The effect of proteinase inhibitors on glomerular albumin permeability induced in vitro by serum from patients with idiopathic focal segmental glomerulosclerosis
Michele Carraro1,
Cristina Zennaro1,3,
Mary Artero1,
Giovanni Candiano2,
Gian Marco Ghiggeri2,
Luca Musante2,
Cristina Sirch1,
Maurizio Bruschi2 and
Luigi Faccini1
1 Department of Medicina Clinica, University of Trieste, Trieste, 2 Unit and Laboratory of Nephrology, Istituto G. Gaslini, Genoa and 3 Department BBCM, University of Trieste, Trieste, Italy
Correspondence and offprint requests to: Michele Carraro, MD, Dipartimento di Medicina Clinica, Università degli Studi di Trieste, Ospedale di Cattinara, Strada di Fiume 447, 34149 Trieste, Italy. Email: m.carraro{at}fmc.units.it
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Abstract
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Background. The putative circulating factor responsible for the glomerular permeability alterations induced in vitro by serum from patients affected by focal segmental glomerulosclerosis (FSGS) remains unidentified. We have observed that a serine proteinase isolated from patient serum increases albumin permeability in isolated glomeruli. The objective of the present study was to determine the effect of various proteinase inhibitors on glomerular albumin permeability (Palb) in isolated glomeruli incubated with FSGS serum.
Methods. The study population consisted of 12 FSGS patients (eight males; mean age: 21±10 years) previously shown to have elevated serum albumin permeability activity. Palb was determined by measuring the change in glomerular volume induced by applying oncotic gradients to isolated healthy rat glomeruli treated with patient serum in comparison to control serum. Solutions of seven different proteinase inhibitors (0.5 mg/ml) were added to the incubation media with the sera (1:1 vol/vol): serine proteinase inhibitors (PMSF, leupeptin, aprotinin, gabexate mesylate), the cysteine proteinase inhibitor E-64, the metalloproteinase inhibitor EDTA and the aspartate proteinase inhibitor pepstatin. Sera from the same patients were also tested with the addition to the incubation media of quinaprilat, an inhibitor of the metalloproteinase angiotensin-converting enzyme.
Results. Mean Palb of the sera was 0.86±0.11, with the addition of PMSF 0.41±0.09, leupeptin 0.30±0.17, aprotinin 0.09±0.14, gabexate mesylate 0.27±0.25, E-64 0.81±0.09, EDTA 0.68±0.10 or pepstatin 0.76±0.11. The mean Palb of the sera combined with quinaprilat was reduced to 0.34±0.35. Thus, only the serine proteinase inhibitors consistently blocked the increased Palb induced by the FSGS sera.
Conclusions. In the cascade of events that lead to the initiation of glomerular fibrosis in FSGS, the putative glomerular permeability factor associated with FSGS may require a serine proteinase to effect its activity.
Keywords: circulating permeability factors; focal segmental glomerulosclerosis; glomerular permeability; proteinase inhibitors; serine proteinase; serum proteinases
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Introduction
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The pathogenesis of both minimal change disease and idiopathic focal segmental glomerulosclerosis (FSGS) is thought to involve a circulating humoral factor which, directly or indirectly, compromises the glomerular permeability barrier to protein [1]. Several investigators have detected permeability alterations induced by patient serum, serum fractions or lymphokines [24] using intact laboratory animals [3,4], isolated kidney preparations [5], isolated glomeruli [2,6,7] or glomerular epithelial cell culture [8] as assay systems. Some research groups have partially characterized [3] or purified [5,7] substances from patient serum which provoke permeability alterations in the same assay systems, although a single permeability factor unique to FSGS patients has not been identified.
