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Handling of low-density lipoprotein by the renal tubule: Release of fragments due to incomplete degradation
Handling of low-density lipoprotein by the renal tubule: Release of fragments due to incomplete degradation ALFREDO A. PEGORARO, KRISHNAMURTHY P. GUDEHITHLU, ERNEST CABRERA, RAVI SHANKAR, JOSE A. L. ARRUDA, GEORGE DUNEA, and ASHOK K. SINGH CHICAGO and MAYWOOD, ILLINOIS
Because the mechanism by which lipoproteins are processed and modified in the renal tubule in patients with nephrosis is not completely understood, we studied the handling of low-density lipoprotein (LDL) in perfused rat kidneys made permeable by protamine. Protamine pretreatment increased the clearance of 125I LDL 25-fold compared to controls, thereby simulating a proteinuric kidney. Similar studies were also conducted in kidneys of rats made proteinuric by the induction of passive Heymann nephritis. Of the perfused iodinated LDL, 5% was localized in the cortex and lesser amounts in the medulla and urine. In the cortex and medulla, iodinated LDL was present mainly in the intact form (90%); just 10% was present in the degraded form. Using horseradish peroxidase conjugated to LDL, we demonstrated specific staining in the proximal tubules, suggesting that specific LDL receptors were present in that location. Although LDL in the tissue was present mostly in the intact form, it was 95% degraded in urine, and the degradation was inhibited by chloroquine, indicating that the lysosomes were the site of LDL metabolism. Gel chromatography and electrophoresis of iodinated LDL in the urine showed the presence of fragments in the range of 5 to 15 kD. We conclude that renal degradation of LDL is incomplete and that the incompletely degraded fragments released into the urine may be toxic to the kidney by virtue of their lipid side-chains. (J Lab Clin Med 2002; 139:372-8) Abbreviations: EDTA ⫽ ethylenediaminetetraacetate; GFR ⫽ glomerular filtration rate; HRP ⫽ horseradish peroxidase; LDL ⫽ low-density lipoprotein; PBS ⫽ phosphate-buffered saline solution; PHN ⫽ passive Heymann nephritis; SDS-PAGE ⫽ sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SEM ⫽ standard error of the mean; TCA ⫽ trichloroacetic acid
From the Division of Nephrology, Cook County Hospital; the Hektoen Institute for Medical Research; the Department of Surgery, Loyola–Veterans Affairs Hines Medical Center; and the Section of Nephrology, University of Illinois at Chicago and Veterans Affairs Medical Center, West Side Division. Supported by the National Kidney Foundation of Illinois. Submitted for publication May 16, 2001; revision submitted February 3, 2002; accepted February 21, 2002. Reprint requests: Ashok K Singh, PhD, Hektoen Institute for Medical Research, 627 South Wood Street, Suite 201, Chicago, IL 60612; e-mail: singashok@aol. Copyright © 2002 by Mosby, Inc. All rights reserved. 0022-2143/2002 $35.00 ⫹ 0 5/1/124201 doi:10.1067/mlc.2002.124201
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he handling of filtered proteins by the renal tubules is not fully understood. Small proteins such as lysozyme are almost completely degraded during their passage through the tubule, but other proteins are excreted intact or degraded only partially.1-5 Far less is known about the fate of complex proteins such as lipoproteins, which are large and contain lipid moieties in their molecules. We chose to study the handling of LDL by the rat kidney because it is one of the common lipoproteins and is present in rat blood and urine.6 Receptors for LDL in the kidneys have been recognized,7 but their role in the handling of
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LDL is unclear. Such understanding may be important because large amounts of urinary lipoproteins are present in nephrotic states and may play a role in tubular damage and interstitial fibrosis, possibly by releasing their lipid moieties. To this end, we first studied the handling of LDL in ex vivo isolated rat kidneys made proteinuric by prior perfusion with protamine, a polycation that increases glomerular permeability to proteins. Similar studies were then conducted in rat kidneys made proteinuric by the induction of PHN. We labeled LDL with iodine 125 so that we might follow its fate after a single-pass perfusion through the kidneys. MATERIALS Preparation of LDL. LDL was prepared from human serum by means of differential centrifugation in accordance with a standard technique.8 In brief, the density of the serum was corrected to 1.019 with NaCl, after which the serum was centrifuged at 29,000 rpm for 16 hours at 10°C. The top layer, consisting of very low density lipoprotein, was removed. The remaining serum was separated from the pellet. Its density was adjusted to 1.063 by the addition of NaCl, after which it was centrifuged at 40,000 rpm for 48 hours at 10°C. The upper yellow layer, consisting of LDL, was recovered and subjected to dialysis against 20 vol of 0.9% NaC1 (pH 8.5) containing 0.02% EDTA. The presence of EDTA was essential to prevent oxidation of LDL. We used the thiobarbuturic acid reactive species assay to determine the content of oxidized LDL in an attempt to confirm that the LDL remained in the unoxidized state after the isolation procedure.9 Protein concentration and cholesterol content were determined with the use of the Bradford method (Bio-Rad Laboratories, Hercules, Calif) and a kit (Sigma Chemical, St Louis, Mo), respectively, in accordance with the manufacturers’ recommended methodologies. Radiolabeling of LDL. We radiolabeled LDL with sodium iodide 125I in accordance with the iodine monochloride method.10 This method was chosen because it is based on an exchange reaction and does not include an oxidation step, thereby avoiding the formation of oxidized LDL. One milligram of LDL was dissolved in 600 L water and placed on ice. One millicurie of sodium iodide 125I (ICN Biomedicals, Irvine, Calif) in 10 L solution was added, followed by 50 L of 250 g/mL solution of iodine monochloride in water. After 60 seconds, another 50 L of iodine monochloride solution was added, and the reaction was terminated at the end of the second minute by the addition of l mL saline solution–EDTA (0.2%). The reaction mixture was subjected to dialyis against saline solution–EDTA at 4°C. The purity and specific activity of the label were tested by means of TCA precipitation analysis and gamma-counting. Covalent conjugation of LDL with HRP for staining of kidney sections. One milligram of HRP (Sigma) was dis-
solved in 100 L PBS adjusted to pH 6.8. Reactive aldehyde groups were generated on HRP with 100 mmol/L sodium metaperiodate (Sigma) oxidation at room temperature for 1
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hour.11 After dialysis against normal saline solution at 4°C, 300 g of LDL was added, and the mixture was incubated at 4°C for 72 hours for conjugation by way of Schiff bonding. Note that the aldehyde groups were generated by the oxidation of the HRP molecule and that the excess oxidant was removed before LDL was added. In this manner, the LDL was prevented from becoming oxidized LDL. Paraffin-embedded sections from normal rat kidneys were layered with the conjugate; this was followed by washing and development with diaminobenzidine-H2O2 solution. The specificity of the label was confirmed by inhibition of the reaction in the presence of excess unlabeled LDL. The slides were examined under light microscopy. Ex vivo perfusion of kidneys with iodinated LDL. SpragueDawley rats weighing 250 to 300 g (Harlan, Indianapolis, Ind) were anesthetized with an intraperitoneal injection of 50 mg/kg sodium pentobarbital and prepared for ex vivo perfusion of isolated kidneys.12 A longitudinal midabdominal incision was made to expose both ureters, which were then cannulated with PE-10 tubing (Intramedic; Clay Adams, Sparks, Md). We isolated the renal vascular bed by ligating the mesenteric vessels and the aorta proximal to the renal arteries. A cannula was placed at the descending aorta below the renal artery and secured with a ligature. An incision was made in the inferior vena cava to permit outflow. The kidneys were initially perfused with PBS (pH 7.4) at 37°C in an attempt to flush blood. The kidneys (n ⫽ 6 in the protamine group, n ⫽ 4 in the PHN group) were then perfused with 10 mL PBS containing 5 g/mL protamine sulfate (Eli Lilly, Indianapolis, Ind) to cause the glomeruli to become permeable. After excess protamine was washed away with PBS, kidneys were perfused with 20 mL PBS containing 125Ilabeled LDL (10-15 ⫻ 106 cpm) at the rate of 2 mL/min, followed by a wash with 20 mL PBS. During perfusion with 125 I-labeled LDL, urine was collected from both ureters and saved. At the end of the experiment, both kidneys were removed, the cortex and medulla separated and their wet weights determined. In a separate group of animals (n ⫽ 3), the kidneys were first perfused with protamine and then perfused with tritiated inulin (ICN, Irvine, CA) for determination of GFR. Similarly, GFR was determined in a separate group of PHN rat kidneys (n ⫽ 3) by means of ex vivo perfusion with [3H]inulin. In another group of animals (n ⫽ 4), the effect of chloroquine was tested. Chloroquine (10 mol/L; Sigma) was added to the protamine solution for perfusion. Tissue processing. We homogenized the cortex and medulla in ice-cold PBS with a polytron homogenizer. The homogenates were centrifuged at 3000 rpm to separate the tissue pellet. Tissue pellets were centrifuged twice with icecold PBS to remove the nonspecifically bound iodinated LDL for further processing. An aliquot of the iodinated LDL containing perfusate, cortex, medulla, and urine was precipitated with 10% TCA and centrifuged at 3000 rpm to separate the TCA-soluble (supernatant containing degraded iodinated LDL) and insoluble (pellet containing intact iodinated LDL) fractions. Both fractions were analyzed in a gamma-counter,
