Department of Nephrology, Prince of Wales Hospital, Randwick, NSW, Australia
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Abstract |
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Methods. The protein fraction of urine samples from nine patients with proteinuria >1.5 g/day was purified. A cell ELISA involving cultured HK-2 PTEC was used to investigate the capacity of urinary protein to promote the deposition of both C3 and C9 on the cell surface. The effect of variations in pH (5.58.0) and in the concentration of urea and ammonia was also examined. C3 was purified and used to further investigate the mechanism of complement deposition.
Results. Urine samples from the majority of patients induced deposition of C3 and C9 on the surface of HK-2 cells via the alternative pathway. This process was maximal at acidic pH values. Preincubation of urinary complement or serum with urea or ammonia inhibited C3 deposition. Purified C3 incubated with HK-2 cells showed no evidence of activation in the absence of other complement components.
Conclusions. These data suggest that bicarbonate protects against complement-mediated damage in the lumen by increasing the local pH, rather than by inhibiting the generation of ammonia. PTEC appear to activate complement through provision of a protected site on their surface, rather than by the activation of C3 by convertase-like protease(s).
Keywords: ammonia; cell ELISA; complement; pH; proximal tubular epithelial cells; urine
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Introduction |
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Complement proteins and their activation products may reach the PTEC via the glomerular filtrate, especially during states of non-selective proteinuria. They are also produced locally by the PTEC and other cells in the kidney [2]. Cultured PTEC synthesize mRNA for all proteins of the alternative complement pathway, including the regulatory factors I, H, and properdin. These form an active system able to generate and specifically cleave C3 to produce C3a [3]. Either filtered or locally synthesized complement may initiate or contribute to pro-inflammatory and metabolic events in the vicinity of PTEC. These events include assembly of the membrane attack complex (MAC) on the cell surface and/or ligation of specific receptors by activation products such as C3a [3,4]. Such processes are selective for the apical cell surface, but may result in secretion of proinflammatory mediators via both the apical and basolateral surfaces.
Many studies since those of Camussi et al. [5] have confirmed that the apical surface of PTEC activates the alternative pathway. Membrane-based inhibitors of complement such as CD46 and CD55 are virtually absent from this surface [6]. Complement activation causes an inflammatory response in PTEC. Binding of C3a to the C3a receptor leads to the production of TGF-ß1 [3,4]. Deposition of the MAC on freshly isolated PTEC induces a time-dependent calcium influx and synthesis of the proinflammatory cytokines IL-6 and TNF- [7]. It also increases fibronectin synthesis via generation of cAMP [8], the production of superoxide anion, chemiluminescent responses, and alterations to the cytoskeleton [4]. Urinary levels of the soluble form of the MAC (MW
106 kDa) are increased in complement-independent glomerular diseases such as diabetic nephropathy. This complex is little filtered at the glomerulus, and is therefore presumed to be generated by local activation of complement within the tubular lumen [9]. These in vitro data, and studies of the protective effect of complement inhibition on puromycin-induced tubulointerstitial disease in rats [10,11], emphasize the potential importance of complement in the progression of tubulointerstitial disease [9,12].
Previous studies of the effect of complement on proximal tubular cells have used diluted serum, rather than urinary samples. While the glomerular filtrate and collected urine differ in many respects, the latter can provide information on the degree of complement activation by filtered proteins, and allow for the contribution of complement proteins synthesized locally in the kidney. The influence of nucleophilic components such as urea or ammonia on urinary complement activity is unclear, although low levels of the latter have been reported to enhance complement activation in serum [13]. This observation suggested that administration of bicarbonate was protective against complement-mediated tubulointerstitial damage in both humans and animals by lowering ammonia levels in the tubular fluid [1315].
The aim of this study was to investigate the capacity of urinary complement components to promote the deposition of both C3 and C9 on the surface of cultured proximal tubular cells. We also investigated the mechanism of this process. Finally, the effect on complement activity of variations in pH and in the concentration of urea and ammonia were examined.
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Subjects and methods |
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Cell culture
The HK-2 PTEC has well-differentiated histochemical and functional characteristics, and shows experimental responses similar to those obtained with freshly isolated proximal tubular cells. In particular, HK-2 cells are epidermal growth factor and anchorage dependent, and show polarized morphology in forming apical microvilli and junctional complexes. HK-2 cells were obtained from the American Type Culture Collection and cultured in Keratinocyte-SFM (Life Technologies; Gaithersburg, MD, USA) supplemented with 2.5 mg/ml bovine pituitary extract and 0.25 µg/ml recombinant epidermal growth factor (Life Technologies). Medium was changed every 23 days.