The mechanism by which FSGS serum overcomes the glomerular permeability barrier has not been studied. It is thought not to involve complement [2] or immunoglobulins, although the factor seems to be retained on protein-A columns during immunoabsorption [9]. Trachtman et al. [10] found that an FSGS serum fraction concentrate added to cultured rat mesangial cells inhibited inducible nitric oxide synthesis, which suggested that the factor might play a dual role in promoting fibrogenesis and inducing permeability alterations, although the latter mechanism was not specifically addressed.
Our interest in the role of serum proteinases in the events that lead to increased glomerular albumin permeability in the presence of FSGS serum was spurred by the observation that the permeability activity was retained on chromatographic columns containing proteinase inhibitors. Subsequently, we purified by two different isolation procedures a mannose-binding lectin (MBL)-associated serine proteinase from FSGS serum which induced increased albumin permeability in isolated glomeruli [7]. However, Sharma et al. [3] found no effect of a cocktail of proteinase inhibitors on FSGS serum permeability activity using the same assay system. Thus, the objective of the present study was to determine the effect of various proteinase inhibitors in vitro, including an inhibitor of the metalloproteinase angiotensin-converting enzyme (ACE), on glomerular albumin permeability activity associated with FSGS serum.
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Subjects and methods
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Patients
Frozen serum samples were available from 12 patients who had presented with steroid-resistant nephrotic syndrome, who subsequently underwent renal biopsy that demonstrated idiopathic FSGS and in who elevated serum permeability activity had previously been demonstrated in the isolated glomeruli assay (see below).
Proteinase inhibitors
The proteinase inhibitors used in the study were purchased from commercial sources and are listed in Table 1 along with their usual substrates. All the inhibitors were dissolved in double distilled water, with the exception of PMSF which was dissolved in ethanol, to a final concentration of 0.5 mg/ml. Quinaprilat was dissolved in absolute methanol (5 ml) and then diluted in distilled water (100 ml) to a final concentration of 104 M.
Determination of glomerular albumin permeability (Palb)
The method for measuring permeability activity was adapted from the original description by Savin et al. [11]. Glomeruli were isolated from the renal cortex of healthy male SpragueDawley rats weighing 200300 g by sieving in isotonic phosphate buffer solution. The pH had been titrated to 7.4. The medium also contained 5 g/dl bovine serum albumin (BSA) as an oncotic agent. The isolated glomeruli were then washed in 1 ml fresh medium.
The solutions of the proteinase inhibitors were mixed with an equal volume of patient serum and left on ice for 10 min. An aliquot of 0.1 ml of the glomeruli was then incubated at 37°C for 10 min in 0.9 ml of medium that included 2% v/v patient or pooled normal human serum or 4% v/v of the inhibitorFSGS serum mixture. The glomeruli were then plated onto a glass coverslip, coated with poly-L-lysine as an adherent and covered with fresh medium. The samples were masked to eliminate operator bias.
The rationale and methodology for the determination of albumin permeability has been described in detail in the literature [2,11]. In brief, each of 1016 glomeruli per test serum was videotaped through an inverted microscope before and after a medium exchange to one containing 1 g/dl BSA. The medium exchange created an oncotic gradient across the basement membrane resulting in a glomerular volume change [
V = (Vfinal Vinitial)/Vinitial], which was measured off-line by a video-based image analysis program (MCID; Imaging Research Inc., St Catharines, Ontario, Canada). The computer program determines the average radius of the glomerulus in two-dimensional space and the volume is derived from the formula V = 4/3
r3. The magnitude of
V was related to the albumin reflection coefficient,
alb, by the following equation: (
alb)experimental =
Vexperimental/
Vcontrol; the
alb of the control glomeruli was assumed to be equal to 1. Palb is defined as (1
alb) and describes the movement of albumin subsequent to water flux. When
alb is zero, albumin moves across the membrane with the same velocity as water and Palb is 1.0. Conversely, when
alb is 1.0, albumin cannot cross the membrane with water and Palb is zero.
