1Endocrine Unit, Austin & Repatriation Medical Centre, Department of Medicine, University of Melbourne, Heidelberg 2Department of Biochemistry and Molecular Biology, Monash University, Clayton 3Department of Nephrology and Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia
Correspondence and offprint requests to: Dr Wayne D. Comper, Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia. Email: wayne.comper{at}med.monash.edu.au
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Abstract |
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Methods. Glomerular permselectivity was measured using tritium-labelled polydisperse Ficoll. In vivo fractional clearance (FC) of albumin, transferrin and immunoglobulin G was measured to include both intact and degraded forms of filtered material. Endogenous protein clearance was analysed using two-dimensional electrophoresis in combination with matrix-assisted laser desorption ionization (MALDI) mass spectrometry.
Results. FCs of proteins and Ficoll in control NAR were similar to those found in SpragueDawley rats. Despite the lack of serum albumin in NAR, proteinuria and morphological changes observed were also similar to those found in SpragueDawley rats, with total protein excretion increasing 6-fold in PAN rats and 4-fold in anti-GBM GN rats with respect to controls. Two-dimensional electrophoresis in combination with MALDI mass spectrometry identified the major proteins being excreted as transferrin and a group of mildly acidic proteins in the MW range 4050 kDa, namely antithrombin III, kininogen, -1-antiproteinase, haemopexin and vitamin D-binding protein.
Conclusions. Both diseases exhibited similar effects to those observed in SpragueDawley rats despite the lack of serum albumin, including inhibition of renal protein degradation. The net changes in protein FC, particularly in the range of radii of 3655 Å, could not be accounted for by changes in size selectivity as Ficoll FC was little affected by the disease states. This emphasizes the need to reassess the relative importance of changes in renal tubular handling vs changes in glomerular capillary barrier in proteinuric states. These studies also demonstrate that albumin is not a critical factor in governing glomerular permselectivity or proteinuria.
Keywords: analbuminaemia; anti-GBM; glomerular permselectivity; glomerulonephritis; PAN; protein clearance
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Introduction |
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The present study investigates the renal processing of proteins by utilizing an environment that is free from albumin. The Nagase analbuminaemic rat (NAR) is a derivative of the SpragueDawley rat that does not produce endogenous serum albumin as a result of abnormal albumin pre-mRNA splicing [4]. The total plasma protein concentration in the NAR is normal, since other individual protein levels in the serum, such as 1-antitrypsin,
2-macroglobulin,
-X protein, transferrin and the immunoglobulins, are elevated [5]. Despite the absence of endogenous serum albumin, proteinuria in NAR has been observed in aging rats [6], after treatment with adriamycin [7] and in rats made nephrotic with Heymann nephritis antigen [8]. Some conflicting studies have been reported, namely Okuda et al. [9] and Abe et al. [10], where only a small increase in protein excretion is observed.
One of the major findings in SpragueDawley rats is that filtered proteins are considerably biochemically modified by lysosomal proteases before excretion, resulting in excretion of <10% intact protein and >90% protein-derived degradation products and not just intact unmodified protein [11]. It has been shown that this process is inhibited in SpragueDawley rats induced with GN.
This study examined the renal processing of serum proteins in terms of renal degradation and permeability in an albumin-deficient environment using NAR. This is also examined in NAR induced with models of proteinuria, namely puromycin aminonucleoside nephrosis (PAN) and anti-glomerular basement membrane GN (anti-GBM GN). PAN and anti-GBM GN are known to produce massive proteinuria in normal SpragueDawley rats [11]. Specifically, we examine the fractional clearance (FC) of carbon-14 labelled albumin, immunoglobulin G (IgG) and transferrin in control NAR as compared to NAR with PAN or anti-GBM GN. As the radioimmunoassay (RIA) method has been shown to detect only intact protein [11], radiolabelled proteins were used in addition to the RIA, as a comparative method of determining FC as radiolabelling allows the detection of both intact and fragmented protein. The FC of Ficolls of equivalent radius to albumin (36 Å), transferrin (48 Å) and IgG (55 Å) were also measured to account for any changes in the permeability properties of the glomerular capillary wall (GCW).
