Abstract
Abstract. A circulating causative factor has been postulated in focal segmental glomerulosclerosis (FSGS). It has been shown that serum or plasma from some FSGS increases glomerular albumin permeability (Palb) in vitro. Palb greater than 0.5 (i.e., FS activity) is associated with recurrence after transplantation. Specimens from 15 FSGS patients were studied to document the presence of a permeability factor, to isolate this factor, to characterize its biochemical properties, and to show its effect in vivo. Total lipids were extracted by chloroform/methanol (2:1); FS activity was absent from total lipid extract. Chylomicrons and lipoproteins were removed from the plasma with dextran sulfate, followed by sequential precipitation of proteins at 50 and 70% ammonium sulfate saturation. FS activity was retained in the 70% ammonium sulfate supernatant and exhibited a 100-fold purification. FS activity was lost after heating at 100°C for 10 min or after protease digestion. Under nondenaturing conditions, electrophoresis of the FSGS 70% supernatant showed a prominent low molecular weight band that was not evident in the 70% supernatant from normal plasma. Dialysis and centrifugation-based membrane ultrafiltration of the FSGS factor indicated a molecular size between 30 and 50 kD. Injection of the 70% FSGS supernatant into rats caused a threefold increase in urine protein in collections from 6 to 24 h after injection. No increase in proteinuria occurred in rats injected with 70% supernatant from normal individuals. It is concluded that the FSGS factor is a low molecular weight protein with the potential to increase Palb in vitro and to cause proteinuria in vivo.
Focal segmental glomerulosclerosis (FSGS) is a glomerular disease characterized by marked proteinuria, steroid resistance, hypertension, and a high incidence of progression to renal failure. It is the most common progressive glomerular disease in children and is the second leading cause of end-stage renal disease in this age group (1). It accounts for 20 to 25% of idiopathic nephrotic syndrome in adults (2,3). Several recent studies report up to an eightfold increase in the incidence of FSGS during the past 20 yr (4,5). The etiology of FSGS is not known and renal biopsy is the only means to confirm the clinical diagnosis (6,7,8,9).
Several observations support the hypothesis that there is a causative factor present in the circulation of some patients with FSGS. (1) Proteinuria recurs in approximately 30% of patients with FSGS who undergo transplantation, and in 80% of those patients who have experienced a previous recurrence (10,11). Recurrence of proteinuria may begin within 1 wk after transplantation. (2) Plasmapheresis (12,13) or immunoadsorption (14,15) have been used successfully to decrease proteinuria and to interrupt the progression of renal insufficiency in recurrent FSGS. (3) Proteinuria has been produced in animals after the injection of whole (16) or fractionated (17) plasma from patients with recurrent FSGS.
Characterization of the circulating factor associated with FSGS (FSGS factor) has been hindered by the lack of a sensitive and reliable assay for its activity (FS activity). We have developed an in vitro bioassay to measure the increase in albumin permeability of isolated rat glomeruli (18) and have used this assay to show that sera from patients with recurrent FSGS cause an immediate and marked increase in the albumin permeability (Palb) of isolated rat glomeruli (19,20).
The in vitro assay has provided a tool to monitor the FS activity in fractions obtained during purification of the possible causative factor. We have previously reported partial purification of the FSGS factor from plasmapheresis fluid obtained from a single patient with recurrent FSGS (21). Present studies were undertaken to determine the nature of this factor, to verify its presence in 15 patients with FSGS, and to document that this factor has the potential to cause proteinuria in vivo. We have developed a biochemical purification strategy that yields a product with a 100-fold increase in FS activity. Our results indicate that the molecule(s) responsible for the FSGS factor activity is associated with a 30- to 50-kD size fraction, sensitive to heat or protease treatment, and causes proteinuria after intravenous injection into rats.
Materials and Methods
Chemicals and Supplies
Bovine serum albumin (35% solution), HPLC grade solvents, analytical grade chemicals for buffers and other reagents, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma Chemical Co. (St. Louis, MO). Spectra/Por dialysis tubing was obtained from Spectrum Medical Industries (Laguna Hills, CA). Centricon tubes with 50- and 30-kD molecular weight cutoff (MWCO) ultrafiltration membranes, and stirred cell concentrator and membrane (3.5-kD MWCO) were obtained from Amicon (Beverly, MA). Bovine serum albumin (BSA) and Coomassie Blue Reagent for protein assay were obtained from Bio-Rad (Hercules, CA). Deionized water was purified using NANOpure ultrapure water system from Barnstead (Dubuque, Iowa). Immobilized Protease Sg (Staphylococcus aureus) bound to cross-linked 6% beaded agarose was obtained from Pierce Chemical Co. (Rockford, IL). Precast 4 to 20% gradient minigels (Novell Experimental Technology, San Diego, CA) were used for electrophoresis on equipment from Hoefer Scientific Instruments (San Francisco, CA).
