstrated [10], Serum albumin was significantly lower in fed PHN rats the recovery of acid hydrolases was significantly higher in the compared to control, reflecting heavy loss of albumin into 'N' fraction in PHN rats compared to control, while the urine, while the concentration of albumin in serum was almost percentage recovery in the other subfractions were not signifiidentical in fasted/food restricted PHN rats compared to control cantly altered for any of these enzymes. (Table 1). The concentration of creatinine in serum was almost Properties of cortical lysosomes. Loss of NAG-latency duridentical in control and PHN rats, while a slight reduction was ing incubation of cortical homogenates from control, fed and noted in FR-PHN rats (Table 1), suggesting that the renal fasted/food restricted PHN rats, was studied as described function was not impaired. The body weight was unaltered in previously [241 in order to compare the properties of lysosomal fed PHN rats versus control, while the kidney weight increased membranes in these various groups of rats. The homogenates 40% in PHN rats (Table 1). All fasted/food restricted PHN rats were preincubated for given lengths of time in isotonic sucrose gained weight during the two week observation period, but the at 37°C pH 7.4, and then immediately assayed for NAG (5 mm
769
Haga et al: Kidney lysosomes in fasted PHN rats
Table 2. Total activities of 5 lysosomal enzymes in homogenates of kidney cortex of glomerulonephritic rats compared to control, and fasted glomerulonephritic rats compared to glomerulonephritic rats Control Total activity N-acetyl-j3-D-glucosaminidase Acid 13-galactosidase Acid /3-glycerophosphatase
Glucronidase Cathepsin D
13.68 2.72 13.29 1.90
0.98
Glomerulonephritic Total P valuea activity
1.17
17.23
1.64
0.29
2.93
0.10
1.57
15.60 1.53 1.52
1.94
0.29 0.29
0.17 0.71
P < 0.001 NS P < 0.01 NS NS
Fasted glomerulonephritic Total P value" activity 1.85
—P < 0.01
13.17 1.79 11.62
0.21
P < 0.001
0.93
0.92
0.11
—P < 0.002 —P < 0.002 —P < 0.05
0.70 0.4
Enzyme activities are expressed as tmol/min/pafr of kidney cortices, while the activity of cathepsn D is expressed as A280/min/pair of kidney cortices. All values represent the mean of 8 experiments SD. a Significance versus control rats b Significance versus glomerulonephritic rats
incubation time) in the presence or absence of 0.00 16 % wt/vol 1,185 gIml. The enzyme profiles of the equilibrium spin from digitonin. The latency of NAG in the homogenates from con- fasted/food restricted PHN rats (Fig. 3C) is almost identical to trol, fed and fasted/food restricted PHN rats was retained for up the enzyme profiles in fed PHN rats (Fig. 3B). to 10 minutes of incubation (a total of 15 mm when including Isopycnic banding of the large granules (fraction 3 1-35 from enzyme assay), followed by a marked drop of latency when the the rate zonal spin) from fed and fasted/food restricted PHN homogenates were incubated for more than 10 minutes exactly rats showed that the studied lysosomal enzymes banded at as described for normal rats [24]. There was, however, no density 1,235 g/ml, which is exactly the same banding density as significant difference between the various experimental groups. found in normal rats [20] and is therefore not shown. Subcellular distribution of endocytosed labelled proteins. In Density gradient centrjfugation.Distribution profiles of protein and lysosomal marker enzymes after rate zonal centrifuga- order to study the tubular uptake and the subcellular distribution of the 'ML' fraction from control (Fig. 2A), PHN rats (Fig. tion of proteins avidly taken up by the tubular cells, 125!.. 2B) and fasted/food restricted PHN rats (Fig. 2C) rats confirm labelled lysozyme and 109Cd-labelled thionein were injected the presence of the three distinct regions previously described (i.v.) into control, fed and fasted/food restricted PHN rats (N =
[20]: a) the peak at fractions 3-4 represents the initial sample position and hence contain soluble protein; b) this is the major protein peak (fraction 12-24) which is composed of mitochondria, brush border membranes, small lysosomes, etc; c) the most rapidly sedimenting material, found in fraction 31-35, contains large lysosomes. As previously demonstrated [20, 21], the distribution of lysosomal enzymes show three essential features. First, each of the acid hydrolases assayed are present in the peak of large lysosomes (fraction 31-35) which is characterized by high purification [20, 21] and high latency of acid hydrolases. The broader band of smaller lysosomes (fractions 12-24) which contains acid P'ase, acid /3-galactosidase and NAG appear to sediment faster than the small lysosomes which contain cathepsin D. Similar distributions were found in the region of small lysosomes of both control, fed and fasted/food
in each group) 30 minutes before sacrifice. The uptake of labelled proteins (Table 3) appears to be slightly increased (although not significantly) in fed and fasted PHN rats com8
pared to the controls. The high recovery of labelled proteins in
the 'ML' fraction (Table 4) indicate the association of the labelled proteins with the endocytic/lysosomal process. Subfractionation of the 'ML' fraction from control (Fig. 2A), fed (Fig. 2B) and fasted PHN rats (Fig. 2C) by rate zonal centrifugation yielded virtually identical distributions of '°9Cd-labelled protein, and identical patterns were achieved for '251-label protein (not shown). The 125! in all the fractions was acid (TCA)
precipitable and thus not due to free or released l25J By isopycnic sedimentation of the small lysosomes (fraction 12-24
in the rate zonal spin), the '°9Cd-labelled protein banded at density 1,200 and 1,235 g!ml in control rats (Fig. 3A), again
restricted PHN rats. The recovery of NAG, acid p'ase and demonstrating the lmCdlabel to be lysosome-associated. In fed cathepsin D in the large lysosomes were higher in the PHN rats
PHN rats the 109Cd-label banded at densities 1,185 and 1,225 g/
compared to control, as previously described [10], while the ml (Fig. 3B), and identical pattern was achieved in fasted/food recovery of acid hydrolases in the large lysosomes from fasted! restricted PHN rats (Fig. 3C). The recovery of labelled proteins food restricted PHN rats (Fig. 2C) was not different from was higher in the 'ML' fraction and lower in the 'Mic' and 'Sup' fractions of fasted/food restricted PHN rats compared to fed control (Fig. 2A). The small lysosome region (fraction 12-24) of the rate zonal PHN rats and control (Table 4).
spin was also pooled and spun to equilibrium in sucrose gradients to identify the banding densities of the small lysosomes [20]. The enzyme profiles from control rats (Fig. 3A)
Discussion
The increase in kidney weight in fed PHN rats as demonstrated in this study is not only caused by osmotic changes normal rat kidney cortex [20]. The enzyme profiles of the causing water retention since total protein is also significantly equilibrium spin from PHN rats (Fig. 3B) demonstrate that all increased. It has been demonstrated that proteinuria induces a confirms our previous findings for lysosomal populations of the
the lysosomal enzymes banded mainly at a density of 1,225 g/ml hypertrophy of the kidney cortex with increase of mitosis in the with a shoulder at density 1,235 g/ml. A significant proportion of epithelium of the convoluted tubules and the glomeruli [26], and
lysosomal enzymes were also recovered at density 1,200 and as cortical hypertrophy is also seen in such varied circum-
770
Haga et a!: Kidney lysosomes in fasted PHN rats A
B 1.30
Cd-thionein
8000
Il IL. J.\
1.20 1.15
4000
10
1.10 1.05
0.
0 0
>.
>
10
20
30
0
40
1.5
0.3
1.0
0.2
0.5
0.1
0.0
0.0
10
20
30
0
40
1.30
Protein A
20
1.25
) 10
.••
1.20 1.15
\ \
20
10
20
30
I' 30
1.05
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2
0
10
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20
30
40
0.3
2.0 1.5
0.2
1.0
0
40
4000
1.10
0.1
0.5
0
Cd-thionein
8000
1.25
10
20
30
40
0.02
0.0
0.0 0
2
10
20
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40
0
Cath.D.
