Kinin system in lupus nephritis

International Immunopharmacology 1 Ž2001. 1889–1896 www.elsevier.comrlocaterintimp

Preliminary StudyrReport

Kinin system in lupus nephritis Renata Dellalibera-Joviliano a , Marina L. Reis b, Eduardo A. Donadi a,) a

DiÕision of Clinical Immunology, Department of Medicine, Faculty of Medicine of Ribeirao ˜ Preto, UniÕersity of Sao ˜ Paulo, AÕenida Bandeirantes 3900, Monte Alegre, 14049-900 Ribeirao ˜ Preto (SP), Brazil b Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, UniÕersity of Sao ˜ Paulo, Ribeirao ˜ Preto (SP), Brazil Received 27 January 2001; accepted 22 May 2001

Abstract There are few studies regarding the evaluation of the kinin system in patients with systemic lupus erythematosus ŽSLE.. In this study, we evaluated the plasma levels of high-molecular weight kininogen ŽHKg., low-molecular weight kininogen ŽLKg. and plasma kallikrein; the plasma activity of tissue kallikrein and kininase II, and urinary kallikrein and kininase II activities in patients presenting with active lupus nephritis. A total of 30 patients Ž29 women. aged 21–62 years Žmedians 39. and 30 controls matched to the patients for sex and age were studied. Patients presenting with other underlying diseases or using drugs, which could interfere with the kinin system, were excluded. HKg and LKg levels were indirectly evaluated by ELISA. Plasma kallikrein, tissue kallikrein, and kininase II were evaluated by their enzymatic activity on selective substrates. The Mann–Whitney test was used for statistical analysis. HKg, LKg and plasma kallikrein levels were significantly increased in patients Ž p - 0.001, for each comparison.. Similarly, tissue kallikrein and kininase II activities were significantly increased in plasma and urine of patients Ž p - 0.001, for each comparison.. In urine, the activities of tissue kallikrein and kininase II were at least seven times higher than those seen in the plasma of patients. These results indicate that the kinin system is involved in the acute manifestations of lupus nephritis. Kinins may facilitate immunecomplex deposition and may induce the release of other pro-inflammatory mediators, including cytokines actively involved in the pathogenesis of lupus nephritis. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Systemic lupus erythematosus ŽSLE.; Kininogens; Kallikreins; Kininase II; Kinins; Lupus nephritis

1. Introduction The kinin system encompasses a series of proteins and proteases with the ability to produce vasoactive peptides by limited proteolysis. High-molecular weight ŽHKg. and low-molecular weight ŽLKg. kininogens are substrates for plasma and tissue

) Corresponding author. Tel.: q55-16-602-2566; fax: q55-16633-6695. E-mail address: [email protected] ŽE.A. Donadi..

kallikreins, yielding the peptides bradykinin ŽBk. and Lys-bradykynin ŽLys-Bk., respectively. Both peptides are short-lived bioactive molecules which are rapidly inactivated by kininases. Two major kinin receptors, B 1 and B 2 , are responsible for most kinin actions. Plasma prekallikrein is a serine protease mainly synthesized by the liver as zymogen. Most plasma prekallikrein circulates complexed with HKg and is activated to kallikrein by negatively charged surfaces w1x. Recently, a cysteine protease present on the membrane of endothelial cells has been reported to

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be a physiological prekallikrein activator. Tissue pro-kallikreins are synthesized by salivary glands, kidneys, brain, pancreas and neutrophils, while the mechanisms of physiological activation of the zymogen are still unknown w1,2x. Kininogens are multifunctional glycoproteins produced, to a major extent, by the liver and secreted into the circulation, and to a lesser extent, by neutrophils, brain, kidneys and platelets w1,3,4x. A single human kininogen gene codes for the synthesis of HKg and LKg via alternative splicing, producing proteins which are distinct in terms of structure and function w5x. Besides kinin precursors, kininogens may inhibit cysteine proteases and down-regulate platelet activity induced by thrombin. HKg may also act as a cofactor for the intrinsic coagulation system, and may bind with endothelial cells impairing neutrophil adhesion w6,7x. Kininases I Žcarboxypeptidases M and N. metabolize Bk and Lys-Bk, removing Arg 9 residue and generating des-Arg 9 Bk or des-Arg 9 Lys-Bk, respectively. These metabolites are specific agonists for B 1 receptors expressed only in pathological states in response to several inflammatory stimuli. Kininase II Ždipeptidylcarboxypeptidase., also known as angiotensin-converting enzyme ŽACE., inactivates Bk and Lys-Bk. Bradykinin and Lys-bradykinin, agonists for B 2 receptors, are constitutively expressed on many cell types, mediating most of the physiological actions of kinins w1,8x. Systemic lupus erythematosus ŽSLE. is an autoimmune disease of unknown etiology. The major manifestations of the disease such as arthritis, serositis and nephritis are reported to be mediated by immunecomplexes w9,10x. Besides being involved in the deposition of circulating immunecomplexes, complement consumption and generation of complement-derived mediators, cytokines and growth factors are also associated with the activity of lupus nephritis w11x. Kinins have been reported to stimulate the activity of cytokines such as tumor necrosis factor and interleukins 1 and 6 from various cell types, and these mediators have been associated with the activity of lupus nephritis w12x. In order to study the contribution of the kinin system to the pathogenesis of SLE, we evaluated kininogens, plasmartissue kallikreins and kininase II in plasma, and tissue kallikrein and kininase II in urine of patients present-