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and the counts were normalized to the final volume of each sample. Receptor-bound iodinated LDL in the tissues was determined on the basis of competitive release as follows: The cortex and medulla homogenates were centrifuged at 3000 rpm. The pellet was suspended in PBS containing excess unlabeled LDL (1 mg/mL). The mixture was vortexed and centrifuged, after which we counted the supernatant to quantify iodinated LDL. The percentage of counts released from the tissue represented the receptor-bound iodinated LDL. To detect fragments of iodinated LDL (3-15 kD) in the urine, we fractionated the TCA-soluble portion in a G-25 Sephadex (Pharmacia, Piscataway, NJ) column. Gamma counts in the fractions immediately after void corresponding to the macromolecular region were considered to represent fragments of iodinated LDL. These data were presented as a percentage of the counts in urine. Induction of PHN. Sprague-Dawley rats weighing 150 to 200 g were injected intraperitoneally with 10 mg (IgG) of rabbit anti-gp600 antibody in 0.5 mL saline solution.13 In a parallel set of control rats, we injected a similar amount of IgG from normal rabbit serum. The animals were tested for proteinuria at day 7 by means of sulfosalicylic acid precipitation.
Table I. Fractional clearance of 125l-labeled LDL in ex vivo–perfused kidneys pretreated with protamine Rat
1 2 3 4 5 6 Mean ⫾ SEM
Fractional clearance of 125l-labeled LDL (%)
0.347 0.577 0.927 0.331 0.541 0.282 0.501 ⫾ 0.098
Molecular exclusion gel chromatography and electrophoresis of rat urine containing filtered iodinated LDL. Gel
chromatography was performed with Biogel P-60 matrix (exclusion limit ⫽ 60 kD, fractionation range ⫽ 3-60 kD; Biorad Laboratories, Richmond, Calif). Standards of purified human serum albumin (66 kD), ovalbumin (45 kD), and lysozyme (14 kD) were used to calibrate the column. The column (1 ⫻ 30 cm) was equilibriated and run in PBS at a flow rate of 5 mL/hour. One-milliliter fractions were collected. SDS-PAGE of the urine samples containing iodinated LDL was performed in accordance with the Laemmli system and the gel subjected to autoradiography in keeping with to standard technique.14 Statistical analyses. Results are presented as mean ⫾ SEM. Unpaired or paired Student’s t test was applied to determine significance of experimental changes with P ⬍ 0.05 considered significant. RESULTS Clearance of LDL. We found the fractional clearance of LDL in normal rat kidneys to be extremely low (0.02% ⫾ 0.012%, n ⫽ 3), consistent with a normally functioning size-selective glomerular basement. The low clearance of LDL in normal kidneys precluded further studies on the fate of the filtered LDL because of the extremely small concentrations present in the urine. This problem was overcome by a brief treatment of the kidney with protamine to increase the clearance of high-molecular-weight molecules. The treatment protocol we used increased the clearance of LDL to 25 times that than untreated kidneys, allowing us to further study the tubular handling of LDL (Table I). We compared the GFR measured on the basis of inulin clear-
Fig 1. Binding of iodinated LDL to kidney tissue after a single pass of ex vivo perfusion. Data presented as mean ⫾ SEM; n ⫽ 6. *P ⬍ 0.05 vs cortex. Iodinated LDL was bound mainly to the cortex, suggesting that it is the main site of LDL uptake and processing.
ance in ex vivo control kidneys and kidneys perfused with protamine. The clearance of inulin was similar in both groups (mean ⫽ 1.04 mL/min/kidney in controls vs 1.2 mL/min/kidney in protamine-perfused group, n ⫽3 ) indicating that protamine treatment did not alter GFR while increasing the permeability to LDL. In addition, the GFR of the ex vivo perfused kidneys was similar to that of intact animals, showing that the ex vivo–perfused kidney preparation was stable during the short experimental period. Distribution of iodinated LDL in the kidney after ex vivo perfusion. We studied the distribution of iodinated LDL
in the kidney after ex vivo perfusion in an effort to determine the region where it would likely be processed. After a single pass of iodinated LDL followed by a 5-minute wash, 5% of the perfused LDL was present in the cortex, with lesser amounts in the medulla and urine (Fig 1) The amount of iodinated LDL in the cortex was three or four times higher than that present in the medulla (Fig 1). TCA precipitation analyis revealed that in both cortex and medulla, the iodinated LDL was mostly (90%) present in the intact
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form, with the remaining 10% in the degraded form (data not shown). Evidence for receptor–mediated binding of iodinated LDL in the kidney. We present two pieces of evidence to
demonstrate the presence of the LDL receptor in the kidney. When homogenates of cortex and medulla containing the bound 125I-labeled LDL were treated with unlabeled LDL to release receptor-bound iodinated LDL, 15% to 35% of the total label present in the homogenates was released in the supernatant, suggesting specific displacement of membrane-bound LDL. The remaining amount of the label may represent trapped LDL present intracellularly. The presence of a receptor was also suggested by the specific staining of LDL-HRP on sections of normal rat kidney (Fig 2, upper panel). The staining was reduced when LDLHRP was incubated in presence of excess unlabeled LDL (Fig 2, lower panel). The localization of LDLHRP was observed mainly in the proximal tubules, with a weaker reaction in the glomerular capillary wall. Staining was also seen in the distal tubules and collecting ducts in the medulla (not shown). Fate of iodinated LDL excreted in urine. In contrast to the state of LDL in the cortex and medulla, where LDL was present mostly in the intact form, almost 95% of iodinated LDL excreted in the urine was in the degraded form (Fig 3, left panel). Pretreatment of kidneys with chloroquine — which increases lysosomal pH — increased the amount of intact iodinated LDL in the urine four- to fivefold (Fig 3, right panel), suggesting that LDL degradation in the urine was the result of lysosomal activity. Presence of fragments of iodinated LDL in urine. We tested for the presence of fragments of iodinated LDL in the urine to determine the extent of degradation of LDL in the urinary space. The quantitation of intact and degraded forms of iodinated LDL was based on the TCA precipitation method, which does not precipitate peptides of less than 15 kD. We therefore tested the TCA-soluble fraction for fragments of LDL in the range of 5 to 15 kD using a size-exclusion column of G-25 Sephadex with an exclusion limit of 5 kD. A substantial proportion (20%) of the urinary iodinated LDL was in the form of LDL fragments of 5 to 15 kD (Table II). Chromatography of iodinated LDL in the perfusate and urine of rats with protamine-induced proteinuria. To fur-
ther examine the fragmentation of iodinated LDL in urine, we subjected the iodinated-LDL preparation and urine samples to chromatography on a gel filtration column. Radiolabeled LDL in the perfusate separated as a single peak, as expected, in the 60-kD–plus region of the chromatogram. A small amount of the label was also observed in the 45-kD region, probably represent-
Fig 2. Binding of HRP-LDL to normal rat kidney. Upper panel: Peroxidase staining with HRP-LDL conjugate. Lower panel: Peroxidase staining with HRP-LDL conjugate and unconjugated LDL. HRP-LDL bound strongly to the proximal tubules and weakly to the glomerular capillary walls. The binding was displaced by unconjugated LDL, demonstrating that the binding was receptor-mediated.