Measurement of urinary complement deposition on PTEC
Effect of pH
The deposition of C3 and C9 on the apical surface of adherent and viable HK-2 cells under various conditions was determined by a cell ELISA. Cells were seeded in flat-bottomed 96-well plates in SFM and assays performed at confluence. Studies of the effect of pH on alternative pathway complement activity were performed within the range of pH 5.58.0 in Tris-maleate buffer (0.05 mol/l), containing 0.1 mol/l NaCl. Additional experiments in phosphate-buffered saline (PBS) at pH 6 or 7 were also performed. In each case, 7 mmol/l MgCl2 and 10 mmol/l ethylene glycol-bis(ß-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) were added to ensure selective measurement of alternative pathway activity. Aliquots of NHS and urine samples were partially purified to remove molecules of low molecular weight using Sephadex G25 columns equilibrated with normal saline. These G25 fractions were then mixed 1:1 with twofold concentrates of Tris-maleate solutions buffered to differing pH values. This allowed the effect of pH on complement activity to be determined independently of the presence of low-molecular-weight nucleophiles such as ammonia or urea. Subsequent experiments comparing complement activity in urinary proteins from nine patients with heavy proteinuria were undertaken at pH 6.
Effect of urea or ammonia
Serum and urine samples were buffer-exchanged into normal saline using Sephadex G25 columns. They were then mixed 1:1 with Tris-maleate buffer containing various amounts of either urea or ammonia at pH 6 to give a final concentration of 0 to 200 mmol/l urea or 0 to 10 mmol/l ammonia. These samples were each preincubated for 3 h at 37°C before exposure to PTEC to allow reactivity between these nucleophiles and complement.
Detection of C3 and C9
Adherent HK-2 cells were washed with PBS, then sites of non-specific binding blocked by incubation in 3% bovine albumin (BSA) in PBS for 60 min at 37°C. Cells were then incubated with diluted aliquots of NHS or urine samples for 45 min at 37°C. Deposition of complement on the apical surface was determined by the addition (30 min, 37°C) of a 1/1000 dilution of a monospecific antibody to C3 (Dako), or to C9 (Calbiochem) in blocking buffer. This was followed, after washing with PBS, by a second species-specific antibody conjugated to alkaline phosphatase, also in blocking buffer. Enzyme activity was detected by the addition of p-nitrophenol phosphate in 0.05 M Tris buffer, pH 9.6. Cells remained adherent on incubation with PBS and viability in the ELISA, as determined by trypan blue exclusion, was 95±1%. Control incubations were performed with sera or urine decomplemented by heating at 56°C for 60 min. (Incubation with ethylene diamino-tetraacetic acid (EDTA), a commonly used inhibitor of complement, led to unacceptable losses of cells).
In further experiments, cells were released from flasks by EDTA, washed with PBS, and incubated in suspension with dilutions of urine proteins purified by G25 column chromatography. Binding of C3 was determined as described above, except that cells were twice washed before resuspension in 10 ml buffer.
Measurement of complement deposition from normal serum
We compared the effects on urinary complement activity of variations in pH, urea and ammonia to their effects on complement activity in NHS. ELISA wells were coated for 1 h at 37°C and overnight at 4°C with 100 µg/ml inulin or lipopolysaccharide (E. coli; both from Sigma). These are activators of the alternative pathway. Non-specific binding was blocked by incubation with 3% BSA (60 min, 37°C). NHS was used at a final dilution of 1/10, and was made to pH 5.58, or preincubated with urea or ammonia at pH 6 as before. These preparations of NHS were incubated in the coated ELISA wells for 30 min at 37°C under conditions allowing only alternative pathway activity. Deposition of C3 and C9 was quantitated essentially as described above. Control experiments, where EDTA was also added, showed quantitative inhibition of C3 and C9 deposition.
Analysis of complement by Western blotting
Proteins from serum or urine were separated by reducing SDSPAGE on 6% or 8% gels followed by Western blotting onto nitro-cellulose membranes. Polyclonal antisera were used to detect C9 (Calbiochem) and C3 (Dako), and any fragments of these molecules. This was followed by the addition of monoclonal second antibodies conjugated to alkaline-phosphatase. Colour development was by nitroblue tetrazolium/bromochoro-indolyl phosphate.