ing with a very specific manifestation of SLE, i.e., lupus nephritis. 2. Materials and methods 2.1. Patients and controls We studied 30 patients Ž29 women. aged 21–62 years Žmedians 39. with lupus nephritis seen at the University Hospital of the School of Medicine of Ribeirao ˜ Preto, University of Sao ˜ Paulo, Brazil. The diagnosis of SLE was based on the criteria of the American College of Rheumatology w13x. The activity of SLE was determined using the SLEDAI criteria w14x, ranging from 23 to 29 scores Žmedians 25.. The presence of active lupus nephritis was based on the detection of proteinuria ŽG 0.5 gr24 h in more than three samples., red blood cell, heme, granular, tubular or mixed casts in urine, decreased levels of C3 and C4, and appearance of antibodies against double-stranded DNA. All patients were submitted to kidney biopsies and nephritis was classified according to the WHO criteria w13,15x. The lupus nephritis activity index ranged from 12 to 20 Žmedians 17., and chronicity index ranged from 0 to 4 Žmedians 2.. Since the presence of antiphospholipid antibodies may interfere with the kinin system w16x, patients possessing these antibodies were excluded. In addition, patients presenting other underlying diseases, which could interfere with the kinin system, such as hypertension, allergic asthmarrhinitis or diabetes, were excluded. Patients were studied during the acute phase or during the relapse of renal disease, before the beginning of specific therapy. Thirty normal individuals matched to the patients for sex and age were also studied. Similarly, control individuals presented no underlying diseases and were not taking drugs that interfere with the kinin system. Control individuals presented no family history of autoimmune disorders. Informed consent was obtained from all individuals, and the study protocol was approved by the local Ethics Committee. 2.2. Blood collection Blood was collected using plastic syringes and placed into plastic tubes using 3.8% sodium citrate

R. Dellalibera-JoÕiliano et al.r International Immunopharmacology 1 (2001) 1889–1896

as anticoagulant and centrifuged at 2000 = g for 15 min at room temperature. The obtained plasma was stored in plastic tubes at y70 8C until use. 2.3. Kininogen leÕel Kininogen concentration was determined in plasma previously treated with kaolin Žlow-molecular weight kininogen. or in untreated plasma Žtotal kininogen.. Plasma samples were submitted to acid denaturation and trypsin hydrolysis by the method of Diniz and Carvalho w17x. The released kinin was measured by ELISA ŽMarkit M, Dainippon Pharmaceutical, Osaka, Japan., as previously described w18,19x. High-molecular weight kininogen was indirectly measured by the difference between the values of total kininogen and low-molecular weight kininogen. The results were expressed as m g Bk Eqrml plasma. 2.4. Plasma kallikrein Prekallikrein circulates in plasma in its inactive form, and once activated into kallikrein, the enzyme is rapidly inactivated by plasma antiproteases, preventing the detection of its activity in vivo. In this assay, we measured the in vitro kallikrein activity as a surrogate marker for plasma prekallikrein levels. Plasma antiproteases were inactivated by acid treatment followed by activation of prekallikrein to kallikrein using a plasma prekallikrein activator ŽChromogenix, Sweden., as described by De La Cadena et al. w20x. The activity of the enzyme on the selective chromogenic substrate H-D-Pro-Phe-Argp-nitroanilide ŽS-2302, Chromogenix. was detected spectrophotometrically at 405 nm. The plasma kallikrein levels were expressed as units ŽU. of kallikreinrml plasma using as standard purified human plasma kallikrein ŽChromogenix.. To assure that the activity observed was that of plasma kallikrein, additional reactions were performed using plasma samples in the presence or absence of soybean trypsin inhibitor ŽSigma, St. Louis, MO.. 2.5. Tissue kallikrein Tissue kallikrein-like activity in plasma and urinary kallikrein activity were measured by amidase hydrolysis of the selective chomogenic substrate