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Subjects and methods |
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Experimental design
Normal or diseased rats were constantly infused with radiolabelled proteins (albumin, transferrin or IgG) or polydisperse Ficoll for a 7 day period via mini-osmotic pumps. Blood and 24 h urine collections were made on day 7. Animals were killed 7 days after pump implantation and the radioactive material in blood, urine and one kidney examined to determine the FC of individual molecules. Ten- to 12-week-old NAR were obtained from the Biological Research Laboratory, Austin & Repatriation Medical Centre (Heidelberg, Victoria, Australia). Throughout the experimental period they were maintained as pairs, housed in rat boxes under a 12 h day/night cycle with free access to standard rat chow and water. Permission to perform all animal experiments was given by the Monash University Animal Ethics Committee.
Induction of PAN
Induction of PAN was performed on 20 rats as previously described [11]. Briefly, rats were immobilized by towel wrapping and an injection of 15 mg/100 g body weight of puromycin aminonucleoside made up as a 3.5% solution in phosphate-buffered saline (PBS) was administered via the tail vein. Age- and weight-matched controls were injected with an equivalent amount of saline. Osmotic pumps were implanted on day 2 after puromycin aminonucleoside administration and the animals were killed 7 days later. The kidneys were removed with one kidney being processed for disease pathology and the other kidney analysed for radioactivity.
Induction of anti-GBM GN
Passive accelerated anti-GBM GN was induced in 14 rats as previously described [12]. Briefly, the rats were immunized subcutaneously with 5 mg normal sheep IgG in Freund's complete adjuvant and 7 days later were injected intravenously with 5 ml/kg sheep anti-rat GBM serum. Osmotic pumps were implanted 3 h before anti-GBM serum administration. Animals were killed 7 days after anti-GBM serum injection, with the kidneys removed. One kidney was processed for disease pathology and the other was analysed for radioactivity. Controls were age- and weight-matched.
Microscopical analysis
Tissues for pathological assessment were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections (2 µm) were stained with haematoxylin and eosin or periodic acidSchiff (PAS) reagents.
Radiolabelling of macromolecules
RSA, transferrin and IgG were labelled with 14C using a reductive methylation technique modified from Eng [13] using [14C]formaldehyde. The specific activities achieved for [14C]RSA, [14C]transferrin and [14C]IgG were 2.39 x 106, 1.67 x 105 and 3.41 x 107 disintegrations per min (d.p.m.)/mg, respectively.
Polydisperse Ficoll 70 was tritiated according to van Damme et al. [14]. The specific activity of polydisperse Ficoll 70 was 2.2 x 107 d.p.m./mg.
All radiolabelled material was checked for integrity by size exclusion chromatography prior to administration.
In vivo FC studies of radiolabelled proteins and polydisperse Ficoll 70 using the osmotic pump method
Alzet osmotic pumps were filled with radiolabelled material ([14C]albumin and [3H]Ficoll, or [14C]transferrin or [14C]IgG) along with 0.02% w/v of thimerosal to inhibit bacterial growth. The Alzet osmotic pumps Model 2001 have a mean filling volume of 221 ± 8 µl, pumping rate of 0.95 ± 0.05 µl/h, length 3 cm, diameter 0.7 cm and empty weight 1.1 g. Once filled, the pumps were incubated for 1 h at 37°C. The concentrations of radioactivity initially in the pump were [14C]RSA 6.38 x 107 d.p.m./ml, [14C]transferrin
5.20 x 108 d.p.m./ml, [14C]IgG
3.41 x 107 d.p.m./ml and [3H]Ficoll
2.54 x 108 d.p.m./ml.
The rats were anaesthetized by inhalation with Forthane, an inhalant isofluorane anaesthetic, and the osmotic pumps implanted subcutaneously between the scapulae using sterile technique. The rats were implanted with one pump filled with [14C]albumin, [14C]transferrin or [14C]IgG and the albumin group were implanted with an additional pump filled with [3H]Ficoll. The rats were then maintained as pairs in rat boxes with free access to food and water at all times, being placed in metabolic cages on days 5 and 7 for 24 h urine collections. On day 7, the animals were culled by decapitation, a plasma sample taken and both kidneys removed for subsequent analysis. Both blood and urine samples were centrifuged for 10 min at 3000 r.p.m. in a KS-5200C Kubota bench top centrifuge (Kubota Corp., Tokyo, Japan) and then analysed for radioactivity. Glomerular filtration rate (GFR) was determined using the creatinine assay and urine flow rate (UFR) calculated from the volume of urine collected in a 24 h period.
Analysis of kidneys for radioactivity
One of the kidneys from each rat was weighed, minced and made up to 3 ml with 1.4 M NaOH in centrifuge tubes. The tubes were covered loosely and suspended in boiling water for 1530 min to allow complete digestion. Four sample aliquots of 100 µl each were taken, then 50 µl hydrogen peroxide was added to decolourize the samples and the volume made up to 1 ml with 850 µl water. Four millilitres of scintillation fluid was added to the samples and they were rested in the dark overnight to reduce chemiluminescence. The samples were counted for radioactivity and the presence of the tracer in the kidneys, determined as d.p.m./g of tissue.