Experimental Animals
Normal male Sprague Dawley rats (120 to 150 g body wt) were maintained on Purina chow with free access to drinking water. Glomeruli were isolated from kidneys removed from rats under Metofane (methoxyflurane; Mallinckrodt Veterinary, Mundelein, IL) anesthesia. Intravenous injection of the 70% supernatant was carried out in rats anesthetized with intraperitoneal Brevital (methohexital sodium, 50 mg/kg; Eli Lilly and Co., Indianapolis, IN).
FSGS Patients and Collection of Plasmapheresis Fluid
Details of the criteria used for the diagnosis of FSGS have been described earlier (13,19). Briefly, diagnosis of FSGS was based on renal biopsies done to evaluate proteinuria or renal insufficiency and on the presence of segmental obliteration of capillaries by increased extracellular matrix in some glomeruli. Mesangial proliferation, segmental deposits of IgM, and/or complement in capillary walls was seen in some biopsy specimens.
Plasma specimens were obtained during the course of therapeutic plasmapheresis from a total of 15 patients. The decision to perform plasmapheresis was made by the primary physician in each case. Eleven patients (nine adults and two children) were being treated for recurrence of FSGS in renal allografts. Of these, seven were male and four were female. An additional three patients were treated with plasmapheresis because of unremitting nephrotic syndrome in their native kidneys. Of these, two were adult males and one was a female child. One plasma sample was obtained from a patient who experienced a fulminant recurrence of FSGS and allograft loss in 1985 and who is treated with three-times-weekly hemodialysis. This patient's serum, plasma, and plasmapheresis fluid have consistently shown high levels of FS activity in the in vitro assay. Plasmapheresis was done using citrate anticoagulation and replacement of removed plasma with 0.9% NaCl and 5% human albumin solutions, and the discarded plasma was shipped to us.
Normal Plasma
Normal plasma was obtained from healthy volunteers or as discarded material from the Blood Center of Southeastern Wisconsin (Milwaukee, WI).
Transport and Storage of Specimens
Plasma was immediately stored at -20°C under sterile conditions or transported to our laboratory on dry ice and stored at -20°C until used. Samples stored at -20°C for up to 3 yr with or without sodium azide and protease inhibitors have been found to retain the FS activity.
Extraction of Total Lipids
In separate experiments, we investigated the possibility that the FSGS factor was a lipid, using lipid extraction from cryoprecipitate-free plasma or after precipitation of chylomicrons and lipoproteins. An aliquot of cryoprecipitate-free plasma from every specimen was extracted twice with 10 vol of a cold chloroform/methanol (2:1) mixture, filtered, and separated into two phases as described by Folch et al. (22). Final upper (aqueous) and lower (chloroform) layers were separated, washed with theoretical lower and upper phases, respectively, and evaporated to dryness under nitrogen. Total lipid residue in the lower (chloroform) phase was reconstituted in the medium containing 5% BSA and used for testing the FS activity (described below). To determine the amount of total lipid removed by dextran sulfate treatment, plasmapheresis fluid was subjected to lipid extraction by chloroform/methanol before and after treatment with dextran sulfate (described below), and the difference in total lipid content was determined.
Enrichment of FSGS Factor
The FSGS factor from patients was concentrated by sequential removal of (1) nonactive cryoprecipitate; (2) lipid-rich lipoproteins and chylomicrons; (3) Ig-rich protein fraction; and (4) albumin-rich protein fraction as described below.
Removal of the Cryoprecipitate. Frozen plasma was allowed to thaw at 4°C, and sodium azide (0.05%) and PMSF (1 mM) were added immediately followed by centrifugation of the plasma at 4°C (5000 × g for 30 min) to remove the cryoprecipitate formed during storage.
Removal of Lipoproteins and Chylomicrons. To remove protein-bound lipids in an aqueous medium, we precipitated lipoproteins and chylomicrons (23,24). An equal volume of 0.1% sodium chloride was added to the cryoprecipitate-free plasma specimen. After mixing, 20 μl/ml of 10% dextran sulfate (molecular weight, 0.5 × 106) was added followed by 100 μl/ml of 1 M calcium chloride. The precipitate was removed by centrifugation at 4°C, and the supernatant was dialyzed against Tris-buffered saline (TBS; 1% sodium chloride in 10 mM Tris-HCl, pH 7.8). The precipitate could not be redissolved for assay of FS activity. Total lipids, protein, and FS activity were determined in the supernatant as described.