ing the radiodamaged fragments of LDL (Fig 4, A and D ). In contrast, urine samples showed an absence of intact LDL, but the presence of smaller fragments of iodinated LDL (Fig 4, B and C), including the 5- to 15-kD fragments measured on the TCA precipitation analysis earlier (Table II), suggesting extensive degradation of iodinated LDL in the urine. Perfusate and urine samples were also analyzed with SDS-PAGE and autoradiography (Fig 5) Intact iodinated LDL in the perfusate remained, as expected, at the origin of the gel; LDL is known to behave anomalously
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Fig 3. Iodinated LDL in urine after a single pass of ex vivo perfusion: effect of chloroquine treatment. Data presented as mean ⫾ SEM; n ⫽ 6 from chloroquine-free group, n ⫽ 4 from chloroquinetreated group. *P ⬍ 0.05 (paired Student t test) vs intact iodinated LDL in the chloroquine-free group. In urine, 95% of the label was degraded; just 5% remained intact. Chloroquine inhibited LDL catabolism, increasing the fraction of intact LDL four- or fivefold. These data suggest that LDL was degraded by the tubules in their lysosomal apparatus.
Table II. Fragments of
Rat
Protamine-pretreated 1 2 3 4 5 6 Mean ⫾ SEM PHN 1 2 3 4 Mean ⫾ SEM
125
l-labeled LDL in urine Radiolabeled LDL fragments (5-15 kD) (% of total urinary 125 l-labeled LDL)
12.2 26.1 25.8 14.2 19.2 22.6 20.0 ⫾ 2.4 31.3 19.9 24.3 21.9 24.3 ⫾ 2.5
as a large molecule on SDS-PAGE as a result of its lipid content (Fig 5, A). In the urine of a protaminetreated rat, the iodinated LDL appeared to be completely fragmented to small peptides in the 15-kD–plus region (Fig 5, B), consistent with the results of gel filtration and TCA analysis. Studies in PHN rat kidneys. To confirm findings in diseased kidneys, similar experiments were also carried out in rats made proteinuric by PHN. The PHN rats had moderate proteinuria (41-78 mg/day, n ⫽ 4) compared with controls (4.8-5.8 mg/day, n ⫽ 3). Fractional clearance of iodinated LDL in the ex vivo–perfused kidneys
Fig 4. Gel-filtration chromatography of intact iodinated LDL in the perfusate and its degradation products in the urine of proteinuric rats. A and D, Iodinated LDL in the perfusate, separated as a single peak, as expected, in the 60-kD–plus region of the chromatogram. A small amount of the label was also observed in the 45-kD region, probably representing the radiodamaged fragments of LDL. B, Urine of protamine-treated rat No. 1; C, urine of protamine-treated rat No. 2. Urine samples showed an absence of intact iodinated LDL but the presence of lower-molecular-weight fragments of iodinatedLDL (5-15 kD), suggesting extensive degradation of iodinated LDL in the urine. E, urine of PHN rat No. 1; F, urine of PHN rat No. 2. Urine from PHN rats showed similar degradation of iodinated LDL.
of the PHN rats was five times higher than that in the untreated controls (0.11 ⫾ 0.04, n ⫽ 4, vs 0.024 ⫾ 0.004, n ⫽ 3) but lower than that of the protaminetreated rat kidneys described in Table I. The TCA precipitability of the iodinated LDL in the PHN urine samples was 3.33% ⫾ 0.19% (n ⫽ 4), similar to that seen in the urine of protamine-treated rats (Fig 3), suggesting extensive degradation of LDL in urine. Also, in the urine of PHN rats the proportion of 5- to 15-kD fragments of iodinated LDL was 24.3%, similar to that seen in the protamine-treated rats (Table II). Chromatography of the PHN urine on gel filtration (Fig 4, E and F) confirmed the degradation of iodinated LDL in the urine to 5- to 15-kD fragments, as was seen in the urine of protamine-treated rats (Fig 4, B and C). SDS-PAGE (Fig 5, C) further confirmed the degradation of iodinated LDL in the urine of PHN rats, similar
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Fig 5. Autoradiograph of SDS-PAGE gel containing intact iodinated LDL and urine samples from perfused rats. A, Intact iodinated LDL. B, urine of protamine-treated rat No. 1; C, urine of PHN rat No. 1. Intact iodinated LDL in the perfusate remained at the origin of the gel; this was expected, for LDL is known to behave anomalously as a large molecule on SDS-PAGE as a result of its lipid content. In the urine of protamine-treated and PHN rats, the iodinated LDL appeared to be completely fragmented to small peptides in the region of less than 15 kD, consistent with the results of gel filtration and TCA analysis.