Glomerular volume response to a different oncotic agent
To confirm that the altered volumetric response reflected altered albumin permeability, we also studied the effect of an impermeable solute, high molecular weight neutral dextran. We did these proofs at concentrations of 1% and 4% (v/v) for both BSA and dextran. We measured the glomerular volume change created by an oncotic pressure gradient on the control glomeruli, the glomeruli incubated with solutions of aprotinin, with human control serum, with patient FSGS serum and solutions of aprotinin and patient FSGS serum.
Inhibitor scavenging capacity
To determine whether the proteinase inhibitors acted on the permeability factor or on the glomerular permeability response in a non-specific way, the response of isolated glomeruli after exposure to superoxide generated by xanthine and xanthine oxidase was studied after the addition of aprotinin, according to the method described by Dileepan et al. [12]. Briefly, isolated glomeruli were incubated in medium containing 0.1 mmol/l xanthine for 10 min at 37°C. Superoxide was generated by the addition of xanthine oxidase 20 U/ml, 10 µl in 1 ml of 5 g/dl BSA, and incubation continued for 10 min. Parallel incubations were run with superoxide dismutase 300 U/ml and aprotinin at a final concentration of 0.5 mg/ml.
Data and statistical analysis
The data are presented as means±SD and comparisons among experiments were done by analysis of variance. A Palb value greater than 0.5 represents glomerular albumin permeability significantly greater than normal [2]. We adhered to the convention of using 0.5 as cut-off (i.e. considering Palb as a discrete variable) because it exceeds the average value derived using sera of control subjects used as a negative control by two SDs and allowed us to exclude non-specific activity with assurance.
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Results
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Patients
Eight of the 12 subjects were males and the mean age at the time of the study was 21.3±9.5 years. Ten patients had undergone renal transplantation and by the time of the study seven had suffered a recurrence of FSGS following transplantation.
Glomerular volume response to a different oncotic agent
The glomerular volume change in response to different oncotic agents (albumin and dextran) are shown in Table 2. The volume increase in response to the dextran gradient (
Vdextran) in experiments 1, 2, 3 and 5 was similar to that observed using albumin (
Valbumin). In experiment 4 (isolated glomeruli incubated with FSGS patient serum) the volume increase to the dextran pressure gradient was higher than the response to the albumin gradient. These data indicate that the altered volumetric responses observed in the study do, indeed, reflect an exclusive alteration to albumin permeability that should provide a sensitive probe for changes in the permeability barrier of the glomerular capillary.
Effect of EDTA on glomerular permeability
The results of the EDTA effect on glomerular volume variation are shown in Figure 1. EDTA was not able to alter albumin permeability per se or increase the damaging effect of FSGS serum. Glomeruli were also incubated with the serine proteinases tested (aprotinin, PMSF, leupeptin and gabexate mesylate) without the addition of FSGS serum in the same manner. All these serine proteinases were not able to alter albumin permeability.

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Fig. 1. Effect of proteinase inhibitors on albumin permeability in isolated glomeruli incubated with FSGS serum, compared with control.
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Inhibitor scavenging capacity
Isolated glomeruli exposed to xanthine/xanthine oxidase showed increased albumin permeability (0.65±0.2; range: 0.630.67). This reaction was abrogated by simultaneous incubation with superoxide dismutase (0.14±0.14; range: 00.28), but not by incubation with aprotinin (0.75±0.01; range: 0.760.74). These data suggest the specific action of aprotinin on the permeability factor(s).
Effect of proteinase inhibitors on Palb
The data regarding the effect of FSGS serum and proteinase inhibitors on Palb are shown in Table 3. Only the serine proteinase inhibitors consistently blocked the increased Palb induced by the FSGS sera. Figure 2 provides a graphic representation of the inhibitor effect.

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Fig. 2. Effect of EDTA of glomerular volume variation. Asterisks indicate experimentals with value significantly different in comparison to another experiment. N, the number of glomeruli studied; Glom, only isolated glomeruli; Glom + Control sera, isolated glomeruli incubated with serum control; Glom + FSGS sera, isolated glomeruli incubated with serum patient FSGS. Twelve measurements were performed for each experiment.
