Departamento de Medicina, 1 Disciplina de Nefrologia and 2 Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, SP, CEP 04023-900, Brazil
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ABSTRACT |
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The activities of serine endopeptidase, prolyl endopeptidase and neutral endopeptidase were determined in tubular fluid collected from several portions of the rat nephron as well as in urine. The enzyme activities were measured by HPLC using bradykinin (BK) as substrate. Free residual peptides of BK obtained by the action of these enzymes on the locally produced BK were also determined. The endopeptidase activities were found to be present throughout the nephron. Equimolar fragments of BK were detected in the early proximal tubule (Arg1-Pro7, Phe8-Arg9, Arg1-Gly4, Phe5-Arg9, and BK), late proximal tubule (Arg1-Phe5, Arg1-Pro7, Gly4-Pro7, Gly4-Arg9, and BK), late distal tubule (Arg1-Gly4, Phe5-Arg9, Arg1-Phe5, Ser6-Arg9, Gly4-Arg9, BK, and [des-Arg9]BK) and urine (Phe8-Arg9, Phe5-Arg9, Arg1-Phe5, Ser6-Arg9, Arg1-Pro7, Gly4-Pro7, Gly4-Arg9, BK, and [des-Arg9]BK). Our data suggest that the endopeptidases and exopeptidases are secreted by the nephron. Early proximal tubules secrete angiotensin converting enzyme and neutral endopeptidase, differing from late distal tubules that produce prolyl endopeptidase, serine endopeptidase, carboxypeptidase, and also neutral endopeptidase. All enzymes detected along the rat nephron were found in the urine. The existence of endopeptidases and carboxypeptidase in the distal nephron may have a potential physiological role in the inactivation of the kinins formed by kallikrein in the kidney and also in the inactivation of additional peptides other than BK.
angiotensin I converting enzyme; bradykinin; prolyl endopeptidase; serine endopeptidase; neutral endopeptidase; exopeptidases; micropuncture; kallikrein-kinin system
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INTRODUCTION |
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MAMMALIAN PROTEASES comprise both the peptidases or exopeptidases, which act at the NH2-terminal or COOH-terminal positions of polypeptides, and the proteinases or endopeptidases, which cleave peptide bonds in the central regions of polypeptides. Kininases are proteolytic enzymes that hydrolyze one or more peptide bonds in the bradykinin molecule (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and in related peptides acting as exopeptidases or endopeptidases. Carboxypeptidases hydrolyze the Phe8-Arg9 bond (kininase I) or the Pro7-Phe8 bond (kininase II), and endopeptidases hydrolyze the Pro3-Gly4, Gly4-Phe5, or Phe5-Ser6 bond in the BK molecule (4).
The kininases present in renal tissue belong either to the carboxypeptidase or to the endopeptidase type of proteinases. Kininase I (arginine carboxypeptidase, EC 3.4.17.3) (11) is a carboxypeptidase B-like enzyme and kininase II (dipeptidyl carboxypeptidase, EC 3.4.15.1) is an angiotensin I converting enzyme (ACE) that, besides converting angiotensin I to angiotensin II, inactivates BK (12). Postproline cleaving enzyme, prolyl endopeptidase, neutral endopeptidase, and serine endopeptidases have been identified in lamb (18), human (15), and rabbit kidney (1), as well as in human urine (8).
In the kidney, the hydrolysis of filtered kinins is now well documented, and most reports indicate that kinin inactivation occurs near the proximal tubule where ACE is present (6). Studies employing stop-flow technique have suggested the presence of an ACE kininase-type activity in the distal nephron, where the urinary kinins are produced by kallikrein (27). Using microdissection of rabbit nephrons, Marchetti et al. (19) showed that in addition to an intense lysyl-BK-hydrolyzing activity in the proximal tubule, a kininase activity was also found in the medullary collecting tubule. The kallikrein-kinin system is located in the distal tubule and cortical and medullary collecting ducts (22). The presence of endopeptidases in these portions of the nephron appears to be physiologically important, because in addition to kinin production, the kinin receptors are also detected.