to that seen in the urine of protamine-treated rats (Fig 5, B). DISCUSSION
The tubular handling of filtered proteins has been studied mostly with low-molecular-weight proteins (lysozyme, cytochrome C) and peptide hormones (angiotensin II, growth hormone, adrenocorticotropic hormone, parathyroid hormone).15 In most cases, the filtered protein is absorbed on the luminal side of the tubule and completely degraded by the lysosomal system of the cell. The one exception is insulin, which is digested both by the peritubular and brush-border sides.16 Large complex proteins have been less thoroughly studied. This may be because the size-selective glomerular barrier normally restricts their entry into the urinary space, so that their concentration in the tubular lumen is very low. In proteinuric states, however, greater amounts of such large proteins leak through the glomerular filter. It is therefore important to understand the tubular handling of complex proteins, such as LDL, which bear toxic lipid moieties that could damage kidney cells,17-21 especially when hyperlipidemia coexists with kidney disease. The tubular handling of large proteins has been best studied with regard to albumin. Albumin in the ultrafiltrate is rapidly taken up by the proximal tubule and internalized in the lysosomal vesicles.22-26 Park and Maack,24 studying the isolated proximal tubule, have shown that albumin is taken up by way of both fluid-
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phase and absorptive endocytosis. Once present in the lysosomal compartment, albumin is broken down more slowly and the degradation products are released into the peritubular side.26 It therefore appears that a large protein such as albumin is processed by the tubule in a manner similar to that of low-molecular-weight peptides. Parallel to these physiological studies is the current literature on the description of a shared tubular receptor for albumin and LDL uptake called megalin.27 Megalin, a 600-kD glycoprotein (previously called gp600 or gp330), is present on the luminal brush border and is associated with another protein, cubulin. Both are involved in albumin binding in the proximal tubule.28-30 The relationship of these receptors to the previously described fluid-phase and absorptive pathways for albumin uptake has not been clarified. Our results suggest a receptor-mediated uptake of LDL by the tubule, indicated by the inhibition of LDL-HRP binding on kidney sections by unlabeled LDL and partial release of bound iodinated LDL in the ex vivo kidney by an excess of unlabeled LDL. Our study did not further characterize the receptor, but the close relationship shown previously between megalin and the LDL receptor27,31,32 strongly suggests that megalin is the receptor responsible for LDL uptake in the tubule. Although the presence of LDL receptors in the kidney has been reported,7 their role in the renal metabolism of LDL is still unknown. Chatterjee et al33,34 described the details of binding, internalization, and degradation of LDL in cultured human proximal tubular cells and suggested that LDL regulates membrane lipid biosynthesis. The LDL was degraded optimally at an acidic pH, suggesting that LDL was digested in the lysosomal system of the proximal tubular cells. These investigators did not analyze the TCA-soluble fractions to determine whether LDL digestion was complete or small fragments of LDL were generated in the process. We found that the renal tubule degraded the LDL molecule, albeit incompletely, and that substantial numbers of partially degraded fragments were released into the urine. Because some of these fragments contain lipid side chains that could be toxic to the tubule, it remains to be determined whether their presence in the urine plays a role in hastening the progression of kidney disease.
REFERENCES
1. Christensen EI, Maunsbach AB. Intralysosomal digestion of lysozyme in renal proximal tubule cells. Kidney Int 1974;6:396407. 2. Maack T. Renal handling of low molecular weight proteins. Am J Med 1975;58:57-64.
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3. Christensen EI. Rapid protein uptake and digestion in proximal tubule lysosomes. Kidney Int 1976;10:301-10. 4. Kau ST, Maack T. Transport and catabolism of parathyroid hormone in isolated rat kidney. Am J Physiol 233;5:F445-54. 5. Maack T, Johnson V, Kau ST, Fiueiredo J, Sigulem D. Renal filtration, transport, and metabolism of low-molecular weight proteins: a review. Kidney Int 1979;16:251-70. 6. Gherardi E, Calandra S. Plasma and urinary lipids and lipoproteins during the development of nephrotic syndrome induced in the rat by puromycin aminonucleoside. Biochim Biophys Acta 1981;710:188-96. 7. Zheng G, Bachinsky DR, Stamenkovic I, Strickland DK, Brown D, Andres G, et al. Organ distribution in rats of two members of the low-density lipoprotein receptor gene family, gp330 and LRP/alpha 2MR, and the receptor-associated protein (RAP). J Histochem Cytochem 1994;42:531-42. 8. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human sera. J Clin Invest 1955;34:1345-52. 9. Sharma P, Reddy K, Franki N, Sanwal V, Sankaran R, Ahuja T, et al. Native and oxidized low density lipoproteins modulate mesangial cell apoptosis. Kidney Int 1996;50:1604-11. 10. Weinstein DB, Carew TE, Steinberg D. Uptake and degradation of low density lipoprotein by swine arterial smooth muscle cells with inhibition of cholesterol biosynthesis. Biochim Biophys Acta 1976;424:404-10. 11. Johnson GD, Holborow EJ. Immunochemistry. In: Weir DM, ed. Handbook of experimental immunology, vol 1. Oxford: Blackwell Scientific Publications, 1973:18.1-18.10. 12. Singh AK. Presence of lysosomal enzymes in the normal glomerular basement membrane matrix. Histochem J 1993;25:562-8. 13. Makker SP, Singh AK. Characterization of the antigen (gp-600) of Heymann nephritis. Lab Invest 1984;50:287-93. 14. Singh AK, Schwartz MM. Circulatory antigen of Heymann nephritis. II. Isolation of 70,000 MW antigen from normal rat serum which cross-reacts with Heymann nephritis antigen. Immunology 1986;59:451-8. 15. Maack T, Park CH, Camargo MJF. Renal filtration, transport, and metabolism of proteins. In: Seldin DW, Giebisch G, eds. The kidney: physiology and pathophysiology. New York: Raven Press, 1992:3005-38. 16. Rabkin R, Kitabchi AE. Factors influencing the handling of insulin by the isolated rat kidneys. J Clin Invest 1978;62:169-75. 17. Humes HD, Nguyen VD, Cielinski DA, Messana JM. The role of free fatty acids in hypoxia-induced injury to renal proximal tubule cells. Am J Physiol 1989;256:F688-96. 18. Thomas ME, Schreiner GF. Contribution of proteinuria to progressive renal injury: consequence of tubular uptake of fatty acid bearing albumin. Am J Nephrol 1993;13:385-98.
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19. Kees-Folts D, Sadow JL, Schreiner GF. Tubular catabolism of albumin is associated with the release of an inflammatory lipid. Kidney Int 1994;45:1697-1709. 20. Schreiner GF. Renal toxicity of albumin and other lipoproteins. Curr Opin Nephrol Hyperten 1995;4:369-73. 21. Thomas ME, Schreiner GF. Contribution of proteinuria to progressive renal injury: consequence of tubular uptake of fatty acid bearing albumin. Am J Nephrol 1993;13:385-98. 22. Maunsbach AB. Absorption of 125I-labeled homologous albumin by rat kidney proximal tubule cells. Ultrastruct Res 1966;15:197241. 23. Bourdeau JE, Carone FA, Ganote CE. Serum albumin uptake in isolated perfused renal tubules. J Cell Biol 1972;54:382-98. 24. Clapp WL, Park H, Madsen KM, Tisher CG. Axial heterogeneity in the handling of albumin by the rabbit proximal tubule. Lab Invest 1988;58:549-58. 25. Park CH, Maack T. Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit. J Clin Invest 1984;73:767-77. 26. Park CH. Time course and vectorial nature of albumin metabolism in isolated perfused rabbit PCT. Am J Physiol 1988;255: F52-8. 27. Raychowdhury R, Niles JL, McCluskey RT, Smith JA. Autoimmune target in Heymann nephritis is a glycoprotein with homology to the LDL receptor. Science 1989;244:1163-5. 28. Makker SP, Singh AK. Characterization of the antigen (gp600) of Heymann nephritis. Lab Invest 1984;50:287-93. 29. Kerjaschki D, Farquhar MG. The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border. Proc Nat Acad Sci U S A 1982;79:5557-61. 30. Birn H, Fyfe JC, Jacobsen C, Mounier F, Verroust PJ, Orskov H, et al. Cubulin is an albumin binding protein important for renal tubular albumin reabsorption. J Clin Invest 2000;105:13353-61. 31. Willnow TE, Goldstein JL, Orth K, Brown MS, Herz J. Low density lipoprotein receptor-related protein and gp 330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance. J Biol Chem 1992;267:26172-80. 32. Saito A, Pietromonaco S, Loo AK-C, Farquhar MG. Complete cloning and sequencing of rat gp 330/‘megalin,’ a distinctive member of the low density lipoprotein receptor gene family. Proc Natl Acad Sci U S A 1994;91:9725-9. 33. Chatterjee S, Clarke KS, Kwiterovich PO. Regulation of synthesis of lactosylceramide and long chain bases in normal and familial hypercholesterolemic cultured proximal tubular cells. J Biol Chem 1986;261:13474-9. 34. Chatterjee S. Role of low density lipoprotein receptors in the regulation of synthesis of lactosylceramide in cultured normal human proximal tubular cells. Ind J Biochem Biophys 1988;25: 85-9.