Removal of Ig-Rich Protein Fraction by Ammonium Sulfate. The plasma specimen, free of the majority of lipoproteins and chylomicrons and the lipids associated with them, was subjected to sequential ammonium sulfate precipitation. First, a 50% saturation of ammonium sulfate was obtained by gradual addition of dry ammonium sulfate with continuous stirring at 4°C. The precipitate was separated by centrifugation at 10,000 × g for 15 min (4°C). The precipitate obtained at 50% ammonium sulfate saturation could not be redissolved completely and was discarded.
Removal of Albumin-Rich Protein Fraction by Ammonium Sulfate. Ammonium sulfate concentration of the supernatant was then increased to 70% saturation, and the resulting precipitate was removed by centrifugation at 20,000 × g for 15 min at 4°C. The precipitate at 70% ammonium sulfate saturation was readily soluble in TBS. Residual protein in the supernatant at 70% ammonium sulfate saturation (70% supernatant) could be precipitated by increasing ammonium sulfate to 80% saturation (70 to 80% precipitate).
Characteristics of the FSGS Factor
The 70% supernatant fraction of the plasma contained very little protein. However, this fraction retained the FS activity and was used to study some of its characteristics, including (1) effect of heat; (2) effect of incubation with a nonspecific proteolytic enzyme; and (3) size.
Heat Inactivation. The 70% supernatant (5 mg protein/ml) was incubated in boiling water for 10 to 40 min, cooled, centrifuged, and the supernatant was tested for FS activity.
Protease Digestion. The 70% supernatant fraction (5 mg protein/ml) was mixed with 1 ml slurry of Protease-Sg immobilized on agarose (2.4 benzoyl arginine ethyl ester U/ml gel; Pierce Chemical Co.) in TBS containing 5 mM calcium chloride. The mixture was incubated overnight at room temperature with gentle shaking. The slurry was washed with TBS and centrifuged. The supernatant was dialyzed against TBS, concentrated under pressure (20 to 25 psi) on an ultrafiltration membrane (3.5 kD MWCO) using an Amicon concentrator (Amicon, Beverly, MA), and tested for FS activity.
Size Determination. Dialysis. Routine dialysis was carried out using 3.5-kD MWCO dialysis membranes with at least three changes of 10 vol of TBS. To determine the molecular size of the active component, the 70% supernatant fraction was dialyzed using 50-kD or 12- to 14-kD MWCO dialysis membranes with three to four changes of 10 vol of TBS. The retentate in the dialysis tube was used to test the FS activity in vitro.
Centrifugation-Based Membrane Ultrafiltration. Centrifuge tubes fitted with 50- or 30-kD MWCO ultrafiltration membranes (Amicon, Beverly, MA) were used for determining the size of the FSGS factor in the 70% supernatant. The 50-kD MWCO membrane was washed thoroughly with TBS, and a 5-ml aliquot of the sample was added to the reservoir held in the filtrate cup. The device was centrifuged on a fixed angle rotor at 5000 × g for 15 min. An aliquot of the filtrate was saved, and the rest of the filtrate was transferred to a thoroughly washed 30-kD MWCO membrane reservoir and spun at 5000 × g for 30 min. The retentate and the filtrate were dialyzed in TBS and used to test the FS activity.
Electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4 to 20% gradient gels in Tris-glycine buffer to detect the protein profile of the fractions obtained during purification. Gels were stained for proteins using Coomassie blue or silver staining method. Electrophoretic pattern of the 70% supernatants from normal and FSGS plasma were also compared under nondenaturing and nonreducing conditions (native conditions).
Protein Determination
Total protein concentration was determined by the Bradford method (25). The Coomassie Blue Reagent (Bio-Rad, Hercules, CA) was added to protein solutions, and the intensity of the color was measured spectrophotometrically at 595 nm.