0.06
0.04
0.01
0.02
0
0.00
0
10
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40
0
10
20
30
40
Fraction number
Protein
10
1.30
0.00
0
10
Fraction number
8000
5
1400:
0
0
NAG
1.0
>-
>
10
20
30
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3-galactosidase
0.10
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0
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20
30
40
Acid Pase
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::fL-T1\¼ 10
20
30
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Protein is presented as mg/fraction, acid /3-glycerophosphatase (acid P'ase), acid /3-galactosidase and N-acetyl-p-D-glucosaminidase (NAG) are presented as mol/min/fraction. Cathepsin D units are expressed
0.03 0.02
as A2/min/fraction. The distribution of '°9Cd-label is given as cpm/0.5 ml. B. Distributions of lysosomal marker enzymes, protein and '00Cd-label after rate sedimentation of the kidney cortex 'ML' fraction from glomerulonephritic rats in an HS zonal rotor. C. Dis-
0.5 0.01
tribution of lysosomal marker enzymes, protein and '°9Cd-label after
0.00
0.0
0
10
20
30
40
Fig. 2A. Distribution of lysosomal marker enzymes, protein and '°9Cd-label after rate sedimentation of a rat kidney cortex 'ML' fraction from control rats in an HS zonal rotor. The rotor contained a 550 ml exponential surcose gradient ranging from 0.5 M to 1.7 M, and 150 ml of 2 M as the cushion. After loading the resuspended 'ML' fraction into the rotor it was spun at 8000 rev/mm for 1 hour.
0
10
20
30
40
Fraction number
stances as unilateral nephrectomy and metabolic acidosis [27— 29], it seems to be a nonspecific response to an increased work load. The increased activity of lysosomal enzymes in the kidney cortex of PHN rats probably also reflects increased work load, emphasizing the importance of the lysosomal system in catabolism of endocytosed proteins. The reduced kidney weight and
rate sedimentation of the kidney cortex 'ML' fraction from fasted! food restricted glomerulonephritic rats in an HS zonal rotor.
administered labelled proteins is slightly greater in kidney cortex of fasted/food restricted PHN rats compared to normal
rats (Table 3), and illustrates the high capacity for reabsorption of filtered proteins on the top of proteinuria. It also suggests that this essential tubular function is not impaired in fed and fasted/food restricted PHN rats, even if other reports [30] might total protein as well as low activity of cortical lysosomal suggest the presence of tubular dysfunctions in glomeruloneenzymes in fasted/food restricted PHN rats follows the body phritic rats with proteinuria. We have previously shown [20] weight and reflects the restriction of food in these rats. In spite that labelled Cd-thionein is rapidly degraded after being enof the reduction of renal mass, the recovery of intravenously docytosed, and that this degradation probably starts in the
771
Haga et al: Kidney lysosomes in fasted PHN rats A
B
Protein
10
r 1.30
B
Cct-thionein
800
61
1
H'
D
1.30
A AL
1.25 1.20
H
5
1.15
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L15 5.
Acid 0.15 1 Pace
c
Protein
10
1.25
0 0
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10
20
30
40
0
Acid
0.50
Pace
0.10
10
20
30
40
Acid RNase
0.50
I
0.10 0.25
0.05
0.00
0
0.00
10
20
30
40
0
10
20
30
40
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Fraction number
BC D
Al1 I
5.
>
Acid Pace
0.10
1.30
.
0.00
i L_ Acid RNase
025
10
20
30
0
Fraction number
30
40
0
10
20
30
40
10
20
30
Fig. 3A. Distributions of protein, lysosomal marker enzymes and
'°9Cd-label after isopycnic banding of the small lysosomes from control rats. The pooled region of the small lysosomes (fraction 12-24
in the rate zonal spin) was spun to equilibrium in a B14 zonal rotor (45,000 rev/mm for 16 hr). Positions marked a, b, c and d indicate densities 1.16, 1.185, 1.200 and 1.235 g/ml, respectively. Details of enzyme activities are as given in Fig. 2A. Acid RN'ase units are expressed as zI A2jmin/fraction. B. Distributions of protein, lysosomal marker enzymes and '°9Cd-label after isopycnic banding of the small lysosomes from glomerulonephritic rats. C. Distributions of protein, lysosomal marker enzymes and 109Cd-label after isopycnic banding of
0.00
0
20
Cd-thionein
1500
0.50
0.05
10
Fraction number
C Protein
0.25 0.00
0.00
40
the small lysosomes from fasted/food restricted glomerulonephritic rats.
Table 3. Recovery of injected t09Cd-labelled thionein and endosome compartment of the kidney cortex. It appears, how'251-labelled lysozyme in homogenates of kidney cortex from control, ever, that the degradation of Cd-thionein is somehow slower in glomerulonephritic and fastedJfood restricted glomerulonephritic rats the large and small lysosomes than in the endosome compart30 mm after i.v. injection ment [20]. Fasted In previous studies [20], three distinct populations of iysoControl Glomerulonephritic glomerulonephritic somes in the 'ML' fraction of the normal rat kidney cortex in 30.5 27.3 33.9 addition to the small acid hydrolase containing particles in the '®Cd 17.8 14.2 13.6 'Mic' fraction were demonstrated. The large, rapidly sediment- 1251 The recovery is expressed as percentage of injected amount, and ing lysosomes (Fig. 2) with a 1,235 gIml density are only seen in the proximal tubule [31], and reabsorbed labelled proteins are represents the average of 8 experiments. rapidly incorporated into these lysosomes (Fig. 2). Consistent with the observations made by Straus [32] in proteinuric rats, acute glomerulonephritis with heavy proteinuria have been slowly sedimenting lysosomes in the 'ML' fraction from normal found to increase the recovery of acid hydrolases in the large rats demonstrate the presence of two distinct populations of lysosomes [10]. This is demonstrated by the significantly in- lysosomes: the small 'dense' (density 1,235 g/ml) and small creased recovery of acid hydrolases in the 'N' fraction, which 'light' (density 1,200 g/ml) lysosomes. We have previously probably reflects an increased recovery of large lysosomes in suggested [201 that the small 'dense' lysosomes most probably this fraction, and also by the increased recovery of acid are located in the proximal tubule being precursors to the large hydrolases in the large lysosomes present in the 'ML' fraction lysosomes, while the small 'light' lysosomes may be derived (Fig. 2B). In spite of significant proteinuria, the recovery of acid from both the proximal and distal tubule. Both populations of hydrolases was not increased in the large lysosomes from small lysosomes are involved in the tubular uptake of proteins, fasted/food restricted PHN rats (Fig. 2C). The recovery of acid and the high concentration of acid hydrolases also indicate that hydrolases in the small lysosomal region (fraction 12-24 in the protein degradation takes place in these lysosomes. The bandrate zonal spin) was almost identical in control, fed and fastedl ing density of lysosomal enzymes and '°9Cd-labelled thionein at food restricted PHN rats. Equilibrium banding of the small, 1,225 g/ml and 1,185 g/ml obtained by equilibrium banding of
772
Haga et a!: Kidney lysosomes in fasted PHN rats Table 4. Recovery of '°9Cd-label and '251-label in the classical subfractions of kidney cortex
N
ML
Mic
Sup
Rec
8.0 7.5 12.6
20.9 25.3 31.7
12.4 14.1
58.0
4.6
51.5
97.3 90.0 92.0
16.1
39.8 50.5 54.6
32.3 29.3 28.8
11.8 6.8 4.5
'°9Cd
Control Glomerulonephntic Fasted glomerulonephritic
53.3
1251
Control Glomerulonephritic Fasted glomerulonephritic
13.4 12.1
88.0 80.8
84.9
Recovery (Rec) is presented as t he percentage of the total homogenate value.