We have previously described the presence of endopeptidases in human and rat urine (8, 7). In the literature, only ACE and neutral endopeptidase has been localized in the kidney (15, 17). Thus the objective of the present study was to localize endopeptidase activity in the tubular fluid collected from different portions of the rat nephron and to localize the different activities using an in vivo model based on the micropuncture technique.
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MATERIALS AND METHODS |
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Tubular fluid samples were collected from different portions of the rat nephron using the micropuncture technique. The activities of prolyl endopeptidase, serine proteinases, neutral endopeptidase, and ACE were measured in the tubular fluid samples and also by incubation of BK with tubular fluid in the presence and absence of protease inhibitors by HPLC.
Micropuncture Technique
Male Munich-Wistar rats weighing 250-300 g were used. The animals were prepared for micropuncture as previously described (5). In summary, rats were anesthetized with Inactin (Byk-Gulden, Constance, Germany), 100 mg/kg body wt ip, and placed on a temperature-regulated table for maintenance of their rectal temperature around 36-37°C. Following tracheotomy, a polyethylene catheter (PE-50) was introduced into the left femoral artery and connected to a direct-writing recorder (model 2.200; Gould, Cleveland, OH) to evaluate mean arterial pressure and to collect blood samples. The left and right jugular veins were catheterized with polyethylene tubing (PE-50) for infusion of 10% inulin at a rate of 1.2 ml/h and of isoncotic rat serum to replace surgical losses to maintain the animals in euvolemic conditions. A catheter (PE-10) was inserted into the left ureter for urine sampling and flow rate determination. The left kidney was dissected from perirenal fat, placed on a plastic support, and fixed with a glass stem for the micropuncture study. After a 45-min equilibration period, the segments of the nephron were identified by the Lissamine green technique (3). Lissamine green solution (5%) was injected in bolus into the jugular vein (0.3 ml). Approximately 3 s later, the dye diffused to the kidney surface and immediately appeared in the superficial loops of the proximal tubules. After 6-9 s, the dye disappeared from the surface to appear again in the superficial distal tubules. Thus it is possible to distinguish both early and distal segments of the proximal tubules as well as the distal nephrons according to the early or late appearance of the dye inside the lumen. Samples from different segments of the nephron were collected: early proximal tubule (EP), late proximal tubule (LP), and late distal tubule (LD). The endopeptidases and the ACE activities were also determined in total urine. The volume of tubular fluid samples was measured with a precalibrated quartz micropipette. The punctured segment was also correlated to the tubular/plasma (TF/P) inulin concentration ratio. Approximately 30-50 nl of tubular fluid was transferred to 50 µl of 0.05 M Tris · HCl buffer, pH 8.0, and stored atInfluence of Time BK Incubation with Tubular Fluid
BK was incubated with tubular fluid at 37°C in 0.05 M Tris · HCl buffer, pH 8.0, for 30, 60, 120, 240, 480, 720, and 840 min. The reaction was stopped by the addition of 10 µl of 10% H3PO4, and the hydrolysis products derived from incubation were chromatographed by HPLC. The results are expressed as % hydrolyzed BK.Measurement of BK and Fragments of BK in Tubular Fluid and Identification of BK Hydrolysis Products after Tubular Fluid Incubation with BK
The tubular fluid and the hydrolysis products derived from incubation of BK with tubular fluid were chromatographed by HPLC and compared with standard peptides as previously described by Casarini et al. (8) in 1992.Incubation of tubular fluid with BK. BK (1 nmol) was incubated for 12 h at 37°C with the tubular fluid solution in 0.05 M Tris · HCl buffer, pH 8.0, in a final volume of 150 µl. The reaction was stopped by the addition of 10 µl of 10% H3PO4. The samples were filtered before injection in HPLC. It is worthwhile to note that part of sample volume was retained in the filtering process. Tubular fluid and BK controls in buffer were studied after the same incubation time.