Measurement of FS Activity
Fractions obtained during purification were tested for their capacity to increase glomerular albumin permeability (Palb) by the method established in our laboratory. Details of the rationale and methodology for using isolated glomeruli, measurement of reflection coefficient, and albumin permeability have been described previously (18,26). Briefly, glomeruli were isolated by sequential sieving in Krebs-Ringer's buffer containing 5 g/dl BSA. Control plasma, FSGS plasma, or its fractions (test samples) were dialyzed (3 × 10 vol) and diluted in TBS to obtain a protein concentration of 4 to 5 mg/ml. For routine bioassay of the activity, 20-μl aliquots of test samples containing 80 to 100 μg of protein were incubated with glomeruli in a final volume of 1 ml at 37°C for 10 min, and the initial image was recorded by videomicroscopy. The medium was changed from 5 g/dl to 1 g/dl BSA to produce an oncotic gradient across the glomerular capillary wall causing movement of fluid into the capillaries and an increase in glomerular size. A second video image was recorded 2 to 3 min after adding 1% BSA. The average diameter of each video image was measured, and the relative volume increase of each glomerulus was calculated using change in volume (ΔV) due to oncotic gradient. Albumin reflection coefficient was calculated as: σalb = ΔVexperimental/ΔVcontrol. Convectional permeability (Palb) was calculated as: Palb = 1 - σalb. When σalb is zero, albumin moves across the membrane at same rate as water and Palb equals 1.0. When σalb is one, albumin cannot cross the membrane with water and Palb equals zero. A Palb value of 0.5 or higher is accepted as positive.
Incubation of glomeruli with medium containing pooled normal serum (1:50 dilution) served as a negative control, and incubation with known active FSGS serum (1:50 dilution) served as a positive control. To determine the lowest concentration of protein required to cause an increase in permeability (Palb ≥ 0.5), test samples were serially diluted with TBS and activity was measured in duplicate experiments.
Injection of 70% Supernatant, Collection of Pre- and Postinjection Urine, and Measurement of Urinary Protein and Creatinine
Seventy percent supernatant from plasma of four patients with recurrent FSGS in renal allografts or from normal pooled plasma were used for intravenous injection into rats. Urine samples were collected on sodium azide (50 μl of 5%) to avoid bacterial contamination. Rats were housed in metabolic cages overnight (about 18 h) before injection to collect urine for preinjection urinary protein determination. The 70% supernatant from normal or FSGS plasma was injected intravenously in anesthetized rats. Rats were allowed to recover from anesthesia and transferred to metabolic cages. Preliminary data showed no difference in urine protein up to 6 h after injection of FSGS plasma preparation, and this duration allowed the animals to recover from anesthesia. Urine samples were collected for 18 h, after the 6-h recovery period; volumes were recorded, centrifuged at 4°C, and supernatants were stored at -20°C.
Creatinine in urine samples was measured using a creatinine/PAP kit (Boehringer Mannheim, Indianapolis, IN). This assay is based on an enzymatic conversion of creatinine to creatine to sarcosine. The latter is oxidized to generate hydrogen peroxide for use in producing a red-colored benzoquinone-imine dye, which is measured spectro-photometrically at 510 nm. Urine protein was measured by the Coomassie Blue dye reagent as described above.
Statistical Analyses
Results are expressed as mean ± SD in Tables 1 and 2. Palb values (mean ± SEM) for FSGS plasma fractions were compared with normal plasma using t test, and P < 0.01 was accepted as significant (see Figure 2, A and B). Values for urinary proteins and creatinine are expressed as mean ± SEM. n represents the number of rats in each group. Values among various groups were compared using one-way ANOVA, and significance was defined as P < 0.01 (see Figure 5).
Demographic characteristics of the subjects included in the study and Palb of plasmapheresis fluida
Effect of removal of lipoproteins and chylomicrons on FS activitya
(A) Albumin permeability (Palb) of isolated rat glomeruli after incubation with fractions of 70% FSGS supernatant obtained by dialysis. Size determination by dialysis through membranes of different molecular weight cutoff (MWCO) was carried out as described. NPP, normal pooled plasma. Values are mean ± SEM (n = 10). *P < 0.01 compared with NPP. 70% FSGS is the supernatant at 70% ammonium sulfate saturation obtained from FSGS plasma. The 12- to 14-kD Retentate and 50-kD Retentate are the residual fractions retained after dialysis in 12- to 14-kD or 50-kD MWCO tube. (B) Palb of isolated rat glomeruli after incubation with fractions of 70% FSGS supernatant obtained by ultrafiltration through membranes. Size determination by ultrafiltration through membranes of different MWCO was carried out as described. Values are mean ± SEM (n = 10 patients). *P < 0.01, significantly different values from NPP. 70% represents the untreated supernatant at 70% ammonium sulfate saturation. 50-kD Retentate and 30-kD Retentate are the residual protein that did not pass through membrane of 50-kD and 30-kD MWCO, respectively. 50-kD Filtrate and 30-kD Filtrate are the protein that passed through 50-kD and 30-kD membrane, respectively.