the small lysosomes in the 'ML' fraction from fed (Fig. 3B) and that the loss of lysosomal enzymes into urine is not caused by fasted (Fig. 3C) PHN rats is significantly lower than the density tubular damage as the urinary activity of j3-glucosidase, which of small 'dense' and 'light' lysosomes in normal rats. It is is mainly located in the cytosol of the tubular epitelium [37], is possible that due to increased protein uptake, the role of distal not increased. The high urinary activity of lysosomal enzymes tubule lysosomes may become more important in the handling in fed PHN rats may reflect elevated fluid phase endocytosis of an extra protein load, and these lysosomes may have lower due to the increase in protein in filtrate. Fusion of primary density [20]. It has recently been shown that proteinuria actu- lysosomes with endosomes may occur before the endocytic ally changes the pattern and activity of lysosomal enzymes vesicle has completely pinched off from the plasma membrane, along the proximal convoluted and straight tubule [33], suggest- and the enzymes may thus leak out before complete closure of ing a greater role of more distal segments in the handling of an the endosomes. It is also possible that increased membrane extra load of protein. It has also been suggested that a high recycling from the lysosome compartment to the cell membrane concentration of acids in the glomerular filtrate of fasted rats results in high urinary excretion of acid hydrolases, or the could impair the reabsorption of proteins in the proximal urinary enzymes may represent newly synthesized forms which convoluted tubule, resulting in reabsorption of protein in lower have entered the secretory route from the Golgi apparatus. The segments activating the lysosomal enzymes in these segments isoenzyme pattern of NAG in urine from fed PHN rats [11], [34]. The appearance of small 'light' (1,225 g/ml and 1,185 g/ml) however, indicates that this enzyme originates from the large
lysosomes may be attributable to increased protein uptake lysosomes in the kidney cortex. The low activity of urinary increasing the membrane turnover, which may result in second-
lysosomal enzymes in FR-PHN rats is not a manifestation of the
ary lysosomes having a higher membrane content which marked decrease in urinary volume as there is no correlation thereby reduces the overall density. between the urinary activity of these enzymes and urinary Although the lysosomes in rat kidney cortex are considerably volume. The prostatic gland may contribute to the urinary more susceptible to degradation when incubated in isotonic sucrose compared to liver [35], there is no indication that the lysosomes are more vulnerable and sensitive to incubation in fed and fasted/food restricted PHN rats compared to normal rats. The measurable latency of NAG in the kidney cortex homogenate appears to be attributable mainly to the large lysosomes [24], however; therefore this experiment gives no
activity of acid phosphatase making this enzyme less suitable as
valid information about the fragility of the small lysosomes. The mechanical fragility of lysosomes is in general indicated by the recovery of acid hydrolases in the particle-free supernatant. In
cant changes in the proximal tubule, as confirmed by the
marker of lysosomal activity. The low activity of urinary lysosomal enzymes in fasted/food restricted PHN rats may simply reflect the low activity of enzymes in kidney cortex and especially the large lysosomes, or may also reflect modified endocytosis or membrane recycling compared to fed PHN rats. In conclusion, PHN with heavy proteinuria induces signifi-
increased recovery of acid hydrolases in the large lysosomes which are found only in this part of the nephron as previously
the rat kidney cortex, however, the recovery of lysosomal described [101. In spite of significant proteinuria, these changes enzymes in the supernatant is not a good indicator of lysosomal were not observed in fasted/food restricted PHN rats, and the rupture during homogenization [24] due to reabsorption of reduced urinary excretion of acid hydrolases in fasted/food released enzymes to particulate material and the presence of restricted PHN rats may also indicate lower recovery of acid
cytosolic iso-enzymes. The increase in proportion of 1251 and
hydrolases in the large lysosomes, which most probably are the
'°9Cd in the lysosome-rich 'ML' fraction and the relative origin of these urinary enzymes. PHN with proteinuria induces decrease in the supernatant (Table 4) indicates that the lysosomes in fasted/food restricted PHN rats are more resistant to mechanical disruption than observed in normal and fed PHN rats. The difference between 125j and 109Cd in the supernatant (Table 4) is due to larger proportion of '°9Cd found in the cytosol [36].
We have previously shown that urinary NAG is of renal origin even in PHN rats with heavy proteinuria [11]. It appears
significant changes in the small lysosomes in both fed and food
restricted rats, and this may be the result of the induction of small lysosomes in the lower part of the nephron due to the handling of an extra load of protein. The uptake of labelled proteins is slightly increased compared to control rats, and the recovery of labelled proteins in the various lysosomal populations is identical in fed and fasted/food restricted PHN rats. Finally, food restriction reduces the recovery of acid hydro-
Haga et a!: Kidney lysosomes in fasted PHN rats
773
lases in the large lysosomes, which are very vulnerable when incubated, and also reduces the proportion of mechanically fragile lysosomes in the kidney cortex when compared to fed PHN rats. As released acid hydrolases from disrupted lysosomes may cause cell injury and be of pathogenic importance in the development of glomerulonephritis, the effect of food re-
14. HULTBERG B, RAVNSKOV U: The excretion of N-acetyl-/3-glucosaminidase in glomerulonephritis. Clin Nephrol 15, 1:33—38, 1981 15. HULTBERG B, NORDEN N, OCKERMAN PA: Urinary excretion of
development of PHN.
18. MICHAELSEN TE, NATVIG JB: Three new fragments, F(ab)2, F(c)2,
acid hydrolases in disease. Scand J Clin Lab Invest 30:215—219, 1972
16. PRICE RG: Urinary enzymes, nephrotoxicity and renal disease. Toxicology 23:99—134, 1982
BM, MATRE R, OFSTAD J: The heterologous immune striction on the lysosomes in the nephron may modify the 17. IvERSEN complex glomerulonephritis. Acta Pathol Microbiol Immunol pathogenic importance of the renal lysosomal system in the Scand (C) 90:241—250, 1982
and Fab/c, obtained by Papain proteolysis of normal human IgG.
Acknowledgments This work was supported financially by the Norwegian Research Council for Science and the Humanities, to whom we are sincerely grateful. Portions of the results in the tables and figures are reprinted with permission from Renal Physiology.
Scand J Immunol 1:255—268, 1972
19. SAVORY I, Pu PH, SUDERMAN FW JR: A biuret method for determination of protein in normal urine. Clin Chem 14:1160-1171, 1968
20. ANDERSEN K-i, HAGA H-J, DOBROTA M: Lysosomes of the renal
cortex. Heterogeneity and role in protein handling. Kidney mt 31: 886—897, 1987
Reprint requests to Hans-Jacob Haga, Dep. of Rheumatology, University of Bergen, Haukeland Sykehus, N-5016 Norway.