Analysis of free tubular fluid. The tubular fluid samples were diluted into 100 µl of column equilibration buffer and submitted to HPLC. For chromatographic identification, 100 µl of the incubation mixture or 100 µl of the diluted tubular fluid was injected into a Milton Roy HPLC system, and the separation was performed through a reverse-phase RP18 Brownlee column (4.6 × 250 mm, Millipore) equilibrated with 0.1% H3PO4 containing 5% acetonitrile (phase A). After 5 min of isocratic elution, peptides were eluted with a 0-35% linear gradient (vol/vol) of 90% acetonitrile in 0.1% H3PO4 (phase B) developed for 20 min at a flow rate of 1.5 ml/min. The effluent was monitored by absorbance at 214 nm.
Influence of inhibitors upon endopeptidases and exopeptidases activities of tubular fluid in the hydrolysis of BK. The tubular fluid solution in 0.05 M Tris · HCl buffer, pH 8.0, was preincubated with 1 mM phenylmethylsulfonyl fluoride (PMSF), a specific inhibitor of serine endopeptidases, 1 mM trans-epoxysuccinyl-leucilamide-(4-guanidino)butano (E-64) and 50 µM EDTA, specific inhibitors of metalloendopeptidases, 3 mM p-hydroxymercurebenzoate (pOHMB) and 1 mM cysteine (Cys), specific inhibitors of thiol endopeptidases, and 1 mM phosphoramidon, an inhibitor of neutral endopeptidase, for 30 min, and the subsequent 12 h later with BK (1 nmol), at 37°C, in a final volume of 200 µl. The inhibitor concentration has been previously determined by Casarini et al. (7). The reaction was stopped by the addition of 10 µl of 10% H3PO4. Tubular fluid and BK controls in buffer were performed at the same incubation time. The samples were analyzed by HPLC under the same conditions as described above.
Amino Acid Analysis of the BK Hydrolysis Products Derived from BK Incubation with Tubular Fluid
The hydrolysis products derived from BK after incubation with tubular fluid from late distal tubules eluted from an HPLC column were submitted to amino acid analysis. BK (20 nmol) was incubated for 12 h at 37°C with 100 nl tubular fluid in 0.05 M Tris · HCl buffer, pH 8.0, in a final volume of 500 µl. The enzymatic reaction was stopped by heating. The incubate was lyophilized and submitted to HPLC separation as described above, and the peptides were eluted for amino acid analysis. The eluted peptides were lyophilized and diluted for sequencing as described by Alonzo and Hirs (2) in 1968. ![]() |
RESULTS |
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The TF/P inulin ratios observed in different segments are shown in
Table 1. As expected, the ratio was
progressively increased from early proximal to late distal portions of
the nephron, indicating that the integrity of the tubules was preserved
and that the samples were not contaminated with interstitial fluid,
thus confirming the correlation between the TF/P ratio and the portion
of the nephron punctured (3).
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The half-time
(t1/2)
of BK is as low as 30 s when infused in vivo (20, 13),
where many kininases exist in constant renovation. In contrast in the
tubular fluid collected, the amount of the enzymes is constant and much
lower than that in the circulation. This fact could explain the
differences between distinct protocols, one with a very high turnover
(systemic circulation) and another with relative fixed volume (tubular
volume collection). Since in our study, the incubation with BK takes
place in the presence of a small volume of tubular fluid, thus
containing small amounts of enzymes, and our assay was based in HPLC,
we determined a single time of incubation (12 h), after
constructing a time course by taking samples at timed intervals
(0 until 12 h), where the linearity with time and enzyme
was confidently established. The use of the single time of incubation,
say 12 h, was possible on the assumption that the time course is linear
up to and beyond that time (Fig. 1).