Activation of C3 by HK-2 cells
The ability of HK-2 cells to cleave purified C3 and to activate the complement cascade was further examined. Human C3 was purified from 200 ml fresh human plasma by ion exchange and size-sorting chromatography in the presence of EDTA, aminocaproic acid, benzamidine and phenylmethylsulphonylfluoride, and aliquots stored at -70°C [16]. The preparation was >90% pure by SDSPAGE, and haemolytically active. This was shown by the ability of complement to specifically cleave the -chain of purified C3. Purified C3 was added to C3 deficient serum (Sigma), and incubated in the presence of Mg/EGTA and zymosan or cobra venom factor to activate the alternative pathway. Under these conditions, only haemolytically active C3 undergoes specific cleavage of the
-chain as demonstrated by SDSPAGE and Western blotting. Cleavage was prevented by the addition of EDTA. The specific activation of C3 in normal serum by HK-2 cells was similarly demonstrated; cells were incubated with 0.030.2% serum in PBS/Mg/EGTA.
The presence of C3-cleaving proteases was determined by incubating 106 HK-2 cells with 130 µl of C3 at 0.1 mg/ml in PBS adjusted to pH 6, or in complement fixation diluent (CFD) at pH 7.2. C3 was exchanged into these buffers by using Sephadex G25 columns. Cells detached by incubation with EDTA and scraping were washed, pelleted, then incubated with C3 in a round-bottomed tube for 2 h at 37°C in a shaking water bath. Similar experiments were performed with adherent cells. These were washed and incubated with C3 for up to 4 h. Samples were then analysed as above for cleavage of C3 by SDS-PAGE and Western blotting. All experiments were repeated a minimum of twice.
Statistical analyses
Data are presented as mean±SEM and were analysed with a two-tailed t-test using P<0.05 as a determinant of significance.
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Results |
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Complement deposition on HK-2 cells
In preliminary experiments, samples 2 and 8 gave the strongest deposition of both C3 and C9 and were used to establish the dose-response parameters of the cell ELISA (Figure 2). Urine samples were buffer-exchanged into saline by passage down a G25 column, resulting in a 1.4-fold dilution. Figure 2
shows that the amount of C3 and C9 deposited on the apical surface of HK-2 cells was proportional to sample dilution up to 1/20 of the G25 fraction of urine. Thus in all subsequent experiments, samples were diluted to a final concentration of 1/20 of a G25-purified fraction. Heat inactivation of both samples (56°C, 60 min), led to >90% inhibition of binding of both C3 and C9.
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The effect of urinary pH on complement deposition on HK-2 cells
The pH dependency of the deposition of urinary C3 and C9 via the alternative pathway is shown in Figure 4. Maximal activity occurred at acidic pH values, and significantly decreased at pH
6.5. Buffering capacity of the system was diminished at pH <5.5, and cell losses greatly increased. Figure 4
also shows a similar dependency on pH for the alternative pathway in NHS. Maximal deposition of C3 occurred at pH 6, and activity significantly fell in more acidic or basic (pH
7.0) conditions. Deposition of C9 also fell significantly at higher pH values (pH
7.5).
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The effect of urea and ammonia on complement activity
Figure 5 shows that preincubation of urinary complement with increasing amounts of both urea and ammonia was associated with a significant decline in C3 deposition on HK-2 cells (P<0.05). Preincubation of normal serum with urea and ammonia also led to a significant, although greater reduction of C3 deposition. This was also true for lipopolysaccharide-coated wells. Ammonia showed greater inhibition of complement than urea at the same concentration.
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Cleavage of C3 by HK-2 cells
The mechanism of complement activation and deposition was then investigated. Figure 6A is a Western blot showing C3 in 0.03% NHS that has been preincubated with HK-2 cells under conditions that allow selective activation of the alternative pathway. C3 present in the same concentration of normal (unactivated) serum is shown for comparison. Specific activation of the molecule is demonstrated by the presence of breakdown products, including a band below the
chain of C3 consistent with the removal of C3a (MW=9000 kDa). Figure 6B
shows the result of incubating purified C3 with a suspension of HK-2 cells. The
and ß chains are visible in purified C3, and these were not altered after incubation with HK-2 cells for 2 h at either pH 6 or pH 7. Adherent cells in culture were also incubated with trace amounts of purified C3 for up to 4 h with similar (negative) results. Importantly, the C3 preparation was haemolytically active. The
chain of C3 was quantitatively lost when it was supplemented with other complement components in the form of C3-deficient serum and activated via the alternative pathway (Figure 6C
, lane 3). This process was prevented when the complement inhibitor EDTA was added to chelate Mg2+ (lane 2).