References 1. Yu BP, MASORO EJ, MURATA I, BERTRAND HA, LYND FT: Life span study of SPF Fischer 344 male rats fed ad libitum or restricted
diets: Longevity, growth, lean body mass and disease. J Gerontol 37:130—141, 1982 2. KLAHR 5, BUERKERT J, PURKERSON ML: Role of dietary factors in
the progression of chronic renal disease. Kidney mt 24:579—587, 1983
3. NEUGARTEN J, FEINER HD, SCHACHT RG, BALDWIN DS: Amelio-
ration of experimental glomerulonephritis by dietary protein restnction. Kidney mt 24:595—601, 1983 4. FARR LE, SMADEL JE: The effect of dietary protein on the course of nephrotoxic nephritis in rats. J Exp Med 70:615—627, 1939 5. ICHIKAWA I, PURKERSON ML, KLAHR S, TROY JL, MARTINEZMALDONADO M, BRENNER BM: Mechanism of reduced glomerular filtration rate in chronic malnutrition. J Clin Invest 65:982—988, 1980 6. BRENNER BM, MEYER TW, HOSTETTER TH: The role of hemody-
namically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in aging, renal ablation and intrinsic renal disease. N Eng! J Med 307:652—659, 1982 7. KARLINSKY ML, HAUT L, BUDDINGTON B, SCHRIER NA, ALFREY
AC: Preservation of renal function in experimental glomerulonephritis. Kidney mt 17:293—302, 1980 8. EL NAHAS AM, MASTERS-THOMAS A, BRADY SA, FARRINGTON K, WILKINSON V, HILSON AJW, VARGHESE Z, MOORHEAD JF: Selec-
tive effect of low protein diets in chronic renal diseases. Brit Med J 289:1337—1341, 1984
9. ANDREWS PM, BATES SB: Dietary protein prior to renal ischemia dramatically affects postischemic kidney function. Kidney mt 30: 299—303, 1986 10. HAGA H-J, ANDERSEN K-J, IvERSEN BM, OFSTAD J, DOBROTA M,
MATRE R: Changes in lysosome populations in the rat kidney cortex induced by Passive Heymann Glomerulonephritis. Renal Physiol 10:249—260, 1987
11. HAGA H-J, ANDERSEN K-J: Isoenzymes of N-acetyl-/3-D-glucosa-
minidase in kidney cortex and urine of normal and nephntic rats. Renal Physiol (Vol. 8) 6:348—356, 1985 12. DAVIES M, BARRET AJ, TRAVIS J, SANDERS E, COLES GA: The degradation of human glomerular basement membrane with purified
lysosomal proteinases. Evidence for pathogenetic role of the polymorphonuclear leucocyte in glomerulonephritis. Clin Sci Mo! Med 54:233—240, 1978
13. DAVIES M, HUGHES KT, THOMAS GJ: Evidence that kidney lysosomal proteinases degrade the collagen of glomerular basement membrane. Renal Physiol 3:116—119, 1980
21. ANDERSEN K-J, HAGA H-i, DOBROTA M: Heterogeneity of rat kidney cortex lysosomes fractionated by gradient centrifugation in zonal rotors. Biochem Soc Trans 8:597—598, 1980 22. BRADFORD MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72:248—254, 1976 23. BOER GJ: A simplified microassay of DNA and RNA using Ethidium Bromide. Anal Biochem 65:225—231, 1975 24. HAGA H-i, ANDERSEN K-J, DOBROTA M: Latency of acid hydrolases in rat kidney cortex. IntJBiochem 19:(1l)l097—1l03, 1987 25. REES VH, FILDES JE, LAURENCE D JR: The dye-binding capacity of human plasma determined fluorometrically and its relation to the determination of plasma albumin. J Gun Pathol 7:336—340, 1954 26. GREGOIRE F: Kidney enzyme changes in experimental proteinuria Lab Invest 25:626—634, 1971 27. ADDIS T, LEW W: The restoration of lost organ tissue. The rate and degree of restoration. J Exp Med 71:325—333, 1940 28. LOTSPEICH WD: Renal hypertrophy in metabolic acidosis and its relation to ammonia excretion. Am J Physiol 208:1135—1142, 1965 29. N0wINSKI WW, PicioN A: The Krebs cycle in glomeruli of normal
rat kidney and in compensatory hypertrophy. J Histochem Cytochem 15:32—37, 1967 30. ZAMLAUSKI-TUCKER Mi, VAN LIEw JB, NOBLE B: Pathophysiol-
ogy of the kidney in rats with Heymann nephritis. Kidney mt 28: 504—512, 1985
31. STRAUS W: Occurence of phagosomes and phago-lysosomes in different segments of the nephron in relations to the reabsorption, transport, digestion, and extrusion of intravenously injected horseradish peroxidase. J Cell Biol 21:295—308, 1964
32. STRAUS W: Changes in droplet' fractions from rat kidney cells after intraperitoneal injection of egg white. J Biophys Biochem Cytol. (Vol. 3) 6:933—947, 1957 33. OLBRICHT Ci, CANNON JK, GARG LC, TISHER CC: Activities of
cathepsins B and L in isolated nephron segments from proteinuric and nonproteinuric rats. Am J Physiol 250:F1055—F1062, 1986 34. LE HIR M, DUBACH UC, GUDER WG: Distribution of acid hydrolases in the nephron of normal and diabetic rats. Int J Biochem 12: 41—45, 1980
35. RUTH RC, WEGLICKI WB: Mechanisms of loss of latency of lysosomal enzymes. Effects of incubation on the properties of lysosomal membranes. Biochem J 186:243—256, 1980 36. DOBROTA M, BONNER FW, CARTER BA: Intracellular redistribution of cadmium-thionein following its uptake into the renal cortex,
in Nephrotoxicity: Assessment and Pathogenesis, edited by BACH PH, BONNER FW, BRIDGES JW, LOCK EA, Chichester, Wiley, 1981, pp. 320—324.
37. PRICE RG, DANCE N, ROBINSON D: A comparision of the /3glucosidase excretion during kidney damage induced by 4-nitrophenyl-arsonic acid and by rabbit anti-rat kidney antibodies. Ear J Clin Invest 2:47—51, 1971
Effect of fasting on lysosomes in kidney cortex of glomerulonephritic rats HANS-JACOB HAGA, KNUT-JAN ANDERSEN, BJARNE M. IVERSEN, JARLE OFSTAD, MILOSLAV DOBROTA, and ROALD MATRE Medical Department A, University of Bergen, Haukeland Sykehus, N-5016, Bergen, Norway; Robens Institute of Industrial and Environmental Health and Safety, University of Surrey, Guildford, United Kingdom; and Broegelman Research Laboratory for Microbiology and Departement of Microbiology, The Gade Institute, University of Bergen, Norway
Effect of fasting on lysosomes in kidney cortex of glomerulonephrltlc rats. The effect of food restriction (FR) on the kidney cortex lysosomes prepared by rate and isopycnic zonal centrifugation was studied in rats with passive Heymann glomerulonephritis (PHN). FR reduced the renal
progression of functional deterioration mainly in patients with tubulointerstitial disease, and that diet has only a marginally beneficial effect in patients with chronic glomerulonephritis [8].
mass by 41%, but the capacity for handling of labelled endocytosed proteins by the lysosomes was not different from fed PHN rats. While
ischemic induced tubular necrosis has also been demonstrated
The dramatic protective effect of dietary protein prior to
PHN with heavy proteinuria increased the recovery of lysosomal in rat models [9]. enzymes in the large lysosomes located in the proximal tubule, no We have previously demonstrated significant effects on the changes were observed in FR-PHN rats in spite of significant proteinuria. The density of the small lysosomes was significantly shifted! lysosomes in the rat kidney cortical tubules induced by acute reduced (from 1,200 and 1,235 g/mI to 1,185 and 1,225 g/ml, respectively) in both fed and FR-PHN rats, suggesting that the handling of extra loads of protein may enhance the absorbtive function of small
lysosomes found in the lower part of the nephron. FR reduced the mechanical fragility of lysosomes in the kidney cortex of PHN-rats. The highly increased urinary excretion of lysosomal enzymes in fed PHN
rats was not observed in FR-PHN rats. As a conclusion, FR reduces both the fragility of lysosomes and the proportion of digestive enzymes in fragile lysosomes. These lysosomal enzymes may be of pathogenic importance in PHN causing cell damage when liberated from disrupted lysosomes.