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The two BK fragments arising from the hydrolysis of a given bond were
detected in approximately equimolar concentrations and are summarized
in Table 2. The results obtained by HPLC
for the early proximal tubule confirmed the presence of ACE and neutral endopeptidase, since in this portion we detected the peptides Arg1-Pro7
and
Phe8-Arg9,
products of the action of ACE on BK, and
Arg1-Gly4
and
Phe5-Arg9,
the products of hydrolysis by neutral endopeptidase. In the late
proximal tubule, we detected
Arg1-Phe5,
Arg1-Pro7,
Gly4-Pro7,
Gly4-Arg9,
and BK, indicating the presence of prolyl endopeptidase and serine
proteinase in addition to that of ACE and neutral endopeptidase type
activities. We did not detect the peptides
Arg1-Gly4
and
Phe8-Arg9
that are normally eluted in a retention time very close
to or even in the same peak injection; however, these peaks can be
easily visualized in the presence of nanomolar concentrations of the peptides. In the present study, we have picomolar concentrations of
these peptides, which sometimes hindered the peak
visualization. However, since we found the complementary peptides
Phe5-Arg9
and
Arg1-Pro7
(Fig. 2), we can deduce that the hydrolysis
of the peptide bond Gly4-Phe5
and
Pro7-Phe8
has occurred.
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The incubation of BK with tubular fluid obtained from late distal tubule produced the following peptides: Arg1-Gly4 and Phe5-Arg9, Arg1-Phe5 and Ser6-Arg9, Gly4-Arg9, BK, and [des-Arg9]BK, suggesting the presence of neutral-, serine-, prolyl-type endopeptidases, and carboxypeptidase able to hydrolyze the peptide bonds Gly4-Phe5, Phe5-Ser6, Pro3-Gly4, and Phe8-Arg9, respectively. In the urine, the following peptides were detected: Arg1-Phe5, Ser6-Arg9, Arg1-Pro7, Phe5-Arg9, Gly4-Pro7, Gly4-Arg9, Phe8-Arg9, and [des-Arg9]BK, suggesting the presence of serine endopeptidase, prolyl endopeptidase, neutral endopeptidase, ACE, and carboxypeptidase (Table 2). Figure 2 shows typical chromatograms with the identification of the cleavage site in BK by the enzymes present in tubular fluid collected from differents portions of rat nephron.
As shown in Table 3, the analysis of
tubular fluid collected into 10%
H3PO4
by HPLC indicated the presence of the peptides Arg1-Pro7,
Phe8-Arg9,
and
Arg1-Gly4
in the early proximal segment, which corresponds to the activity of ACE
and neutral endopeptidase. In the late proximal tubule, enzymes are
able to hydrolyze the peptide bonds
Phe5-Ser6,
Gly4-Phe5,
and
Pro3-Gly4,
identified by the peptides
Arg1-Gly4,
Arg1-Phe5,
Ser6-Arg9,
Gly4-Arg9,
Phe8-Arg9,
and BK. The late distal portion of the nephron contains endopeptidases able to hydrolyze the peptide bonds
Pro3-Gly4,
Gly4-Phe5,
and
Phe5-Ser6
in the BK molecule, because we could detect the peptides
Gly4-Arg9,
Arg1-Gly4,
Arg1-Phe5,
Ser6-Arg9,
BK, and [des-Arg9]BK.
Finally, all of the above peptides were found in urine, confirming the
presence of endopeptidases able to hydrolyze the peptide bonds Pro3-Gly4,
Gly4-Phe5,
Phe5-Ser6,
Pro7-Phe8,
and
Phe8-Arg9
of BK. The peptides detected in the tubular fluid collected from all
portions of the nephron are reported in Fig.
3. Figure 4
shows the elution diagram of the standards. When the tubular fluid was preincubated with a pool of inhibitors and then incubated with BK, the
same quantities of free peptides as those found in the control fluid
were detected. The amount of BK added to the incubate was also
recovered for the most part, with the difference corresponding to the
loss during the filtration process (Table
4).