Proteinuria in rats after intravenous injection of 70% supernatant from FSGS plasma. Effects of injecting 70% supernatant from normal pooled plasma or FSGS plasma on rat urinary protein are shown. Preinjection urine protein and creatinine were determined in overnight (18 h) collection for each rat. Rats were anesthetized and injected intravenously with 1 ml of 70% supernatant containing 12 mg of protein from normal or FSGS plasma. Postinjection urine samples were collected from 6 to 24 h after injection, constituting an 18-h collection period. Seventy percent supernatants from four FSGS plasma samples were used in separate experiments. Pre- and postinjection urinary protein (mg protein/mg creatinine) were compared. Values represent mean ± SEM. n represents the number of animals studied, and P < 0.01 was accepted as significant.
Results
Documentation of FS Activity in Plasma of FSGS Patients
Demographic characteristics of the patients studied and the initial Palb of the plasmapheresis samples are shown in Table 1. Plasma from each patient with FSGS caused a significant increase in Palb. The volume of plasma obtained from plasma-pheresis ranged from 960 to 4450 ml (typically about 4000 ml). It is important to note that plasmapheresis fluid contains both patient plasma and the human albumin that was used as a replacement solution. Total protein in the samples ranged from 30 to 170 g.
Effect of Removal of Lipids by Organic Solvents
To determine whether the FSGS factor is a lipid, we used an aliquot of cryoprecipitate-free plasma and extracted total lipids by chloroform/methanol (2:1) as described. The amount of total lipids in the plasma specimens averaged 570 mg/dl (Table 2). The lower chloroform phase contained 570 ± 210 mg/dl lipids and no detectable protein. The lipids extracted by this method did not increase Palb of isolated rat glomeruli.
Enrichment of FSGS Factor
Precipitation of Lipoproteins and Chylomicrons. Initial treatment of plasma with high molecular weight dextran sulfate and calcium chloride removed 83.5% of total lipids and 15.5% of the total protein without affecting the FS activity (Table 2).
Precipitation of Proteins by Ammonium Sulfate. The lipoprotein and chylomicron-free supernatant was further enriched by a two-stage precipitation of plasma proteins with ammonium sulfate. In the first step, ammonium sulfate was added to 50% saturation, which precipitated 38% of the proteins. This precipitate did not redissolve completely and was discarded. The supernatant obtained after centrifugation contained about 62% of the initial plasma protein and tested positive for the FS activity.
Increasing the concentration of ammonium sulfate in the supernatant from 50 to 70% resulted into precipitation of the majority of plasma proteins. After centrifugation, a supernatant containing 1.8% of the initial plasma protein (Figure 1) was obtained. The precipitate at 70% ammonium sulfate saturation could be redissolved and did not show FS activity. The residual protein in the supernatant at 70% ammonium sulfate saturation (70% supernatant) could be precipitated by increasing ammonium sulfate to 80% saturation (70 to 80% precipitate). The 70 to 80% precipitate had FS activity in that it increased glomerular albumin permeability in the in vitro bioassay. The supernatant at 80% ammonium sulfate saturation did not contain any detectable protein (data not shown) and tested negative for FS activity. As shown in Table 3, we achieved a 100-fold concentration of the FSGS factor by sequential removal of the cryoprecipitate, lipoproteins, and most immunoglobulins and albumin from the plasma of FSGS patients by following the protocol described above. No further enrichment was afforded by precipitation at 80% ammonium sulfate saturation.
Protein removal by sequential precipitation. Plasmapheresis fluid from individual focal segmental glomerulosclerosis (FSGS) patients was processed separately according to the methods described in the text. Average values of the percent protein removed from the plasma are presented as mean ± SD (n = 15 patients). Values for protein in supernatants after lipoprotein removal (Post Lipo) at 50 and 70% ammonium sulfate saturation are shown. The amount of protein in the supernatant at 80% saturation of ammonium sulfate was calculated as: Protein in the 70% supernatant — The amount of protein in the 80% precipitate. The number above each bar represents the percent of protein removed from the plasma specimen.
Change in total protein and FS activity with purificationa
Characteristics of the FSGS Factor
Every plasma specimen caused an increase in Palb of isolated rat glomeruli. The activity was not found to alter after storage up to 3 yr at -20°C. Addition of the protease inhibitor PMSF or a cocktail of inhibitors also did not make any discernible difference in activity (data not shown). Thus, we have not found any obvious difference in FS activity due to prolonged storage of samples at -20°C or by addition of protease inhibitors.
Some biochemical characteristics of the enriched FS activity in the 70% supernatant fraction are shown in Table 4 and Figure 2, A and B. Heating the 70% supernatant for 10 min or longer at 100°C abolished its effect on glomerular albumin permeability. The addition of protease also eliminated the FS activity (Table 4).