passive Heymann glomerulonephritis (PHN) [10, 11]. PHN induced a significant increase in the cortical activity of lysosomal enzymes mainly in the large lysosomes or 'protein droplets' in the proximal tubule [10]; PHN also induced significant changes in various other populations of small lysosomes found in the cortical nephron, probably reflecting an increased reabsorption of protein caused by proteinuria. Lysosomal en-
zymes are considered to be of pathogenic importance in the development of glomerulonephritis [12, 13], and increase in
urinary lysosomal enzyme activity has been widely accepted as indicative of kidney disease or kidney damage [14—16]. We have previously demonstrated increased urinary activity of several Food restriction, which involves either low protein diet or lysosomal enzymes in rats with acute passive Heymann glomeraccess to food on alternate days, has been shown to extend the ulonephritis (PHN) [10, 11]. Since food restriction reduces the life span of normal rats [1], and to retard the progression of urinary excretion of protein in glomerulonephritic rats [3—6], the nephntis in rat models and man [2—4]. It has been postulated favorable effect of food restriction on the development of
that the effect of food restriction may be caused by altered
glomerular flow reducing the progression of glomeruloscierosis [5, 6], or may be directly attributable to restriction of protein [3, 4] or dietary phosphorous [7]. However, the exact mechanisms by which food restriction influences the course of nephritis are still unknown. Most of the studies concerning the protective effect of foodl protein restriction have been devoted to hemodynamic changes
glomerulonephritis may be due to lysosomal changes in the nephron because of a reduced protein load. The particular aim
of this study was therefore to examine the effect of food restriction on the lysosomes in the kidney cortical tubules of PHN rats having a high urinary excretion of protein. Methods
Preparation of acute Heymann glomerulonephritis
in the glomeruli resulting in reduced urinary excretion of
Antiserum to Fx1A was raised in rabbits as previously
protein and retarded progression of glomerulosclerosis, but a few recent studies have demonstrated that protein restriction described [17]. Rabbit IgG was prepared/purified by ion-exmay also have significant effects on the tubules. It has, for change chromatography on DEAE- cellulose (Whatman DE-52) instance, been demonstrated that protein restriction retards the equilibrated with 0.015 M phosphate buffer pH 7.6, followed by a gel filtration on a Sephadex G-200 Column equilibrated with 0.015 M phosphate buffered saline pH 7.2 (PBS) [18]. IgG prepared from normal rabbits served as control. Male Wistar rats (weight 130 to 170 g) used in the experiments
Received for publication January 28, 1988 and in revised form July 25, 1988
were fed on Brood Stock Feed R3. This diet is composed of
© 1988 by the International Society of Nephrology
21.0% crude protein, 19.0% pepsin digested protein, 6% crude 766
Haga et a!: Kidney lysosomes in fasted PHN rats
767
fat, 52.0% carbohydrates, 1.2% calcium, 1.0%. phosphorous, of the eight homogenates thus obtained were kept for further 0.09% sodium and 0.7% potassium. Forty-two percent of the assays of enzyme activities and protein before the homogenates total fatty acids consisted of linoleic acid (C18:2) and 8.2% of were pooled and further separated into the nuclear 'N', mitolinolenic acid (C18:3). Feed consumption was 10 to 15 glrat/day chondrial-lysosomal 'ML', microsomal 'Mic' and cytosolic and metabolizable energy was 3.10 Meal/kg. In this experiment supernatant 'Sup' fractions by differential pelleting [20, 21]. we studied three groups of rats with eight rats in each group: The 'ML' fraction was further subfractionated by rate and control rats, passive Heymann glomerulonephritic (PHN) rats isopycnic centrifugation techniques as previously described in and food restricted/fasted (FR) PHN rats. PHN was induced by detail by Andersen et al [20]. a single intravenous injection of 7.3 mg IgG with an anti-Fx1A titer of 1:2048. Control rats received 7.3 mg normal rabbit IgG Chemical and enzyme assay in homogenates and subcellular without detectable antibodies against Fx1A. Antibodies against fractions glomerular basement membrane were not detectable in either of Protein (mg per ml) was measured by the protein dye method the IgG preparations [171. described by Bradford [22] using bovine serum albumin as a The effect of food restriction was studied by feeding PHN standard. DNA was measured as described by Boer [23] and rats every alternate day from induction of glomerulonephritis to was expressed as g. The lysosomal enzymes, N-acetyl-/3-Dsacrifice 14 days later, and all rats were allowed to drink tap glucosaminidase (NAG), acid /3-galactosidase, acid /3-glycerowater ad libitum during the days without food. The glomerulo- phosphatase, acid RNA'ase and cathepsin D were assayed as nephritic model was not found to cause infiltration of white previously described [20]. To minimize lysosomal autolysis blood cells in the renal parenchyma [17]. during incubation, latency measurements were performed for only 10 minutes at 37°C. Latency measurements were perImmunofluorescence technique formed as previously described [24]. Cathepsin D units are Kidney specimens from rats in the experimental groups were expressed as A280/min/fraction and acid ribonuclease frozen in liquid nitrogen. Sections 4 m thick were cut on a (RNA'ase) as zi A2/min/fraction. The other enzyme activities cryostat and placed on microscopic slides. Air dried sections are expressed as pmol/min/fraction. were incubated for 34 minutes in a humid atmosphere with Labelling of '251-lysozyme and '°9Cd-thionein was performed twofold dilutions of FITC-labelled swine anti-rabbit IgG. The as previously described [20]. The labelled lysozyme (18 Ci per sections were then washed in PBS for 30 minutes, mounted in animal) and labelled Cd-thionein (5 pCi per animal) were glycerol-PBS and examined in a Leitz Orthoplan microscope injected intravenously into each rat (N = 8) 30 minutes before with a HBO-200 mercury lamp. sacrifice in two different experiments. Fluorescine-isothiocyanate (FITC) labelled swine IgG was Serum albumin was estimated fluorometrically using rat purchased from DAKO Immunoglobulin A/S, Copenhagen, albumin as standard [25]. Creatinine in serum was measured by Denmark. an automated version of the picrate method [17], and expressed as tmol/liter. Collection and preparation of urine Urine was collected on ice at the height of proteinuria 14 days Statistical methods following injection of IgG [17]. The rats were housed in metaStudents t-test for non-paired and paired observations was bolic cages where they were without admission to food but had used for statistical evaluation. All P values were considered free access to tap water during the 24-hour collection period. significant when smaller than 0.05. All urine were centrifuged (2000 rev/mm for 5 mm) and diafiltered in an Amicon UF 12 cell (Amicon Corp., Danvers, Results Massachusetts, USA) at 50 psi using an Um-lO filter [11] to Glomerulonephritis remove enzyme inhibitors of low molecular weight. Glomerulonephritis was confirmed by direct fluorescence Chemical and enzyme assay in urine staining of cryosections from kidneys (Fig. 1) 14 days after Urinary protein was measured by the biuret method [19] induction of passive Heymann glomerulonephritis (PHN). Food using bovine serum albumin as standard. The acid hydrolases restriction had no effect on the fluoroscine staining in glomeruli N-acetyl-3-D-glucosaminidase, acid-3-galactosidase, acid phos- of PHN rats. No fluorescence was observed in kidneys of phatase and /3-glucosidase were assayed by a fluorometric assay control rats. As previously described [101 PHN induced highly [10], and enzyme activities were expressed as nmol/min per 24 increased urinary excretion of protein and urinary activity of all hours. three lysosomal enzymes studied (NAG, acid 3-galactosidase and acid phosphatase; Table 1) 2 weeks after induction. The Tissue homogenization and fractionation urinary excretion of protein was significantly lower in FR-PHN Male Wistar rats were starved 24 hours before sacrifice at day rats compared to fed PHN rats, and the urinary activity of NAG 14 after injection of IgG, and anesthetized with ether before the and acid /3-galactosidase was significantly lower in food rekidneys were removed after bleeding out the animal. Blood stricted compared to fed PHN rats two weeks after induction of samples were collected for determination of albumin and cre- PHN (Table 1). The partial correlation test demonstrated that in atinine. Individual pairs of renal cortices were homogenized in control rats (N = 38) no correlation was found between the 0.25 M sucrose with 5 m Tris-HC1 as previously described [20, urinary activity of NAG, acid /3-galactosidase, acid phospha21]. The homogenates were enriched in tubular material as the tase and urinary volume, while in PHN rats (N = 40) a slight glomeruli were retained on the coarse filter. Samples from each correlation was observed between the urinary activity of acid
Haga et a!: Kidney lysosomes in fasted PHN rats
768
Table 1. The excretion of protein and activity of 4 urinary enzymes in glomerulonephritic rats compared to control, and in fasted glomerulonephritic compared to fed glomerulonephritic rats
Control Activity and
amount
Urinary volume Urinary protein N-acetyl-fl-glucosaminidase Acid 13-galactosidase
Acid phosphatase /3-glucosidase
60.8
40,9
10.0 17.3 15.9
5.3 10.6
74.6 3.2 53.9 35.5
6.8 35.3 1.0
Fasted glomerulonephritic Activity and amount P valueb
Glomerulonephritic Activity and P valuea amount 52.4 341.5 53.8 77.2 368.4 3.4 54.0 24.6 232.3 2.2 230.9
29.9 119.7
24.2 30.3 124.8 1.9
NS P < 0.001 P < 0.01 P < 0.001 P < 0.05 NS NS
11.3
9.1
7.3 16.4
4.2
63.2 37.6 110.1
10.7 76.1
0.8 0.6
—P < 0.01 —P < 0.001 —P < 0.001
P < 0.001 NS NS —P < 0.05 P < 0.01
Serum creatinine 6.6 8.8 43.4 4.7 Serum albumin 2.0 6.3 —P < 0,002 34.6 4.0 190.3 23.2 23.4 P < 0.02 144.9 19.5 —P < 0.001 Total protein P < 0.05 1.6 0.3 0.8 1.3 0.3 —P < 0.05 Kidney weight 217.5 15.5 10.7 NS 180.1 17.9 —P < 0.001 Body weight Excretion of urinary protein is expressed as mg/24 hours, and enzyme activity as nmol/min per 24 hours. Serum albumin is expressed as mg/ml, serum creatinine as mol/liter, total protein in mg/pair of kidney cortices, and kidney (pair) and body weights in grams. All values represent mean of 8 experiments SD. a Significance versus control rats b Significance versus glomerulonephritic rats
body weight was significantly lower than for fed PHN rats at the
time of sacrifice 14 days after induction of PHN. The kidney weights and indeed the amount of kidney cortex protein of fasted/food restricted PHN rats were also significantly lower than for fed PHN and control rats (Table 1). While the amount of DNA in kidney cortex did not increase significantly in fed PHN rats compared to control, food restriction reduced the DNA content from 888.0 sg to 637.3 pg (—P < 0.005) in PHN rats. The lysosomal system
Activities of five lysosomal enzymes in cortical homogenate.