Because the mechanism by which lipoproteins are processed and modified in the renal tubule in patients with nephrosis is not completely understood, we studied the handling of low-density lipoprotein (LDL) in perfused rat kidneys made permeable by protamine. Protamine pretreatment increased the clearance of 125I LDL 25-fold compared to controls, thereby simulating a proteinuric kidney. Similar studies were also conducted in kidneys of rats made proteinuric by the induction of passive Heymann nephritis. Of the perfused iodinated LDL, 5% was localized in the cortex and lesser amounts in the medulla and urine. In the cortex and medulla, iodinated LDL was present mainly in the intact form (90%); just 10% was present in the degraded form. Using horseradish peroxidase conjugated to LDL, we demonstrated specific staining in the proximal tubules, suggesting that specific LDL receptors were present in that location. Although LDL in the tissue was present mostly in the intact form, it was 95% degraded in urine, and the degradation was inhibited by chloroquine, indicating that the lysosomes were the site of LDL metabolism. Gel chromatography and electrophoresis of iodinated LDL in the urine showed the presence of fragments in the range of 5 to 15 kD. We conclude that renal degradation of LDL is incomplete and that the incompletely degraded fragments released into the urine may be toxic to the kidney by virtue of their lipid side-chains. (J Lab Clin Med 2002; 139:372-8) Abbreviations: EDTA ⫽ ethylenediaminetetraacetate; GFR ⫽ glomerular filtration rate; HRP ⫽ horseradish peroxidase; LDL ⫽ low-density lipoprotein; PBS ⫽ phosphate-buffered saline solution; PHN ⫽ passive Heymann nephritis; SDS-PAGE ⫽ sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SEM ⫽ standard error of the mean; TCA ⫽ trichloroacetic acid
From the Division of Nephrology, Cook County Hospital; the Hektoen Institute for Medical Research; the Department of Surgery, Loyola–Veterans Affairs Hines Medical Center; and the Section of Nephrology, University of Illinois at Chicago and Veterans Affairs Medical Center, West Side Division. Supported by the National Kidney Foundation of Illinois. Submitted for publication May 16, 2001; revision submitted February 3, 2002; accepted February 21, 2002. Reprint requests: Ashok K Singh, PhD, Hektoen Institute for Medical Research, 627 South Wood Street, Suite 201, Chicago, IL 60612; e-mail: singashok@aol. Copyright © 2002 by Mosby, Inc. All rights reserved. 0022-2143/2002 $35.00 ⫹ 0 5/1/124201 doi:10.1067/mlc.2002.124201
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he handling of filtered proteins by the renal tubules is not fully understood. Small proteins such as lysozyme are almost completely degraded during their passage through the tubule, but other proteins are excreted intact or degraded only partially.1-5 Far less is known about the fate of complex proteins such as lipoproteins, which are large and contain lipid moieties in their molecules. We chose to study the handling of LDL by the rat kidney because it is one of the common lipoproteins and is present in rat blood and urine.6 Receptors for LDL in the kidneys have been recognized,7 but their role in the handling of
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LDL is unclear. Such understanding may be important because large amounts of urinary lipoproteins are present in nephrotic states and may play a role in tubular damage and interstitial fibrosis, possibly by releasing their lipid moieties. To this end, we first studied the handling of LDL in ex vivo isolated rat kidneys made proteinuric by prior perfusion with protamine, a polycation that increases glomerular permeability to proteins. Similar studies were then conducted in rat kidneys made proteinuric by the induction of PHN. We labeled LDL with iodine 125 so that we might follow its fate after a single-pass perfusion through the kidneys. MATERIALS Preparation of LDL. LDL was prepared from human serum by means of differential centrifugation in accordance with a standard technique.8 In brief, the density of the serum was corrected to 1.019 with NaCl, after which the serum was centrifuged at 29,000 rpm for 16 hours at 10°C. The top layer, consisting of very low density lipoprotein, was removed. The remaining serum was separated from the pellet. Its density was adjusted to 1.063 by the addition of NaCl, after which it was centrifuged at 40,000 rpm for 48 hours at 10°C. The upper yellow layer, consisting of LDL, was recovered and subjected to dialysis against 20 vol of 0.9% NaC1 (pH 8.5) containing 0.02% EDTA. The presence of EDTA was essential to prevent oxidation of LDL. We used the thiobarbuturic acid reactive species assay to determine the content of oxidized LDL in an attempt to confirm that the LDL remained in the unoxidized state after the isolation procedure.9 Protein concentration and cholesterol content were determined with the use of the Bradford method (Bio-Rad Laboratories, Hercules, Calif) and a kit (Sigma Chemical, St Louis, Mo), respectively, in accordance with the manufacturers’ recommended methodologies. Radiolabeling of LDL. We radiolabeled LDL with sodium iodide 125I in accordance with the iodine monochloride method.10 This method was chosen because it is based on an exchange reaction and does not include an oxidation step, thereby avoiding the formation of oxidized LDL. One milligram of LDL was dissolved in 600 L water and placed on ice. One millicurie of sodium iodide 125I (ICN Biomedicals, Irvine, Calif) in 10 L solution was added, followed by 50 L of 250 g/mL solution of iodine monochloride in water. After 60 seconds, another 50 L of iodine monochloride solution was added, and the reaction was terminated at the end of the second minute by the addition of l mL saline solution–EDTA (0.2%). The reaction mixture was subjected to dialyis against saline solution–EDTA at 4°C. The purity and specific activity of the label were tested by means of TCA precipitation analysis and gamma-counting. Covalent conjugation of LDL with HRP for staining of kidney sections. One milligram of HRP (Sigma) was dis-
solved in 100 L PBS adjusted to pH 6.8. Reactive aldehyde groups were generated on HRP with 100 mmol/L sodium metaperiodate (Sigma) oxidation at room temperature for 1
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hour.11 After dialysis against normal saline solution at 4°C, 300 g of LDL was added, and the mixture was incubated at 4°C for 72 hours for conjugation by way of Schiff bonding. Note that the aldehyde groups were generated by the oxidation of the HRP molecule and that the excess oxidant was removed before LDL was added. In this manner, the LDL was prevented from becoming oxidized LDL. Paraffin-embedded sections from normal rat kidneys were layered with the conjugate; this was followed by washing and development with diaminobenzidine-H2O2 solution. The specificity of the label was confirmed by inhibition of the reaction in the presence of excess unlabeled LDL. The slides were examined under light microscopy. Ex vivo perfusion of kidneys with iodinated LDL. SpragueDawley rats weighing 250 to 300 g (Harlan, Indianapolis, Ind) were anesthetized with an intraperitoneal injection of 50 mg/kg sodium pentobarbital and prepared for ex vivo perfusion of isolated kidneys.12 A longitudinal midabdominal incision was made to expose both ureters, which were then cannulated with PE-10 tubing (Intramedic; Clay Adams, Sparks, Md). We isolated the renal vascular bed by ligating the mesenteric vessels and the aorta proximal to the renal arteries. A cannula was placed at the descending aorta below the renal artery and secured with a ligature. An incision was made in the inferior vena cava to permit outflow. The kidneys were initially perfused with PBS (pH 7.4) at 37°C in an attempt to flush blood. The kidneys (n ⫽ 6 in the protamine group, n ⫽ 4 in the PHN group) were then perfused with 10 mL PBS containing 5 g/mL protamine sulfate (Eli Lilly, Indianapolis, Ind) to cause the glomeruli to become permeable. After excess protamine was washed away with PBS, kidneys were perfused with 20 mL PBS containing 125Ilabeled LDL (10-15 ⫻ 106 cpm) at the rate of 2 mL/min, followed by a wash with 20 mL PBS. During perfusion with 125 I-labeled LDL, urine was collected from both ureters and saved. At the end of the experiment, both kidneys were removed, the cortex and medulla separated and their wet weights determined. In a separate group of animals (n ⫽ 3), the kidneys were first perfused with protamine and then perfused with tritiated inulin (ICN, Irvine, CA) for determination of GFR. Similarly, GFR was determined in a separate group of PHN rat kidneys (n ⫽ 3) by means of ex vivo perfusion with [3H]inulin. In another group of animals (n ⫽ 4), the effect of chloroquine was tested. Chloroquine (10 mol/L; Sigma) was added to the protamine solution for perfusion. Tissue processing. We homogenized the cortex and medulla in ice-cold PBS with a polytron homogenizer. The homogenates were centrifuged at 3000 rpm to separate the tissue pellet. Tissue pellets were centrifuged twice with icecold PBS to remove the nonspecifically bound iodinated LDL for further processing. An aliquot of the iodinated LDL containing perfusate, cortex, medulla, and urine was precipitated with 10% TCA and centrifuged at 3000 rpm to separate the TCA-soluble (supernatant containing degraded iodinated LDL) and insoluble (pellet containing intact iodinated LDL) fractions. Both fractions were analyzed in a gamma-counter,