Effect of heat and protease treatment on FSGS activitya
As shown in Figure 2A, FS activity in the 70% supernatant was retained in the 12- to 14-kD MWCO dialysis tube but not in the 50-kD MWCO membrane. However, FS activity was recovered from the retentate when untreated FSGS plasma was dialyzed in a 50-kD membrane.
Centrifugation-based membrane ultrafiltration of the 70% supernatant from FSGS plasma showed that the FS activity passed through a 50-kD membrane and was recovered in the 50-kD filtrate. However, the FS activity did not pass through 30-kD MWCO membrane and was recovered in the 30-kD retentate (Figure 2B). These results suggest that the FS activity in the 70% supernatant can be recovered from a 30- to 50-kD fraction.
Electrophoresis
SDS-PAGE of plasma and its fractions showed several bands corresponding to proteins of a wide molecular size range. Figure 3 represents a typical SDS-PAGE separation of proteins in different fractions obtained from FSGS plasma. A comparison of initial plasma protein and the 70% ammonium sulfate fractions showed a decrease in high molecular weight proteins consistent with the removal of proteins such as immunoglobulins and lipoproteins.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of proteins at different stages of purification of the FSGS factor. An equal amount of total protein from each stage of purification was analyzed by SDS-PAGE on a 4 to 20% gradient Tris-glycine gel and developed by Coomassie-G-250 staining. Lanes on the gel were designated as follows: STD, wide range standard protein mixture was used as a reference; lane 1, serum from FSGS patient; lane 2, FSGS plasmapheresis fluid; lane 3, supernatant after precipitation with dextran sulfate; lane 4, supernatant at 50% ammonium sulfate saturation; lane 5, supernatant at 70% ammonium sulfate saturation.
Figure 4 illustrates electrophoretic separation of the proteins in 70% supernatants from normal (lane A) and plasma from two FSGS patients who experienced recurrence of FSGS in renal allografts (lanes B and C) under nondenaturing conditions. Lane B is from an anuric patient on hemodialysis, and lane C is from a patient with nephrotic syndrome due to recurrent FSGS. An increase in the low molecular weight protein fraction is evident in both lanes B and C compared with lane A.
Polyacrylamide gel electrophoresis of 70% supernatants from normal and FSGS plasma under nondenaturing conditions. Electrophoretic analysis of total protein in the 70% supernatants using a 4 to 20% gradient Tris-glycine gel under nondenaturing conditions followed by silver staining. Supernatants at 70% ammonium sulfate saturation containing equal amounts of protein from three patients were used for electrophoresis in separate lanes. Lanes on the gel were designated as follows: Lane A, normal pooled plasma; lane B, plasma from a patient currently on hemodialysis with end-stage renal disease after recurrent FSGS; lane C, plasma from recurrent FSGS patient.
In Vivo Effect of the 70% Supernatant
Amount of urinary protein (mg protein/mg of creatinine) before and after intravenous injection of 70% supernatant from normal or FSGS plasma into rats is shown in Figure 5. Injection of FSGS 70% supernatant caused a significant increase in urinary protein from 3.1 ± 1.8 (preinjection) to 9.4 ± 2.4 mg (6 to 24 h postinjection). There was no increase in urinary protein (mg protein/mg creatinine) in rats injected with normal 70% supernatant. As shown, the increase in urinary protein caused by the injection of FSGS 70% supernatant from four patients with recurrent FSGS was consistently present, suggesting that the increase in urine protein by FSGS 70% supernatant is not a patient-specific phenomenon.
Discussion
Massive proteinuria and altered glomerular histology are characteristic features of FSGS. These changes indicate a damaged glomerular filtration barrier. A circulating factor has been postulated to be the causative agent. The in vitro bioassay used in our laboratory is based on the observation that a substance (FSGS factor) in the plasma or serum of some patients with FSGS increases glomerular albumin permeability (Palb), whereas normal pooled human serum or sera from patients with other renal diseases do not increase Palb (20). This method allows us to study the direct effect of a given agent on the filtration barrier of glomeruli without the involvement of hemodynamic parameters, or cellular or humoral/immune responses (18,26). An adaptation of this method has been reported by other investigators (27). Using this bioassay, we have shown that the presence of a high level of activity (FS activity) in serum from patients with primary FSGS before transplantation is strongly associated with subsequent recurrence of proteinuria. This activity is diminished after plasmapheresis and is recoverable from discarded plasma (20).
The data obtained in the present studies were collected using discarded plasmapheresis fluid from 14 patients with a history of FSGS recurrent proteinuria, and one patient who is currently on hemodialysis due to prior allograft loss because of recurrence. Because FSGS has been found to be prevalent among children (1) and adults (2,3), and its incidence has been reported to have increased during the past two decades (4,5), we included adult and pediatric patients of both genders, as well as patients with different racial backgrounds. The plasma from each of these patients with FSGS caused an increase in Palb (Table 1). Purification of protein from each of these samples was carried out individually.