The total activities of all five lysosomal enzymes (but not
Fig. 1. Direct fluorescence staining in cryostat sections from kidney 14 days after induction of glomeru!onephritis. Granular deposits of rabbit anti-Fx1A IgG can be seen in glomeruli.
glucuronidase) increased in PHN rats compared to control two weeks after induction, but only the activities of NAG and acid /3-glycerophosphatase were significantly increased as ps eviously described (Table 2) [101. The total activities of all five enzymes studied were significantly lower in fasted/food restricted compared to fed PHN rats, and the activities of all the enzymes in fasted/food restricted PHN rats were even lower than in control rats (Table 2). The distribution profiles for protein in the classical 'N', 'ML',
'Mic' and 'Sup' fractions confirms our findings in previous
studies [20], and was almost identical in control and fed PHN rats. The recovery of protein was higher in the 'ML' fraction phosphatase and urine volume (r = 0.40; P < 0.05). The urinary from fasted/food restricted PHN rats (33.1%) compared to fed activity of /3-glucosidase did not increase in any of the experi- PHN rats (27.3%), however, with a concomitant lower recovery mental rats compared to control. in the 'N' and 'Sup' fractions. As previously demonstrated [10], Serum albumin was significantly lower in fed PHN rats the recovery of acid hydrolases was significantly higher in the compared to control, reflecting heavy loss of albumin into 'N' fraction in PHN rats compared to control, while the urine, while the concentration of albumin in serum was almost percentage recovery in the other subfractions were not signifiidentical in fasted/food restricted PHN rats compared to control cantly altered for any of these enzymes. (Table 1). The concentration of creatinine in serum was almost Properties of cortical lysosomes. Loss of NAG-latency duridentical in control and PHN rats, while a slight reduction was ing incubation of cortical homogenates from control, fed and noted in FR-PHN rats (Table 1), suggesting that the renal fasted/food restricted PHN rats, was studied as described function was not impaired. The body weight was unaltered in previously [241 in order to compare the properties of lysosomal fed PHN rats versus control, while the kidney weight increased membranes in these various groups of rats. The homogenates 40% in PHN rats (Table 1). All fasted/food restricted PHN rats were preincubated for given lengths of time in isotonic sucrose gained weight during the two week observation period, but the at 37°C pH 7.4, and then immediately assayed for NAG (5 mm
769
Haga et al: Kidney lysosomes in fasted PHN rats
Table 2. Total activities of 5 lysosomal enzymes in homogenates of kidney cortex of glomerulonephritic rats compared to control, and fasted glomerulonephritic rats compared to glomerulonephritic rats Control Total activity N-acetyl-j3-D-glucosaminidase Acid 13-galactosidase Acid /3-glycerophosphatase
Glucronidase Cathepsin D
13.68 2.72 13.29 1.90
0.98
Glomerulonephritic Total P valuea activity
1.17
17.23
1.64
0.29
2.93
0.10
1.57
15.60 1.53 1.52
1.94
0.29 0.29
0.17 0.71
P < 0.001 NS P < 0.01 NS NS
Fasted glomerulonephritic Total P value" activity 1.85
—P < 0.01
13.17 1.79 11.62
0.21
P < 0.001
0.93
0.92
0.11
—P < 0.002 —P < 0.002 —P < 0.05
0.70 0.4
Enzyme activities are expressed as tmol/min/pafr of kidney cortices, while the activity of cathepsn D is expressed as A280/min/pair of kidney cortices. All values represent the mean of 8 experiments SD. a Significance versus control rats b Significance versus glomerulonephritic rats
incubation time) in the presence or absence of 0.00 16 % wt/vol 1,185 gIml. The enzyme profiles of the equilibrium spin from digitonin. The latency of NAG in the homogenates from con- fasted/food restricted PHN rats (Fig. 3C) is almost identical to trol, fed and fasted/food restricted PHN rats was retained for up the enzyme profiles in fed PHN rats (Fig. 3B). to 10 minutes of incubation (a total of 15 mm when including Isopycnic banding of the large granules (fraction 3 1-35 from enzyme assay), followed by a marked drop of latency when the the rate zonal spin) from fed and fasted/food restricted PHN homogenates were incubated for more than 10 minutes exactly rats showed that the studied lysosomal enzymes banded at as described for normal rats [24]. There was, however, no density 1,235 g/ml, which is exactly the same banding density as significant difference between the various experimental groups. found in normal rats [20] and is therefore not shown. Subcellular distribution of endocytosed labelled proteins. In Density gradient centrjfugation.Distribution profiles of protein and lysosomal marker enzymes after rate zonal centrifuga- order to study the tubular uptake and the subcellular distribution of the 'ML' fraction from control (Fig. 2A), PHN rats (Fig. tion of proteins avidly taken up by the tubular cells, 125!.. 2B) and fasted/food restricted PHN rats (Fig. 2C) rats confirm labelled lysozyme and 109Cd-labelled thionein were injected the presence of the three distinct regions previously described (i.v.) into control, fed and fasted/food restricted PHN rats (N =
[20]: a) the peak at fractions 3-4 represents the initial sample position and hence contain soluble protein; b) this is the major protein peak (fraction 12-24) which is composed of mitochondria, brush border membranes, small lysosomes, etc; c) the most rapidly sedimenting material, found in fraction 31-35, contains large lysosomes. As previously demonstrated [20, 21], the distribution of lysosomal enzymes show three essential features. First, each of the acid hydrolases assayed are present in the peak of large lysosomes (fraction 31-35) which is characterized by high purification [20, 21] and high latency of acid hydrolases. The broader band of smaller lysosomes (fractions 12-24) which contains acid P'ase, acid /3-galactosidase and NAG appear to sediment faster than the small lysosomes which contain cathepsin D. Similar distributions were found in the region of small lysosomes of both control, fed and fasted/food
in each group) 30 minutes before sacrifice. The uptake of labelled proteins (Table 3) appears to be slightly increased (although not significantly) in fed and fasted PHN rats com8
pared to the controls. The high recovery of labelled proteins in
the 'ML' fraction (Table 4) indicate the association of the labelled proteins with the endocytic/lysosomal process. Subfractionation of the 'ML' fraction from control (Fig. 2A), fed (Fig. 2B) and fasted PHN rats (Fig. 2C) by rate zonal centrifugation yielded virtually identical distributions of '°9Cd-labelled protein, and identical patterns were achieved for '251-label protein (not shown). The 125! in all the fractions was acid (TCA)
precipitable and thus not due to free or released l25J By isopycnic sedimentation of the small lysosomes (fraction 12-24
in the rate zonal spin), the '°9Cd-labelled protein banded at density 1,200 and 1,235 g!ml in control rats (Fig. 3A), again
restricted PHN rats. The recovery of NAG, acid p'ase and demonstrating the lmCdlabel to be lysosome-associated. In fed cathepsin D in the large lysosomes were higher in the PHN rats
PHN rats the 109Cd-label banded at densities 1,185 and 1,225 g/
compared to control, as previously described [10], while the ml (Fig. 3B), and identical pattern was achieved in fasted/food recovery of acid hydrolases in the large lysosomes from fasted! restricted PHN rats (Fig. 3C). The recovery of labelled proteins food restricted PHN rats (Fig. 2C) was not different from was higher in the 'ML' fraction and lower in the 'Mic' and 'Sup' fractions of fasted/food restricted PHN rats compared to fed control (Fig. 2A). The small lysosome region (fraction 12-24) of the rate zonal PHN rats and control (Table 4).