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Table 4 contains the data regarding the effect of quinaprilat and FSGS serum on Palb. The ACE inhibitor significantly reduced Palb in 8 of the 12 sera with a coefficient of variation of 103%.
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Discussion
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The present study demonstrated that serine proteinase inhibitors effectively block the glomerular permeability alterations induced in vitro by FSGS serum. A previous study by Sharma et al. [3] using the same method to determine permeability activity, however, showed that PMSF, a serine proteinase inhibitor, and a mixture of inhibitors did not alter the Palb of the FSGS serum. In the present study, PMSF did inhibit serum permeability activity, although it was numerically the least effective of the serine proteinase inhibitors studied, barely reducing the Palb below significant levels (0.41±0.09). Another difference between the two studies may be related to the technique used to prepare the inhibitors, which was not described in the previous paper. Finally, the components of the inhibitor cocktail were not characterized, thus, precluding comparison with the present study.
FSGS is a common histopathological entity and is described as a primary or idiopathic form or a secondary phenomenon associated with other diseases, such as reflux nephropathy, obesity, heroin nephropathy, etc. The patients included in the present study were representative of the fraction of patients with primary FSGS who have high serum permeability activities and are the most likely to have recurrence of the disease following renal transplantation. The findings of our study should be considered relevant to this group of patients and not to the entire spectrum of diseases characterized by FSGS.
Tissue proteinases may be divided in four major families of enzymes based on the residues found in their active sites [13]: aspartate, cysteine, serine or zinc (metallo-type). Aspartate and cysteine proteinases function at low pH and are found almost exclusively in the intracellular environment. In fact, the cysteine inhibitor E-64 and the aspartate inhibitor pepstatin used in the neutral pH conditions of our study did not alter the permeability activity of the FSGS serum. In the current literature, interest in proteinases and their inhibitors in the glomerulopathies centres on the metalloproteinases, both for their role in degradation and remodelling of extracellular matrix components and for the well-known affect of ACE inhibitors to reduce proteinuria in a variety of glomerulopathies, particularly diabetic nephropathy, either by a local or systemic haemodynamic effect on angiotensin or bradykinin. In the present study, EDTA, which non-specifically chelates the zinc moiety of metalloproteinases by a concentration-dependent mechanism, was ineffective in blocking the permeability activity of the FSGS serum. EDTA alone had no effect on albumin permeability and was not able to increase the effect. The same control was not applied to glomeruli incubated with the serine proteinase inhibitors.
On the other hand, the metallo-ACE inhibitor quinaprilat significantly reduced Palb, albeit with a high coefficient of variation. In the isolated glomeruli assay that we employed, haemodynamic alterations should not have played a role and, thus, a protective effect of the metalloproteinase inhibitor would have been strictly local. In a recent in vitro study of the effect of quinaprilat on Palb in isolated glomeruli from diabetic rats [14], we showed that very low concentrations of quinaprilat were effective in reducing Palb, lower than the concentration required to inhibit kidney ACE. It should be emphasized that in the present experiment the inhibitors were first left on ice to interact with the FSGS serum, rather than with the glomeruli before the serum was added, since our principal interest was focused on the FSGS serum components that induce permeability alterations. Thus, if a role exists for metalloproteinases in the series of events that lead to glomerular permeability alterations induced by FSGS serum, it is likely non-specific and might not involve kidney ACE. Before drawing definitive conclusions regarding metalloproteinases in the present pathological model, other specific inhibitors should be tested in a range of concentrations.