In the absence of any information on the chemical nature of the active factor, we used classical techniques of biochemical analysis and purification to enrich the substance. First, we addressed the possibility that the effector molecule could be a lipid. Total lipids from an aliquot of the cryoprecipitate-free plasma were extracted by chloroform/methanol (2:1) and tested for activity. The lipid extract did not increase Palb of isolated rat glomeruli. Although this technique provides a good means to extract total lipids, it denatures most plasma proteins irretrievably and is unsuitable for purification of unknown biologically active proteins. Preliminary work showed that large amount of lipids in FSGS plasma interfered with sedimentation of protein precipitates in viscous solutions at higher concentrations of ammonium sulfate. Thus, to remove the majority of lipids in an aqueous milieu without the use of organic solvents, high molecular weight dextran sulfate was used to selectively precipitate lipoproteins and chylomicrons from the plasma (23, 24). The precipitate accounted for 83.5% of total lipids and 15.5% of total proteins. It had to be discarded without testing for FS activity as it could not be redissolved (Table 2).
Plasma from which cryoprecipitate, lipoproteins, and chylomicrons had been removed was subjected to sequential precipitation of proteins by ammonium sulfate (28, 29). At 50% ammonium sulfate saturation, known to precipitate the majority of immunoglobulins, the supernatant retained FS activity and approximately 62% of the total protein. We could not determine the activity in the precipitate because it did not redissolve into a clear solution. The precipitate obtained at 70% saturation of ammonium sulfate is known to contain most of the plasma albumin (29). Redissolved precipitate did not increase glomerular albumin permeability in vitro and was discarded. The 70% supernatant contained FS activity and about 1.8% of the total plasma protein. This methodology was repeated with plasma from each of the 15 FSGS patients, and there was a consistent 100-fold increase in the specific activity. Although the active fraction could be precipitated by raising ammonium sulfate to 80% saturation, this step removed only 0.37% of the proteins. We decided not to include this step in our purification protocol because it did not remove significant amounts of inactive proteins from 70% supernatant, and considerable loss of the active material was observed during centrifugation of the 80% ammonium sulfate supernatant.
Several observations in the present studies suggest that the FSGS factor is a protein. (1) FS activity is present in the supernatant obtained after precipitation of chylomicron-lipoproteins fraction and is absent in the total lipid extract. (2) The active component could be fractionated by ammonium sulfate-precipitation, which is a common technique for protein purification. (3) The active substance is denatured by heat. (4) Immobilized protease abolishes FS activity.
We used electrophoresis to follow the changes in protein profiles at different stages of purification. A comparison of the results of SDS-PAGE of proteins at different stages of fractionation corroborates the removal of a large percentage of proteins from the plasma specimens. Certain low molecular weight proteins, faintly noticeable in the unprocessed plasma, appeared more prominently in the 70% supernatant (Figure 3). We followed this observation by comparing electrophoretic separation of 70% supernatants from normal and FSGS plasma under nondenaturing conditions and developed by silver staining. Results indicate that a lower molecular weight protein fraction was more abundant in the FSGS preparation compared with the normal plasma. Since electrophoretic separation under native conditions is influenced by size as well as the charge, components of this band correspond to proteins with low molecular weight and greater negative charge (Figure 4). Further analysis of this fraction is in progress.
Previously, we had found that the FS activity in the untreated FSGS plasma was associated with a 50- to 100-kD fraction as determined by size exclusion chromatography (20). Similarly, results of the current studies show that the active substance in the whole FSGS plasma does not pass through a 50-kD dialysis membrane. However, activity was found to be associated with a 30- to 50-kD fraction in the 70% supernatant prepared from FSGS plasma (Figure 2, A and B). This apparent discrepancy could be due to degradation of the active substance during purification or aggregation of molecules in the whole plasma. As mentioned earlier, we have not found any obvious loss of activity in the plasma samples stored up to 3 yr or fractionated without sodium azide and protease inhibitors. Thus, the difference in size estimates of the FSGS factor from whole plasma and the 70% supernatant appears to be a result of the complexity of native plasma, which allows noncovalent associations among molecules in the absence of detergents or chaotropic agents. Since the in vitro bioassay of FS activity relies on permeability characteristics of the glomerular membranes, which are greatly influenced by detergents, we have carried out all steps of purification without detergents. Size determination experiments will be repeated with pure preparations of the FSGS factor in the presence of detergents and chaotropic agents.