spin was also pooled and spun to equilibrium in sucrose gradients to identify the banding densities of the small lysosomes [20]. The enzyme profiles from control rats (Fig. 3A)
Discussion
The increase in kidney weight in fed PHN rats as demonstrated in this study is not only caused by osmotic changes normal rat kidney cortex [20]. The enzyme profiles of the causing water retention since total protein is also significantly equilibrium spin from PHN rats (Fig. 3B) demonstrate that all increased. It has been demonstrated that proteinuria induces a confirms our previous findings for lysosomal populations of the
the lysosomal enzymes banded mainly at a density of 1,225 g/ml hypertrophy of the kidney cortex with increase of mitosis in the with a shoulder at density 1,235 g/ml. A significant proportion of epithelium of the convoluted tubules and the glomeruli [26], and
lysosomal enzymes were also recovered at density 1,200 and as cortical hypertrophy is also seen in such varied circum-
770
Haga et a!: Kidney lysosomes in fasted PHN rats A
B 1.30
Cd-thionein
8000
Il IL. J.\
1.20 1.15
4000
10
1.10 1.05
0.
0 0
>.
>
10
20
30
0
40
1.5
0.3
1.0
0.2
0.5
0.1
0.0
0.0
10
20
30
0
40
1.30
Protein A
20
1.25
) 10
.••
1.20 1.15
\ \
20
10
20
30
I' 30
1.05
40
2
0
10
20
30
40
10
20
30
40
20
30
40
0.3
2.0 1.5
0.2
1.0
0
40
4000
1.10
0.1
0.5
0
Cd-thionein
8000
1.25
10
20
30
40
0.02
0.0
0.0 0
2
10
20
30
40
0
Cath.D.
0.06
0.04
0.01
0.02
0
0.00
0
10
20
30
40
0
10
20
30
40
Fraction number
Protein
10
1.30
0.00
0
10
Fraction number
8000
5
1400:
0
0
NAG
1.0
>-
>
10
20
30
40
3-galactosidase
0.10
0.5
0.0
0
10
20
30
40
Acid Pase
1.0
::fL-T1\¼ 10
20
30
40
Protein is presented as mg/fraction, acid /3-glycerophosphatase (acid P'ase), acid /3-galactosidase and N-acetyl-p-D-glucosaminidase (NAG) are presented as mol/min/fraction. Cathepsin D units are expressed
0.03 0.02
as A2/min/fraction. The distribution of '°9Cd-label is given as cpm/0.5 ml. B. Distributions of lysosomal marker enzymes, protein and '00Cd-label after rate sedimentation of the kidney cortex 'ML' fraction from glomerulonephritic rats in an HS zonal rotor. C. Dis-
0.5 0.01
tribution of lysosomal marker enzymes, protein and '°9Cd-label after
0.00
0.0
0
10
20
30
40
Fig. 2A. Distribution of lysosomal marker enzymes, protein and '°9Cd-label after rate sedimentation of a rat kidney cortex 'ML' fraction from control rats in an HS zonal rotor. The rotor contained a 550 ml exponential surcose gradient ranging from 0.5 M to 1.7 M, and 150 ml of 2 M as the cushion. After loading the resuspended 'ML' fraction into the rotor it was spun at 8000 rev/mm for 1 hour.
0
10
20
30
40
Fraction number
stances as unilateral nephrectomy and metabolic acidosis [27— 29], it seems to be a nonspecific response to an increased work load. The increased activity of lysosomal enzymes in the kidney cortex of PHN rats probably also reflects increased work load, emphasizing the importance of the lysosomal system in catabolism of endocytosed proteins. The reduced kidney weight and
rate sedimentation of the kidney cortex 'ML' fraction from fasted! food restricted glomerulonephritic rats in an HS zonal rotor.
administered labelled proteins is slightly greater in kidney cortex of fasted/food restricted PHN rats compared to normal
rats (Table 3), and illustrates the high capacity for reabsorption of filtered proteins on the top of proteinuria. It also suggests that this essential tubular function is not impaired in fed and fasted/food restricted PHN rats, even if other reports [30] might total protein as well as low activity of cortical lysosomal suggest the presence of tubular dysfunctions in glomeruloneenzymes in fasted/food restricted PHN rats follows the body phritic rats with proteinuria. We have previously shown [20] weight and reflects the restriction of food in these rats. In spite that labelled Cd-thionein is rapidly degraded after being enof the reduction of renal mass, the recovery of intravenously docytosed, and that this degradation probably starts in the
771
Haga et al: Kidney lysosomes in fasted PHN rats A
B
Protein
10
r 1.30
B
Cct-thionein
800
61
1
H'
D
1.30
A AL
1.25 1.20
H
5
1.15
Fib
L15 5.
Acid 0.15 1 Pace
c
Protein
10
1.25
0 0
>
10
20
30
40
0
Acid
0.50
Pace
0.10
10
20
30
40
Acid RNase
0.50
I
0.10 0.25
0.05
0.00
0
0.00
10
20
30
40
0
10
20
30
40
0
Fraction number
BC D
Al1 I
5.
>
Acid Pace
0.10
1.30
.
0.00
i L_ Acid RNase
025
10
20
30
0
Fraction number
30
40
0
10
20
30
40
10
20
30
Fig. 3A. Distributions of protein, lysosomal marker enzymes and
'°9Cd-label after isopycnic banding of the small lysosomes from control rats. The pooled region of the small lysosomes (fraction 12-24
in the rate zonal spin) was spun to equilibrium in a B14 zonal rotor (45,000 rev/mm for 16 hr). Positions marked a, b, c and d indicate densities 1.16, 1.185, 1.200 and 1.235 g/ml, respectively. Details of enzyme activities are as given in Fig. 2A. Acid RN'ase units are expressed as zI A2jmin/fraction. B. Distributions of protein, lysosomal marker enzymes and '°9Cd-label after isopycnic banding of the small lysosomes from glomerulonephritic rats. C. Distributions of protein, lysosomal marker enzymes and 109Cd-label after isopycnic banding of
0.00
0
20
Cd-thionein
1500
0.50
0.05
10
Fraction number
C Protein
0.25 0.00
0.00
40
the small lysosomes from fasted/food restricted glomerulonephritic rats.
Table 3. Recovery of injected t09Cd-labelled thionein and endosome compartment of the kidney cortex. It appears, how'251-labelled lysozyme in homogenates of kidney cortex from control, ever, that the degradation of Cd-thionein is somehow slower in glomerulonephritic and fastedJfood restricted glomerulonephritic rats the large and small lysosomes than in the endosome compart30 mm after i.v. injection ment [20]. Fasted In previous studies [20], three distinct populations of iysoControl Glomerulonephritic glomerulonephritic somes in the 'ML' fraction of the normal rat kidney cortex in 30.5 27.3 33.9 addition to the small acid hydrolase containing particles in the '®Cd 17.8 14.2 13.6 'Mic' fraction were demonstrated. The large, rapidly sediment- 1251 The recovery is expressed as percentage of injected amount, and ing lysosomes (Fig. 2) with a 1,235 gIml density are only seen in the proximal tubule [31], and reabsorbed labelled proteins are represents the average of 8 experiments. rapidly incorporated into these lysosomes (Fig. 2). Consistent with the observations made by Straus [32] in proteinuric rats, acute glomerulonephritis with heavy proteinuria have been slowly sedimenting lysosomes in the 'ML' fraction from normal found to increase the recovery of acid hydrolases in the large rats demonstrate the presence of two distinct populations of lysosomes [10]. This is demonstrated by the significantly in- lysosomes: the small 'dense' (density 1,235 g/ml) and small creased recovery of acid hydrolases in the 'N' fraction, which 'light' (density 1,200 g/ml) lysosomes. We have previously probably reflects an increased recovery of large lysosomes in suggested [201 that the small 'dense' lysosomes most probably this fraction, and also by the increased recovery of acid are located in the proximal tubule being precursors to the large hydrolases in the large lysosomes present in the 'ML' fraction lysosomes, while the small 'light' lysosomes may be derived (Fig. 2B). In spite of significant proteinuria, the recovery of acid from both the proximal and distal tubule. Both populations of hydrolases was not increased in the large lysosomes from small lysosomes are involved in the tubular uptake of proteins, fasted/food restricted PHN rats (Fig. 2C). The recovery of acid and the high concentration of acid hydrolases also indicate that hydrolases in the small lysosomal region (fraction 12-24 in the protein degradation takes place in these lysosomes. The bandrate zonal spin) was almost identical in control, fed and fastedl ing density of lysosomal enzymes and '°9Cd-labelled thionein at food restricted PHN rats. Equilibrium banding of the small, 1,225 g/ml and 1,185 g/ml obtained by equilibrium banding of
772
Haga et a!: Kidney lysosomes in fasted PHN rats Table 4. Recovery of '°9Cd-label and '251-label in the classical subfractions of kidney cortex
N
ML
Mic
Sup
Rec
8.0 7.5 12.6
20.9 25.3 31.7
12.4 14.1
58.0
4.6
51.5
97.3 90.0 92.0
16.1
39.8 50.5 54.6
32.3 29.3 28.8
11.8 6.8 4.5
'°9Cd
Control Glomerulonephntic Fasted glomerulonephritic
53.3
1251
Control Glomerulonephritic Fasted glomerulonephritic
13.4 12.1
88.0 80.8
84.9
Recovery (Rec) is presented as t he percentage of the total homogenate value.