Column chromatography
Plasma and urine samples were analysed using a Sephadex G-100 column (column dimensions: 1.7 x 60 cm2) or a Sephacryl S-300 column (column dimensions: 2.1 x 65 cm2). The columns were run at 4°C with a flow rate of 20 ml/h in PBS containing 2 mg/ml BSA and 0.02% sodium azide, pH 7.4. The columns were calibrated using blue dextran and tritiated water to determine the void and total volume, respectively. Ninety-five fractions of 1.71 ml (G-100) or 100 fractions of
1.66 ml (S-300) were collected with recoveries between 95 and 105% obtained. Kav was determined with the formula:
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IgG RIA
The RIA utilized [125I]-labelled rat serum IgG, prepared according to the chloramine-T method [15] using 10 µl of a 1 mg/ml IgG standard, rabbit antiserum (polyclonal) to rat IgG, sheep anti-rabbit antibodies and non-immune rabbit serum. The detection limit of the assay was 31.2 ng/ml. The standard curve was prepared using a rat serum IgG standard (1 mg/ml) that was diluted to give a range of 400031.2 ng/ml.
Transferrin RIA
The RIA utilized [125I]-labelled rat serum transferrin, prepared according to the chloramine-T method [15] using 10 µl of a 1 mg/ml transferrin standard, rabbit antiserum (polyclonal) to rat transferrin, sheep anti-rabbit antibodies and non-immune rabbit serum. The detection limit of the assay was 6 ng/ml. The standard curve was prepared using a rat serum IgG standard (1 mg/ml) that was diluted to give a range of 16006 ng/ml.
Total protein assay
Total urinary protein was determined using the biuret assay.
Identification of major urine proteins
Five of the major proteins in the NAR proteinuric urine were identified using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (performed by the Australian Proteome Analysis Facility, Sydney, Australia). Urinary protein was separated using two-dimensional electrophoresis (first dimension pH 410 isoelectric focusing for 35^ 000 Vh, second dimension on an 818%T criterion format polyacrylamide slab gel) and visualized using a SYPRO Ruby fluorescent stain. A plasma sample from a control animal was also analysed and used as a reference. The key proteins present in the proteinuric urine sample were selected to undergo in gel tryptic digestion and subsequent MALDI peptide analysis.
Counting of radioactivity
Tritium and [14C] radioactivity was determined using a 1 : 3 sample to Optiphase scintillation fluid ratio and recorded on a Wallac 1409 liquid scintillation counter (Wallac, Finland).
Statistical analysis
All data are expressed as means ± SD, with n representing the number of determinations. Significance of results was determined using the Student's t-test. A P-value of <0.05 was considered to be statistically significant.
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Results |
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Characteristics of protein excretion
Protein excretion in control animals did not differ significantly from pre-experimental values (75.9 ± 13.0 and 79.8 ± 18.8 mg/24 h, respectively). Proteinuria was evident in both disease states after 7 days (609.8 ± 140.4 mg/24 h in PAN and 371.0 ± 149.7 mg/24 h in anti-GBM GN).
The nature of the excreted protein from NAR was examined by two-dimensional electrophoresis and MALDI mass spectrometry. Figure 2a and b are two-dimensional electrophoresis profiles of plasma and urine samples from a NAR. The urine was collected from a NAR with PAN. The plasma is from a control NAR and is provided as a reference. Transferrin can be seen in both the plasma and urine at approximately pH 7.0. Five major proteins present in the urine (marked on Figure 2b) were analysed by mass spectrometry and identified as antithrombin III (48.9 kDa, pI 5.93, 26.1% protein sequence coverage), kininogen (45.7 kDa, pI 5.90, 17.2%), -1-antiproteinase (42.7 kDa, pI 5.60, 52.5%), haemopexin (48.9 kDa, pI 7.33, 28.4%) and vitamin D-binding protein (51.8 kDa, pI 5.41, 37.0%). MW and pI values are based on database information for the identifications made. The percentage value refers to the percentage of the total protein sequence covered by the peptides.