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Discussion |
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It is clear that sufficient active components of the alternative pathway of complement remain in the glomerular filtrate to allow deposition of C3 and C9 on the surface of proximal tubular cells. However, Table 1 shows that significant tubulointerstitial fibrosis may occur without the deposition of C3 and C9 (as detected in the cell ELISA; samples 46, 9). Tubulointerstitial disease was absent in patient 8, despite deposition of C3 and C9. Previous studies showing complement activation by PTEC used serum as a source of complement [4,7,17]. Our data demonstrate the efficiency of activation of low concentrations of complement proteins by PTEC; specific cleavage of C3 occurred in as little as 0.03% of NHS (
0.02 mg protein/ml).
Evidence suggests that complement activation in the urine of patients or animals with non-selective proteinuria contributes to tubulointerstitial disease and progressive loss of renal function. Thus in a puromycin-induced model of interstitial nephritis, rats injected with the complement inhibitors cobra venom factor or soluble complement receptor type 1 (sCR1) showed significantly improved tubulointerstitial pathology and para-aminohippurate clearance despite having the same degree of proteinuria [10]. The damage was at least partially MAC dependent [11]. Studies in the same model showed more severe injury in animals in which tubular expression of Crry, a complement regulatory protein, had been inhibited [18].
In patients with proteinuria, the excretion of complement activation products decreased significantly 2 weeks after sodium bicarbonate administration, while levels of proteinuria or of complement-activation products in the plasma were not affected [15]. Another study showed oral bicarbonate delayed the progression of renal disease [14].
Increased urinary levels of ammonia are present in patients with proteinuria [19], and it has been proposed that bicarbonate is protective against alternative-pathway-mediated damage to tubules by lowering the formation of ammonia and thus activation of the alternative pathway [13]. Such nucleophiles inactivate C3 producing a C3b-like molecule able to act as a nucleus for the formation of the alternative complement pathway convertase. The convertase, however, activates only native C3. In our study, both urea and ammonia had an inhibitory effect on the deposition of C3 and C9 on the surface of the proximal tubular cell. They had a similar although greater inhibitory effect on C3 deposition from serum. This may reflect relative differences in the proportions of each complement component present in urine compared with serum. For example, the concentration of factor D of the alternative pathway in serum is rate limiting, but factor D is much increased in the urine of patients with chronic renal failure [20].
We were unable to reproduce the biphasic effect of ammonia on the activity of the alternative pathway of complement in serum reported previously [13]. The latter study used the lysis of rabbit erythrocytes as an end-point, and reported a small (15%) increase in haemolytic activity at low concentrations of ammonia, and dose-responsive inhibition at higher concentrations. HK-2 cells were not lysed under the conditions of these experiments and, in terms of the deposition of C3 and C9, both ammonia and urea were inhibitory at all concentrations in a dose-responsive manner. The mechanism of complement inhibition by these nucleophiles may include inactivation of the thiol ester bond of C3, and interference with other components of complement.
Conversely, the activity of complement in urine samples fluctuated according to pH, with values >7 being strongly inhibitory. Higher pH values were also inhibitory to deposition of C3 and C9 from NHS, showing this pH dependence is a general property of complement. Differences between the relative activities of the alternative pathway in urine and serum at pH 5.5 may reflect the more complex and varied species of molecules on the HK-2 cell surface available to form a protected site in comparison with inulin-coated wells. These data suggest the administration of bicarbonate protects against complement-mediated damage in animals [13] and complement activation in the tubular lumen in human subjects [15] by directly inhibiting complement activity via raised pH, rather than indirectly through inhibiting the generation of ammonia. While administration of bicarbonate significantly reduces the urinary ammonia concentration [14], it is likely that inhibitory concentrations of urea and other nucleophiles would remain.
The evidence suggests that proximal tubular cells activate the alternative pathway of complement through provision of a protected site on the surface of the HK-2 cell, which would allow the assembly of an alternative pathway convertase in an environment protected from inhibitors of complement such as factor H. An alternative mechanism, namely the cleavage of C3 to form a C3b-like molecule (i.e. by convertase-like protease(s) associated with the membrane of, or secreted by, HK-2 cells) was shown to be less likely. These cells were unable to cleave purified C3 alone, but activated the same molecule with the addition of other components of the alternative pathway.
Our data show that deposition of C3 and C9 on the tubular surface is inhibited by alkaline pH, and thus reinforce previous reports that alkalinizing agents such as bicarbonate should be exploited in the treatment of tubulointerstitial damage in patients with glomerular disease and proteinuria. The potential of other inhibitors of complement, including sCR1 and the peptide compstatin, should be further examined.
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Notes |
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References |
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