the small lysosomes in the 'ML' fraction from fed (Fig. 3B) and that the loss of lysosomal enzymes into urine is not caused by fasted (Fig. 3C) PHN rats is significantly lower than the density tubular damage as the urinary activity of j3-glucosidase, which of small 'dense' and 'light' lysosomes in normal rats. It is is mainly located in the cytosol of the tubular epitelium [37], is possible that due to increased protein uptake, the role of distal not increased. The high urinary activity of lysosomal enzymes tubule lysosomes may become more important in the handling in fed PHN rats may reflect elevated fluid phase endocytosis of an extra protein load, and these lysosomes may have lower due to the increase in protein in filtrate. Fusion of primary density [20]. It has recently been shown that proteinuria actu- lysosomes with endosomes may occur before the endocytic ally changes the pattern and activity of lysosomal enzymes vesicle has completely pinched off from the plasma membrane, along the proximal convoluted and straight tubule [33], suggest- and the enzymes may thus leak out before complete closure of ing a greater role of more distal segments in the handling of an the endosomes. It is also possible that increased membrane extra load of protein. It has also been suggested that a high recycling from the lysosome compartment to the cell membrane concentration of acids in the glomerular filtrate of fasted rats results in high urinary excretion of acid hydrolases, or the could impair the reabsorption of proteins in the proximal urinary enzymes may represent newly synthesized forms which convoluted tubule, resulting in reabsorption of protein in lower have entered the secretory route from the Golgi apparatus. The segments activating the lysosomal enzymes in these segments isoenzyme pattern of NAG in urine from fed PHN rats [11], [34]. The appearance of small 'light' (1,225 g/ml and 1,185 g/ml) however, indicates that this enzyme originates from the large
lysosomes may be attributable to increased protein uptake lysosomes in the kidney cortex. The low activity of urinary increasing the membrane turnover, which may result in second-
lysosomal enzymes in FR-PHN rats is not a manifestation of the
ary lysosomes having a higher membrane content which marked decrease in urinary volume as there is no correlation thereby reduces the overall density. between the urinary activity of these enzymes and urinary Although the lysosomes in rat kidney cortex are considerably volume. The prostatic gland may contribute to the urinary more susceptible to degradation when incubated in isotonic sucrose compared to liver [35], there is no indication that the lysosomes are more vulnerable and sensitive to incubation in fed and fasted/food restricted PHN rats compared to normal rats. The measurable latency of NAG in the kidney cortex homogenate appears to be attributable mainly to the large lysosomes [24], however; therefore this experiment gives no
activity of acid phosphatase making this enzyme less suitable as
valid information about the fragility of the small lysosomes. The mechanical fragility of lysosomes is in general indicated by the recovery of acid hydrolases in the particle-free supernatant. In
cant changes in the proximal tubule, as confirmed by the
marker of lysosomal activity. The low activity of urinary lysosomal enzymes in fasted/food restricted PHN rats may simply reflect the low activity of enzymes in kidney cortex and especially the large lysosomes, or may also reflect modified endocytosis or membrane recycling compared to fed PHN rats. In conclusion, PHN with heavy proteinuria induces signifi-
increased recovery of acid hydrolases in the large lysosomes which are found only in this part of the nephron as previously
the rat kidney cortex, however, the recovery of lysosomal described [101. In spite of significant proteinuria, these changes enzymes in the supernatant is not a good indicator of lysosomal were not observed in fasted/food restricted PHN rats, and the rupture during homogenization [24] due to reabsorption of reduced urinary excretion of acid hydrolases in fasted/food released enzymes to particulate material and the presence of restricted PHN rats may also indicate lower recovery of acid
cytosolic iso-enzymes. The increase in proportion of 1251 and
hydrolases in the large lysosomes, which most probably are the
'°9Cd in the lysosome-rich 'ML' fraction and the relative origin of these urinary enzymes. PHN with proteinuria induces decrease in the supernatant (Table 4) indicates that the lysosomes in fasted/food restricted PHN rats are more resistant to mechanical disruption than observed in normal and fed PHN rats. The difference between 125j and 109Cd in the supernatant (Table 4) is due to larger proportion of '°9Cd found in the cytosol [36].
We have previously shown that urinary NAG is of renal origin even in PHN rats with heavy proteinuria [11]. It appears
significant changes in the small lysosomes in both fed and food
restricted rats, and this may be the result of the induction of small lysosomes in the lower part of the nephron due to the handling of an extra load of protein. The uptake of labelled proteins is slightly increased compared to control rats, and the recovery of labelled proteins in the various lysosomal populations is identical in fed and fasted/food restricted PHN rats. Finally, food restriction reduces the recovery of acid hydro-
Haga et a!: Kidney lysosomes in fasted PHN rats
773
lases in the large lysosomes, which are very vulnerable when incubated, and also reduces the proportion of mechanically fragile lysosomes in the kidney cortex when compared to fed PHN rats. As released acid hydrolases from disrupted lysosomes may cause cell injury and be of pathogenic importance in the development of glomerulonephritis, the effect of food re-
14. HULTBERG B, RAVNSKOV U: The excretion of N-acetyl-/3-glucosaminidase in glomerulonephritis. Clin Nephrol 15, 1:33—38, 1981 15. HULTBERG B, NORDEN N, OCKERMAN PA: Urinary excretion of
development of PHN.
18. MICHAELSEN TE, NATVIG JB: Three new fragments, F(ab)2, F(c)2,
acid hydrolases in disease. Scand J Clin Lab Invest 30:215—219, 1972
16. PRICE RG: Urinary enzymes, nephrotoxicity and renal disease. Toxicology 23:99—134, 1982
BM, MATRE R, OFSTAD J: The heterologous immune striction on the lysosomes in the nephron may modify the 17. IvERSEN complex glomerulonephritis. Acta Pathol Microbiol Immunol pathogenic importance of the renal lysosomal system in the Scand (C) 90:241—250, 1982
and Fab/c, obtained by Papain proteolysis of normal human IgG.
Acknowledgments This work was supported financially by the Norwegian Research Council for Science and the Humanities, to whom we are sincerely grateful. Portions of the results in the tables and figures are reprinted with permission from Renal Physiology.
Scand J Immunol 1:255—268, 1972
19. SAVORY I, Pu PH, SUDERMAN FW JR: A biuret method for determination of protein in normal urine. Clin Chem 14:1160-1171, 1968
20. ANDERSEN K-i, HAGA H-J, DOBROTA M: Lysosomes of the renal
cortex. Heterogeneity and role in protein handling. Kidney mt 31: 886—897, 1987
Reprint requests to Hans-Jacob Haga, Dep. of Rheumatology, University of Bergen, Haukeland Sykehus, N-5016 Norway.
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