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and the counts were normalized to the final volume of each sample. Receptor-bound iodinated LDL in the tissues was determined on the basis of competitive release as follows: The cortex and medulla homogenates were centrifuged at 3000 rpm. The pellet was suspended in PBS containing excess unlabeled LDL (1 mg/mL). The mixture was vortexed and centrifuged, after which we counted the supernatant to quantify iodinated LDL. The percentage of counts released from the tissue represented the receptor-bound iodinated LDL. To detect fragments of iodinated LDL (3-15 kD) in the urine, we fractionated the TCA-soluble portion in a G-25 Sephadex (Pharmacia, Piscataway, NJ) column. Gamma counts in the fractions immediately after void corresponding to the macromolecular region were considered to represent fragments of iodinated LDL. These data were presented as a percentage of the counts in urine. Induction of PHN. Sprague-Dawley rats weighing 150 to 200 g were injected intraperitoneally with 10 mg (IgG) of rabbit anti-gp600 antibody in 0.5 mL saline solution.13 In a parallel set of control rats, we injected a similar amount of IgG from normal rabbit serum. The animals were tested for proteinuria at day 7 by means of sulfosalicylic acid precipitation.
Table I. Fractional clearance of 125l-labeled LDL in ex vivo–perfused kidneys pretreated with protamine Rat
1 2 3 4 5 6 Mean ⫾ SEM
Fractional clearance of 125l-labeled LDL (%)
0.347 0.577 0.927 0.331 0.541 0.282 0.501 ⫾ 0.098
Molecular exclusion gel chromatography and electrophoresis of rat urine containing filtered iodinated LDL. Gel
chromatography was performed with Biogel P-60 matrix (exclusion limit ⫽ 60 kD, fractionation range ⫽ 3-60 kD; Biorad Laboratories, Richmond, Calif). Standards of purified human serum albumin (66 kD), ovalbumin (45 kD), and lysozyme (14 kD) were used to calibrate the column. The column (1 ⫻ 30 cm) was equilibriated and run in PBS at a flow rate of 5 mL/hour. One-milliliter fractions were collected. SDS-PAGE of the urine samples containing iodinated LDL was performed in accordance with the Laemmli system and the gel subjected to autoradiography in keeping with to standard technique.14 Statistical analyses. Results are presented as mean ⫾ SEM. Unpaired or paired Student’s t test was applied to determine significance of experimental changes with P ⬍ 0.05 considered significant. RESULTS Clearance of LDL. We found the fractional clearance of LDL in normal rat kidneys to be extremely low (0.02% ⫾ 0.012%, n ⫽ 3), consistent with a normally functioning size-selective glomerular basement. The low clearance of LDL in normal kidneys precluded further studies on the fate of the filtered LDL because of the extremely small concentrations present in the urine. This problem was overcome by a brief treatment of the kidney with protamine to increase the clearance of high-molecular-weight molecules. The treatment protocol we used increased the clearance of LDL to 25 times that than untreated kidneys, allowing us to further study the tubular handling of LDL (Table I). We compared the GFR measured on the basis of inulin clear-
Fig 1. Binding of iodinated LDL to kidney tissue after a single pass of ex vivo perfusion. Data presented as mean ⫾ SEM; n ⫽ 6. *P ⬍ 0.05 vs cortex. Iodinated LDL was bound mainly to the cortex, suggesting that it is the main site of LDL uptake and processing.
ance in ex vivo control kidneys and kidneys perfused with protamine. The clearance of inulin was similar in both groups (mean ⫽ 1.04 mL/min/kidney in controls vs 1.2 mL/min/kidney in protamine-perfused group, n ⫽3 ) indicating that protamine treatment did not alter GFR while increasing the permeability to LDL. In addition, the GFR of the ex vivo perfused kidneys was similar to that of intact animals, showing that the ex vivo–perfused kidney preparation was stable during the short experimental period. Distribution of iodinated LDL in the kidney after ex vivo perfusion. We studied the distribution of iodinated LDL
in the kidney after ex vivo perfusion in an effort to determine the region where it would likely be processed. After a single pass of iodinated LDL followed by a 5-minute wash, 5% of the perfused LDL was present in the cortex, with lesser amounts in the medulla and urine (Fig 1) The amount of iodinated LDL in the cortex was three or four times higher than that present in the medulla (Fig 1). TCA precipitation analyis revealed that in both cortex and medulla, the iodinated LDL was mostly (90%) present in the intact
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form, with the remaining 10% in the degraded form (data not shown). Evidence for receptor–mediated binding of iodinated LDL in the kidney. We present two pieces of evidence to
demonstrate the presence of the LDL receptor in the kidney. When homogenates of cortex and medulla containing the bound 125I-labeled LDL were treated with unlabeled LDL to release receptor-bound iodinated LDL, 15% to 35% of the total label present in the homogenates was released in the supernatant, suggesting specific displacement of membrane-bound LDL. The remaining amount of the label may represent trapped LDL present intracellularly. The presence of a receptor was also suggested by the specific staining of LDL-HRP on sections of normal rat kidney (Fig 2, upper panel). The staining was reduced when LDLHRP was incubated in presence of excess unlabeled LDL (Fig 2, lower panel). The localization of LDLHRP was observed mainly in the proximal tubules, with a weaker reaction in the glomerular capillary wall. Staining was also seen in the distal tubules and collecting ducts in the medulla (not shown). Fate of iodinated LDL excreted in urine. In contrast to the state of LDL in the cortex and medulla, where LDL was present mostly in the intact form, almost 95% of iodinated LDL excreted in the urine was in the degraded form (Fig 3, left panel). Pretreatment of kidneys with chloroquine — which increases lysosomal pH — increased the amount of intact iodinated LDL in the urine four- to fivefold (Fig 3, right panel), suggesting that LDL degradation in the urine was the result of lysosomal activity. Presence of fragments of iodinated LDL in urine. We tested for the presence of fragments of iodinated LDL in the urine to determine the extent of degradation of LDL in the urinary space. The quantitation of intact and degraded forms of iodinated LDL was based on the TCA precipitation method, which does not precipitate peptides of less than 15 kD. We therefore tested the TCA-soluble fraction for fragments of LDL in the range of 5 to 15 kD using a size-exclusion column of G-25 Sephadex with an exclusion limit of 5 kD. A substantial proportion (20%) of the urinary iodinated LDL was in the form of LDL fragments of 5 to 15 kD (Table II). Chromatography of iodinated LDL in the perfusate and urine of rats with protamine-induced proteinuria. To fur-
ther examine the fragmentation of iodinated LDL in urine, we subjected the iodinated-LDL preparation and urine samples to chromatography on a gel filtration column. Radiolabeled LDL in the perfusate separated as a single peak, as expected, in the 60-kD–plus region of the chromatogram. A small amount of the label was also observed in the 45-kD region, probably represent-
Fig 2. Binding of HRP-LDL to normal rat kidney. Upper panel: Peroxidase staining with HRP-LDL conjugate. Lower panel: Peroxidase staining with HRP-LDL conjugate and unconjugated LDL. HRP-LDL bound strongly to the proximal tubules and weakly to the glomerular capillary walls. The binding was displaced by unconjugated LDL, demonstrating that the binding was receptor-mediated.