Once it was known that elastase, a polymorphonuclear serine proteinase, could induce proteinuria when infused into the rat renal artery [13], serine proteinases were considered potential mediators of glomerular disease. In the present work, all the serine proteinase inhibitors studied were effective in blocking the permeability activity of the FSGS serum, particularly aprotinin, a specific inhibitor used clinically to promote haemostasis during cardiac surgery. Leupeptin is described as a serinecysteine proteinase inhibitor and functions on substrates of both enzyme families, although not those located in lysosomes or polymorphonuclear granules. PMSF and gabexate mesylate (also known as FOY) are both well-known synthetic serine proteinase inhibitors. Our interest in the study of proteinases as possible mediators in the pathogenesis of idiopathic FSGS was stimulated by the observation that the permeability activity of the serum was retained on chromatographic columns containing proteinase inhibitors. We recently isolated a MBL-associated serine proteinase, using two different chromatographic techniques, which induced increased glomerular albumin permeability in isolated glomeruli [7]. MBL is a serum oligopeptide that activates the classical complement pathway independent of C1q [15]. Lhotta et al. [16] measured MBL levels in serum from patients with a variety of glomerulonephritides as well as in renal biopsy specimens [16], including FSGS. Whereas MBL was detected in focal segmental deposits, there were no significant differences of MBL serum levels between patients and controls.
How might serine proteinases function in the pathogenesis of FSGS? One possibility is that they provoke glomerular damage directly, compromising the filtration barrier to protein, which subsequently leads to inflammation and fibrosis. Alternatively, in plasma they might activate, or be activated by, the so-called permeability factor hypothesized to play a role in the recurrence of FSGS following renal transplantation. It should be remembered that all tissue proteinases are synthesized as inactive pro-enzymes and the processes that lead to activation are normally tightly regulated. The pathogenesis of FSGS is also emerging as a complex process involving not only permeability factors but also naturally occurring inhibitors, inhibitor loss in the urine and genetic components [7,17,18], perhaps, resulting in a cascade of events that ultimately damages the glomerular visceral epithelial cell, which is thought to represent the key component of the filtration barrier to protein. Thus, it is not surprising that at some point in the cascade, serine proteinases would play a role. Interestingly, in a previous study by Sharma et al. [19], much longer incubation times with elastase or matrix metalloproteinase (stromelysin-1) were required before Palb increased in isolated glomeruli untreated with FSGS serum. Thus, the brief incubation times used in the present study suggest that a proteinase is not directly involved in provoking the glomerular damage. It should also be kept in mind that proteinase activity might play a non-specific part of increased glomerular permeability after a variety of insults, not only that induced by the hypothesized permeability factor.
Is there therapeutic potential for serine proteinase inhibitors in the course of idiopathic FSGS? Since we hypothesize that the step at which serine proteinases would have a crucial role in the pathogenesis of FSGS is at the beginning of the cascade, before significant fibrosis has occurred, such therapy might best be suited to patients with high risk of recurrence following renal transplantation. Aprotinin and gabexate mesylate have been used extensively for the treatment of human subjects for, respectively, haemostasis in cardiac surgery patients [20] and for the treatment of pancreatitis and disseminated intravascular coagulation [21]. Leupeptin is being considered as a therapy in neurodegeneration disorders [22]. A drawback of these drugs is that they must be administered intravenously. Aprotinin, in particular, may cause anaphylactic reactions with a second dose and it is not available in Europe since it is extracted from beef lung. Unfortunately, a simple animal model of recurrent FSGS (i.e. associated with elevated Palb activity) in which drug testing can be performed does not yet exist. Given the wide range of systemic and collateral effects produced by serine proteinase inhibitors, studies to determine their efficacy in the treatment of primary or recurrent FSGS must await a formulation that can be safely and conveniently administered repetitively.
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Acknowledgments
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We are indebted to Prof. R. Gusmano for her valuable contribution. The study was supported by continuous funding from the Fondo Malattie Renali del Bambino and by a grant from the Italian Ministry of Health (Progetto Finalizzato Patogenesi delle malattie della barriera di filtrazione glomerulare).
Conflict of interest statement. None declared.
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Received for publication: 12. 8.03
Accepted in revised form: 6. 5.04