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The incubate of BK and tubular fluid from late distal tubule
was cromatographed by HPLC. The peptides eluted after
acid hydrolysis were analyzed with an amino acid analyzer. The results
are shown in Table 5.
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The sites of hydrolysis of peptide bonds in BK by endopeptidases and
exopeptidases determined by HPLC fragment identification are reported
in Table 6.
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DISCUSSION |
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The main objectives of the present study included the determination of the potential sites along the nephron where BK can be metabolized and the enzymes that are involved in this process. Endopeptidase and exopeptidase activities were evaluated in tubular fluid obtained from different nephron segments by micropuncture technique using BK as substrate and also by analysis of the free peptides detected in the tubular fluid by HPLC.
We report here for the first time the identification of free BK fragments in tubular fluid and also HPLC determination of the hydrolysis products after incubation of tubular fluid with BK. It is interesting to note that a long period of time is needed to metabolize 90% of BK in vitro. Under the conditions of the present protocol, as observed in Fig. 1, 12 h of incubation were employed as a consequence of the small volume of tubular fluid used (30-50 nl) for the reaction and thus with low concentrations of the enzymes able to metabolize BK.
The kallikrein-kinin system is a complex series of enzymes and bioactive peptides that acts as a paracrine/autocrine system, with its major effects on collecting tubule function. Studies have been performed to localize the components of the kallikrein-kinin system in the kidney. Kallikrein has been found in several species in the cortical segment between the distal convoluted tubule and cortical collecting duct (16). A close anatomic relationship between the distal tubule and the afferent arteriole has been suggested and supports the hypothesis that the kallikrein-kinin system is functionally linked to the renin-angiotensin system and modulates tubuloglomerular feedback (24). Proud et al. (21) have localized a low-molecular-weight kininogen, the substrate for kallikrein, in a region from the late distal tubule through the cortical and medullary collecting ducts in the human kidney.
Kininase II converts angiotensin I to angiotensin II and is considered the major degradative enzyme in the kidney, cleaving kinins filtered across the glomerulus (10). This enzyme has shown to be located in the proximal tubules on the surface of the brush border, like aminopeptidases (26, 28). Recently we demonstrated that ACE is present and secreted into the collecting duct (9). Also neutral endopeptidase (EC 24.11) is located in the kidney, associated with renal brush border structures (15, 17).
In the present study, we measured the kininase activities after incubation of tubular fluid collected from various segments of the nephron with BK and also BK peptides free in the tubular fluid by HPLC. Endopeptidase activities that hydrolyze BK in the Pro3-Gly4 (prolyl endopeptidase), Gly4-Phe5 (neutral endopeptidase), Phe5-Ser6 (serine endopeptidase), and Phe8-Arg9 (carboxypeptidase) bonds were found in late distal portion of the nephron. It should be pointed out that incubation of BK with tubular fluid collected from all portions of the nephron (with the exception of the early distal nephron) resulted in the hydrolysis of BK, indicating the presence of active enzymes at each of these sites. However, since urine circulates within the nephron, especially for distal samples, it contains fragments of both proximal and distal origin, and urinary samples also contain fragments from the entire nephron. Thus it is interesting to emphasize that despite the presence of enzymes that metabolize BK in all segments, the origin of these enzymes is not necessarily from the site of the sample collection.
Activities such as prolyl endopeptidase, neutral endopeptidase, and serine endopeptidase, but not carboxypeptidase, were also found in the late proximal portion of the nephron. In the early proximal portion, we confirmed the presence of ACE and neutral endopeptidase. Urine collected by micropuncture contained all activities described above, and this result confirms our previous data of the purification and identification of endopeptidases and carboxypeptidases in human urine (8) (Table 6).
To confirm the existence of these endopeptidases, tubular fluid was preincubated with a pool of inhibitors followed by incubation with BK and the same picomolar quantities of peptides as detected in the analysis of tubular fluid without incubation, and the BK added in the incubate was recovered. These studies demonstrated that the endopeptidases were inhibited by the specific inhibitors, confirming the class of these enzymes.