ing the radiodamaged fragments of LDL (Fig 4, A and D ). In contrast, urine samples showed an absence of intact LDL, but the presence of smaller fragments of iodinated LDL (Fig 4, B and C), including the 5- to 15-kD fragments measured on the TCA precipitation analysis earlier (Table II), suggesting extensive degradation of iodinated LDL in the urine. Perfusate and urine samples were also analyzed with SDS-PAGE and autoradiography (Fig 5) Intact iodinated LDL in the perfusate remained, as expected, at the origin of the gel; LDL is known to behave anomalously
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Fig 3. Iodinated LDL in urine after a single pass of ex vivo perfusion: effect of chloroquine treatment. Data presented as mean ⫾ SEM; n ⫽ 6 from chloroquine-free group, n ⫽ 4 from chloroquinetreated group. *P ⬍ 0.05 (paired Student t test) vs intact iodinated LDL in the chloroquine-free group. In urine, 95% of the label was degraded; just 5% remained intact. Chloroquine inhibited LDL catabolism, increasing the fraction of intact LDL four- or fivefold. These data suggest that LDL was degraded by the tubules in their lysosomal apparatus.
Table II. Fragments of
Rat
Protamine-pretreated 1 2 3 4 5 6 Mean ⫾ SEM PHN 1 2 3 4 Mean ⫾ SEM
125
l-labeled LDL in urine Radiolabeled LDL fragments (5-15 kD) (% of total urinary 125 l-labeled LDL)
12.2 26.1 25.8 14.2 19.2 22.6 20.0 ⫾ 2.4 31.3 19.9 24.3 21.9 24.3 ⫾ 2.5
as a large molecule on SDS-PAGE as a result of its lipid content (Fig 5, A). In the urine of a protaminetreated rat, the iodinated LDL appeared to be completely fragmented to small peptides in the 15-kD–plus region (Fig 5, B), consistent with the results of gel filtration and TCA analysis. Studies in PHN rat kidneys. To confirm findings in diseased kidneys, similar experiments were also carried out in rats made proteinuric by PHN. The PHN rats had moderate proteinuria (41-78 mg/day, n ⫽ 4) compared with controls (4.8-5.8 mg/day, n ⫽ 3). Fractional clearance of iodinated LDL in the ex vivo–perfused kidneys
Fig 4. Gel-filtration chromatography of intact iodinated LDL in the perfusate and its degradation products in the urine of proteinuric rats. A and D, Iodinated LDL in the perfusate, separated as a single peak, as expected, in the 60-kD–plus region of the chromatogram. A small amount of the label was also observed in the 45-kD region, probably representing the radiodamaged fragments of LDL. B, Urine of protamine-treated rat No. 1; C, urine of protamine-treated rat No. 2. Urine samples showed an absence of intact iodinated LDL but the presence of lower-molecular-weight fragments of iodinatedLDL (5-15 kD), suggesting extensive degradation of iodinated LDL in the urine. E, urine of PHN rat No. 1; F, urine of PHN rat No. 2. Urine from PHN rats showed similar degradation of iodinated LDL.
of the PHN rats was five times higher than that in the untreated controls (0.11 ⫾ 0.04, n ⫽ 4, vs 0.024 ⫾ 0.004, n ⫽ 3) but lower than that of the protaminetreated rat kidneys described in Table I. The TCA precipitability of the iodinated LDL in the PHN urine samples was 3.33% ⫾ 0.19% (n ⫽ 4), similar to that seen in the urine of protamine-treated rats (Fig 3), suggesting extensive degradation of LDL in urine. Also, in the urine of PHN rats the proportion of 5- to 15-kD fragments of iodinated LDL was 24.3%, similar to that seen in the protamine-treated rats (Table II). Chromatography of the PHN urine on gel filtration (Fig 4, E and F) confirmed the degradation of iodinated LDL in the urine to 5- to 15-kD fragments, as was seen in the urine of protamine-treated rats (Fig 4, B and C). SDS-PAGE (Fig 5, C) further confirmed the degradation of iodinated LDL in the urine of PHN rats, similar
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Fig 5. Autoradiograph of SDS-PAGE gel containing intact iodinated LDL and urine samples from perfused rats. A, Intact iodinated LDL. B, urine of protamine-treated rat No. 1; C, urine of PHN rat No. 1. Intact iodinated LDL in the perfusate remained at the origin of the gel; this was expected, for LDL is known to behave anomalously as a large molecule on SDS-PAGE as a result of its lipid content. In the urine of protamine-treated and PHN rats, the iodinated LDL appeared to be completely fragmented to small peptides in the region of less than 15 kD, consistent with the results of gel filtration and TCA analysis.
to that seen in the urine of protamine-treated rats (Fig 5, B). DISCUSSION
The tubular handling of filtered proteins has been studied mostly with low-molecular-weight proteins (lysozyme, cytochrome C) and peptide hormones (angiotensin II, growth hormone, adrenocorticotropic hormone, parathyroid hormone).15 In most cases, the filtered protein is absorbed on the luminal side of the tubule and completely degraded by the lysosomal system of the cell. The one exception is insulin, which is digested both by the peritubular and brush-border sides.16 Large complex proteins have been less thoroughly studied. This may be because the size-selective glomerular barrier normally restricts their entry into the urinary space, so that their concentration in the tubular lumen is very low. In proteinuric states, however, greater amounts of such large proteins leak through the glomerular filter. It is therefore important to understand the tubular handling of complex proteins, such as LDL, which bear toxic lipid moieties that could damage kidney cells,17-21 especially when hyperlipidemia coexists with kidney disease. The tubular handling of large proteins has been best studied with regard to albumin. Albumin in the ultrafiltrate is rapidly taken up by the proximal tubule and internalized in the lysosomal vesicles.22-26 Park and Maack,24 studying the isolated proximal tubule, have shown that albumin is taken up by way of both fluid-
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phase and absorptive endocytosis. Once present in the lysosomal compartment, albumin is broken down more slowly and the degradation products are released into the peritubular side.26 It therefore appears that a large protein such as albumin is processed by the tubule in a manner similar to that of low-molecular-weight peptides. Parallel to these physiological studies is the current literature on the description of a shared tubular receptor for albumin and LDL uptake called megalin.27 Megalin, a 600-kD glycoprotein (previously called gp600 or gp330), is present on the luminal brush border and is associated with another protein, cubulin. Both are involved in albumin binding in the proximal tubule.28-30 The relationship of these receptors to the previously described fluid-phase and absorptive pathways for albumin uptake has not been clarified. Our results suggest a receptor-mediated uptake of LDL by the tubule, indicated by the inhibition of LDL-HRP binding on kidney sections by unlabeled LDL and partial release of bound iodinated LDL in the ex vivo kidney by an excess of unlabeled LDL. Our study did not further characterize the receptor, but the close relationship shown previously between megalin and the LDL receptor27,31,32 strongly suggests that megalin is the receptor responsible for LDL uptake in the tubule. Although the presence of LDL receptors in the kidney has been reported,7 their role in the renal metabolism of LDL is still unknown. Chatterjee et al33,34 described the details of binding, internalization, and degradation of LDL in cultured human proximal tubular cells and suggested that LDL regulates membrane lipid biosynthesis. The LDL was degraded optimally at an acidic pH, suggesting that LDL was digested in the lysosomal system of the proximal tubular cells. These investigators did not analyze the TCA-soluble fractions to determine whether LDL digestion was complete or small fragments of LDL were generated in the process. We found that the renal tubule degraded the LDL molecule, albeit incompletely, and that substantial numbers of partially degraded fragments were released into the urine. Because some of these fragments contain lipid side chains that could be toxic to the tubule, it remains to be determined whether their presence in the urine plays a role in hastening the progression of kidney disease.