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Integrity of excreted protein
Size-exclusion chromatography of the urine (Figure 3) showed that all tracers were excreted as degraded fragments in control NAR. The profiles seen with NAR depict typical renal processing of filtered proteins seen in control SpragueDawley rats [11]. The radiolabelled material remains intact in the plasma (data not shown), so the fragmentation of the tracers can be attributed to degradation by the kidney. We have previously demonstrated that degradation is not the result of enzymes in the urine or the result of extrarenal degradation before filtration [11]. Further, small fragments seen in the plasma at 7 days have very low clearance due to binding to plasma proteins [16] and would not be expected to contribute significantly to their appearance in urine.
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Protein FC
Table 2 shows the concentration of radiolabelled material in the kidneys on day 7 of the osmotic pump studies. For all of the tracers there is no more than a 23-fold increase in radioactivity from the plasma to the kidneys, which is representative of the normal flux through the kidney rather than accumulation of the tracer. In both control and diseased states the amount of tracer appearing in the urine was <1% of the infusion rate. For both transferrin and IgG, the amount of labelled protein being infused into the plasma was negligible when compared with the plasma protein concentration.
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Discussion |
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The similarities of SpragueDawley rats [11] and NAR suggest that, irrespective of the nature of filtered protein, the same underlying mechanisms of filtration and cellular processing of filtered protein are involved. No significant increase in Ficoll clearance was observed in proteinuric NAR, although it cannot be ruled out that relatively minor changes in glomerular permselectivity occur. The moderate increases in FC of proteins and protein-derived material as measured by radioactivity in GN were observed in both NARs and SpragueDawley rats. Charge selectivity was demonstrated to be negligible in SpragueDawley rats [11] and evidence for the charge selectivity concept has now been seriously questioned [17]. In terms of cellular processing, extensive fragmentation has been identified for filtered and excreted albumin, transferrin and IgG in both control SpragueDawley rats and NAR. Both strains exhibit significant inhibition of the fragmentation process in GN. These results demonstrate that the intrinsic cellular machinery for processing proteins distal to the GBM is essentially the same in both strains.
Previous studies have suggested that albumin may influence the permselectivity of the GCW. The lack of albumin in NAR did not influence size selectivity as there was no significant difference in the FC of Ficoll in the NAR compared with SpragueDawley rats [11]. Exogenous radioactive albumin FC, as measured by RIA, in NAR was 7.7 ± 6.1 x 105, which is considerably higher than that measured in SpragueDawley rats (4.4 ± 2.6 x 106). However, when all the albumin-derived material in urine is taken into account, the FC in NAR was lower (3.8 ± 4.3 x 104) than that in SpragueDawley rats (1.7 ± 0.4 x 103). Previous efforts to determine glomerular permeability of proteins in NAR have employed SDSPAGE [18], which will only detect intact protein. Clearly, assays that only detect intact protein cannot yield true FC of proteins as they fail to detect fragments.
Emori et al. [5] have shown that to compensate for the lack of albumin in the plasma, other serum proteins are elevated. This group of plasma proteins constitutes a major fraction of the major proteins excreted in both PAN and GN. As we have demonstrated in this study, these proteins consist of transferrin and a group of relatively small, slightly acidic proteins with a molecular weight range of 4050 kDa. The average FC of these proteins is 0.001. Studies on similar macromolecules, such as neutral and acidic horseradish peroxidase, yield FCs in the range of 0.010.07 [17]. This would suggest that filtered proteins in NAR are processed in a special manner. We have previously provided evidence that in SpragueDawley rats, filtered albumin is retrieved by cells distal to the GBM and returned to the blood supply intact [19]. Similar mechanisms may be operative for filtered plasma proteins in NAR.
Transferrin has been thought to play a role in tubular toxicity in diabetic patients [20] and may have been the cause of tubulointerstitial injury observed in NAR in the absence of albumin. In addition, the other proteins identified in the proteinuric urine may have contributed to the nephrotoxic effect.
Studies of the renal handling of proteins in NAR offer an interesting comparison to renal handling in SpragueDawley rats and in humans. The major conclusions of this study are that GN is manifested in a very similar fashion in both NAR and SpragueDawley rats, despite the marked difference in plasma protein composition. GCW permeability also seems to be essentially independent of plasma protein composition. Post-glomerular tissue injury was observed in NAR, thus demonstrating that albumin is not crucial for glomerular permselectivity or eliciting tubular damage. The renal degradation pathway that operates on filtered proteins prior to excretion was evident in both rat species and was inhibited in renal disease. There is also evidence that, as for SpragueDawley rats where most of filtered albumin is retrieved, it is likely that a special mechanism for renal processing of the bulk of filtered plasma proteins in NAR may be operative.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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