Several investigators have described vascular permeability factors in the context of minimal change nephrotic syndrome or FSGS (30,31,32,33,34,35). None of these factors has been characterized fully. The known characteristics of the FSGS factor that we are studying appear to be different from permeability factors under investigation by others. The vascular permeability factor described by Lagrue and coworkers is a 12-kD protein that is elaborated by concanavalin A-stimulated T lymphocytes of patients with nephrotic syndrome (30, 31). Vascular permeability factor causes increased permeability in skin capillaries, although in our preliminary studies, the FSGS factor did not alter permeability of skin capillaries in rabbits. Bakker and colleagues have studied a factor with an apparent molecular weight of 100 kD that neutralizes glomerular anionic sites, increases permeability of skin capillaries and monolayers of endothelial cells, and activates macrophages to produce superoxide (32). Levin et al. have described a cationic protein of more than 150 kD size in the urine and serum of patients with steroid-responsive nephrotic syndrome (33). Alterations in glomerular histology or permeability by plasma of nephrotic patients or by lymphocyte products have been reported since 1981, but the active substances have not been identified (34, 35). We have yet to study the effect of the FSGS factor on glomerular structure.
We have demonstrated that systemic injection of the 70% supernatant of FSGS plasma caused an increase in urinary protein excretion in rats, whereas injection of normal 70% supernatant had no significant effect on urinary protein excretion. The proteinuria caused by FSGS 70% supernatant was not evident during the first 6 h after injection but was present in every rat between 6 and 24 h. Detailed studies on the time course of proteinuria are in progress. Previous attempts to induce proteinuria in animals by injecting patient plasma (16) or its fractions (14) have not yielded consistent results. Several reports of proteinuria in experimental animals after injection of FSGS serum or plasma have been published (16, 36). However, subsequent studies have not documented a consistent response of individual rats to injection with FSGS serum or plasma or of material derived from these sources. Dantal et al. (14) documented an increase in albumin excretion after injecting FSGS plasma fraction as determined by RIA but not total urinary protein. We believe that our in vivo results are a consequence of using a preparation that has been significantly purified, and thus provide a higher dose of the FSGS factor.
It is possible that the observed proteinuria induced in rats is not a unique effect of the 70% supernatant from FSGS plasma. We will test identical preparations of plasma from patients with other renal diseases after establishing a complete protocol for purification of FSGS factor. It is unlikely that these preparations will cause proteinuria, as we have previously reported that sera from patients with corticosteroid-sensitive nephrotic syndrome, hypertensive nephropathy, membranous nephropathy, and polycystic kidney disease do not increase glomerular albumin permeability in our in vitro bioassay (20).
The mechanism by which the FSGS factor increases glomerular albumin permeability in vitro or in vivo remains to be elucidated. Increased glomerular albumin permeability after 10 min of incubation in vitro raises the possibility that the FSGS factor interacts with membrane components of the exposed glomerular epithelial cells. Glomerular epithelial cells have been shown to play a crucial role in maintaining the filtration barrier (37, 38). The magnitude of the effect of FSGS sera on glomerular permeability in vitro was comparable to that of anti-Fx 1a antibody (39), superoxide (40), hydroxyl ion (41), or tumor necrosis factor-α (42). The rapidity of increase in glomerular permeability suggests that the immediate effect of the FSGS factor is unlikely to be mediated by metalloproteinase-3 (43) or through charge neutralization by protamine (18), as these agents required prolonged incubations of more than 4 h and 1 h, respectively.
In summary, our results strongly indicate the presence of a factor in the plasma of some patients with FSGS that can increase glomerular albumin permeability in vitro and that this factor can be recovered and enriched from the plasma of these patients. Additionally, we have demonstrated that injection of the enriched preparation of this factor causes proteinuria in rats. Further purification of the FSGS factor is ongoing.
Acknowledgments
Acknowledgments
This work was supported by National Institutes of Health Grant RO1 AM-22040 and a grant from the American Heart Association, Wisconsin Affiliate. We thank Ms. Xiu Li Ge and Ms. Chen Xu for technical assistance. We also thank Dr. R. Sreenivas Reddy for performing microsurgical procedures on rats, and our numerous colleagues who provided us with plasma samples and clinical information regarding FSGS patients. We are also indebted to our colleagues Drs. William G. Couser, Jared J. Grantham, and Billy G. Hudson for their support and advice, to all of our patients for their unflagging determination, and particularly to Mr. Todd Whitlock.
Footnotes
American Society of Nephrology
Preliminary results of this study were presented at the 28th annual meeting of the American Society of Nephrology, November 1995, San Diego, CA.
- © 1999 American Society of Nephrology