REFERENCES
1. Christensen EI, Maunsbach AB. Intralysosomal digestion of lysozyme in renal proximal tubule cells. Kidney Int 1974;6:396407. 2. Maack T. Renal handling of low molecular weight proteins. Am J Med 1975;58:57-64.
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3. Christensen EI. Rapid protein uptake and digestion in proximal tubule lysosomes. Kidney Int 1976;10:301-10. 4. Kau ST, Maack T. Transport and catabolism of parathyroid hormone in isolated rat kidney. Am J Physiol 233;5:F445-54. 5. Maack T, Johnson V, Kau ST, Fiueiredo J, Sigulem D. Renal filtration, transport, and metabolism of low-molecular weight proteins: a review. Kidney Int 1979;16:251-70. 6. Gherardi E, Calandra S. Plasma and urinary lipids and lipoproteins during the development of nephrotic syndrome induced in the rat by puromycin aminonucleoside. Biochim Biophys Acta 1981;710:188-96. 7. Zheng G, Bachinsky DR, Stamenkovic I, Strickland DK, Brown D, Andres G, et al. Organ distribution in rats of two members of the low-density lipoprotein receptor gene family, gp330 and LRP/alpha 2MR, and the receptor-associated protein (RAP). J Histochem Cytochem 1994;42:531-42. 8. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human sera. J Clin Invest 1955;34:1345-52. 9. Sharma P, Reddy K, Franki N, Sanwal V, Sankaran R, Ahuja T, et al. Native and oxidized low density lipoproteins modulate mesangial cell apoptosis. Kidney Int 1996;50:1604-11. 10. Weinstein DB, Carew TE, Steinberg D. Uptake and degradation of low density lipoprotein by swine arterial smooth muscle cells with inhibition of cholesterol biosynthesis. Biochim Biophys Acta 1976;424:404-10. 11. Johnson GD, Holborow EJ. Immunochemistry. In: Weir DM, ed. Handbook of experimental immunology, vol 1. Oxford: Blackwell Scientific Publications, 1973:18.1-18.10. 12. Singh AK. Presence of lysosomal enzymes in the normal glomerular basement membrane matrix. Histochem J 1993;25:562-8. 13. Makker SP, Singh AK. Characterization of the antigen (gp-600) of Heymann nephritis. Lab Invest 1984;50:287-93. 14. Singh AK, Schwartz MM. Circulatory antigen of Heymann nephritis. II. Isolation of 70,000 MW antigen from normal rat serum which cross-reacts with Heymann nephritis antigen. Immunology 1986;59:451-8. 15. Maack T, Park CH, Camargo MJF. Renal filtration, transport, and metabolism of proteins. In: Seldin DW, Giebisch G, eds. The kidney: physiology and pathophysiology. New York: Raven Press, 1992:3005-38. 16. Rabkin R, Kitabchi AE. Factors influencing the handling of insulin by the isolated rat kidneys. J Clin Invest 1978;62:169-75. 17. Humes HD, Nguyen VD, Cielinski DA, Messana JM. The role of free fatty acids in hypoxia-induced injury to renal proximal tubule cells. Am J Physiol 1989;256:F688-96. 18. Thomas ME, Schreiner GF. Contribution of proteinuria to progressive renal injury: consequence of tubular uptake of fatty acid bearing albumin. Am J Nephrol 1993;13:385-98.
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19. Kees-Folts D, Sadow JL, Schreiner GF. Tubular catabolism of albumin is associated with the release of an inflammatory lipid. Kidney Int 1994;45:1697-1709. 20. Schreiner GF. Renal toxicity of albumin and other lipoproteins. Curr Opin Nephrol Hyperten 1995;4:369-73. 21. Thomas ME, Schreiner GF. Contribution of proteinuria to progressive renal injury: consequence of tubular uptake of fatty acid bearing albumin. Am J Nephrol 1993;13:385-98. 22. Maunsbach AB. Absorption of 125I-labeled homologous albumin by rat kidney proximal tubule cells. Ultrastruct Res 1966;15:197241. 23. Bourdeau JE, Carone FA, Ganote CE. Serum albumin uptake in isolated perfused renal tubules. J Cell Biol 1972;54:382-98. 24. Clapp WL, Park H, Madsen KM, Tisher CG. Axial heterogeneity in the handling of albumin by the rabbit proximal tubule. Lab Invest 1988;58:549-58. 25. Park CH, Maack T. Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit. J Clin Invest 1984;73:767-77. 26. Park CH. Time course and vectorial nature of albumin metabolism in isolated perfused rabbit PCT. Am J Physiol 1988;255: F52-8. 27. Raychowdhury R, Niles JL, McCluskey RT, Smith JA. Autoimmune target in Heymann nephritis is a glycoprotein with homology to the LDL receptor. Science 1989;244:1163-5. 28. Makker SP, Singh AK. Characterization of the antigen (gp600) of Heymann nephritis. Lab Invest 1984;50:287-93. 29. Kerjaschki D, Farquhar MG. The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border. Proc Nat Acad Sci U S A 1982;79:5557-61. 30. Birn H, Fyfe JC, Jacobsen C, Mounier F, Verroust PJ, Orskov H, et al. Cubulin is an albumin binding protein important for renal tubular albumin reabsorption. J Clin Invest 2000;105:13353-61. 31. Willnow TE, Goldstein JL, Orth K, Brown MS, Herz J. Low density lipoprotein receptor-related protein and gp 330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance. J Biol Chem 1992;267:26172-80. 32. Saito A, Pietromonaco S, Loo AK-C, Farquhar MG. Complete cloning and sequencing of rat gp 330/‘megalin,’ a distinctive member of the low density lipoprotein receptor gene family. Proc Natl Acad Sci U S A 1994;91:9725-9. 33. Chatterjee S, Clarke KS, Kwiterovich PO. Regulation of synthesis of lactosylceramide and long chain bases in normal and familial hypercholesterolemic cultured proximal tubular cells. J Biol Chem 1986;261:13474-9. 34. Chatterjee S. Role of low density lipoprotein receptors in the regulation of synthesis of lactosylceramide in cultured normal human proximal tubular cells. Ind J Biochem Biophys 1988;25: 85-9.