The results concerning enzyme activity demonstrated that, when tubular fluid was incubated with BK in the absence of inhibitors, the amount of free peptides increased and the amount of BK decreased (Table 4). BK was inactivated by 85%, 96%, 97%, and 75% by endopeptidases present in fluid collected from early proximal tubules, late proximal tubules, late distal tubules, and urine, respectively. In contrast, the incubation of BK with tubular fluid in the presence of inhibitors (Table 2) resulted in the recovery of similar amounts of free peptides found in normal tubular fluid and urine (Table 3) and a higher amount of intact BK, indicating that BK was not further metabolized and therefore that inhibition occurred. The hydrolysis of BK was inhibited by about 60%, 50%, 60%, and 65% in early proximal tubules, late proximal tubules, late distal tubules, and urine, respectively. The concentrations of these inhibitors were based on a previous study (8), where the following seven enzymes were characterized from human urine: carboxypeptidases (similar to carboxypeptidase N), serine endopeptidases (H1 and H2, chymotrypsin-like), prolyl endopeptidase (H), and neutral endopeptidase-like (similar to neutral endopeptidase). Thus the nature of the kininases present in the different portions of the nephron might resemble the type of kinin-hydrolyzing enzymes, which are sensitive to PMSF (serine endopeptidases), phosphoramidon (neutral endopeptidase), pOHMB and Cys (prolyl endopeptidase), and E64 and EDTA (ACE and carboxypeptidases).
Additionally, we further analyzed the different enzymes based on the peptide bond hydrolyzed in the BK molecule (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9), as deduced from the data obtained in Table 2. Equimolar concentrations of peptides were detected after incubation with BK. For example, in the late distal tubule, Arg1-Phe5 (110 pmol) and the complementary fragment Ser6-Arg9 (100 pmol) were obtained, indicating that the peptide bond hydrolyzed was Phe5-Ser6. Taken together, these results suggest the presence of different classes of enzymes in tubular fluid samples (Table 6).
The amino acid sequence of peptides eluted from an HPLC column after incubation of BK and tubular fluid from late distal tubule was determined (Table 5). The sequence obtained for the peptides confirms the peptide bond hydrolyzed by the enzymes in this portion, since, for example, the peptide Arg1-Phe5 and the complementary peptide Ser6-Arg9 were found to be present in equimolar concentration.
The presence of endopeptidases (kininases) in the distal nephron might be physiologically important, because this segment is a target site for the kinins' actions. Several degradative pathways for kinins exist, and these enzymes may be involved in hydrolysis of the kinins formed by the kidney, limiting the action of these peptides to the site of their formation. In the literature, neutral endopeptidase appears to be at least as important as kininase II in degrading kinins in the nephron (23). In vivo, when phosphoramidon inhibited neutral endopeptidase activity, 77% of kininase activity was reduced and the kinin excretion was increased by 73%. 125I-labeled tyrosine-BK infused arterially failed to appear in urine during simultaneous phosphoramidon and captopril infusion, suggesting the presence of other potent kininases.
Therefore, in addition to the localization of ACE and neutral endopeptidase, serine endopeptidase, and prolyl endopeptidase, enzymatic activity was detected in the tubular fluid, which can be responsible for the hydrolysis of BK and other peptides that have action at the late distal tubule level. However, further studies are needed for a better understanding of the physiological events induced by the actions of these enzymes on BK and other peptides in the renal vasculature, interstitium, and other tubular segments and of their respective impact in normal as well as in pathophysiological conditions.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. Schor, Professor of Medicine, Nephrology Division, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Botucatu, 740, 04023-900, São Paulo, SP, Brazil (E-mail: nschor.dmed{at}epm.br).
Received 5 March 1998; accepted in final form 19 March 1999.
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