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H-D-Val-Leu-Arg-paranitroanilide ŽS2266, Chromogenix. as described by Amundsen et al. w21x. Plasma and urine reactions were developed in the presence or absence of aprotinin ŽSigma., an inhibitor of tissue kallikrein. The paranitroaniline ŽpNa. formed in these reactions was detected at 405 nm. The difference between these two evaluations corresponded to the activity of tissue kallikrein. The results were expressed as m mol pNarml plasma or m mol pNarml urine. 2.6. Kininase II Kininase II activity in plasma and urine was determined using the selective substrate HippurylHys-Leu ŽSigma. in a fluorimetric assay as described by Cushman and Cheung w22x. The results were expressed as m mol Hys–Leurml plasma or Hys– Leurml urine. 2.7. Statistical analysis Data were analyzed using the non-parametric Mann–Whitney test. Correlations were estimated by Spearman rank correlation coefficient. Differences were considered significant at p - 0.05.

3. Results 3.1. HKg and LKg Table 1 and Fig. 1 show HKg and LKg concentrations in plasma of patients with lupus nephritis. HKg

Table 1 Analysis of the plasma concentration of high-molecular weight ŽHKg. and low-molecular weight ŽLKg. kininogens, and plasma kallikrein levels seen in a series of 30 patients with systemic lupus erythematosus presenting with nephritis and 30 controls LKg Ž m g Bk Eqrml plasma.

HKg Ž m g Bk Eqrml plasma.

Plasma kallikrein ŽUrml plasma.

Patients 4.24 Ž2.81–6.06. 1.83 Ž0.76–3.34. 3.58 Ž2.98–4.99. Controls 0.78 Ž0.43–1.32. 0.49 Ž0.27–0.79. 1.68 Ž1.32–2.13. Ž p- 0.001. Ž p- 0.001. p value Ž p- 0.001. Data are expressed as median and range Ž . values.

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Fig. 1. High-molecular weight ŽHKg. and low-molecular weight ŽLKg. kininogen concentrations in plasma of patients with systemic lupus erythematosus presenting with nephritis and of controls. Data are expressed as m g Bradykinin ŽBk. Equivalents ŽEq.rml plasma. Horizontal bars indicate median values.

Fig. 2. Tissue kallikrein-like activity and plasma kallikrein levels in plasma of patients with systemic lupus erythematosus presenting with nephritis and of controls. Data are expressed as units Urml plasma Žplasma kallikrein. and m mol paranitroanilinerml plasma Žtissue kallikrein.. Horizontal bars indicate median values.

R. Dellalibera-JoÕiliano et al.r International Immunopharmacology 1 (2001) 1889–1896 Table 2 Analysis of the activity of tissue kallikrein and kininase II in plasma Ž ns 30. and urine Ž ns8. of patients with systemic lupus erythematosus presenting with nephritis, and in plasma and urine of controls Plasma

Urine

Tissue kallikrein ( m mol pNaa r ml . ŽA. Patients 1.95 15.49 Ž1.53–3.10. Ž13.9–16.8. ŽB. Controls 1.58 6.45 Ž1.03–2.13. Ž5.00–7.00. A=B A=B p- 0.001 p- 0.001 Kininase II ( m molr ml) ŽA. Patients 1.75 Ž1.25–2.30. ŽB. Controls 0.55 Ž0.49–0.72. A=B p- 0.001

Plasma versus urine comparisons ps 0.002 ps 0.002

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in patients Žmedians 15.49. was significantly increased in relation to controls Žmedians 6.45, p 0.001.. In addition, amidase activity of urinary kallikrein was significantly higher than that observed in plasma. These results are shown in Table 2. The correlation between the activity of tissue kallikrein and the concentration of its natural substrate ŽLKg. showed no significant result. Similarly, no significant correlation was observed in comparing the tissue kallikrein-like activity in plasma and the urinary kallikrein activity.

3.4. Kininase II 15.41 Ž13.94–17.8. 0.74 Ž0.63–0.81. A=B p- 0.001

ps 0.002 ps 0.002

Data are expressed as median and range Ž . values. a pNa—paranitroaniline.

Kininase II activity in plasma Žmedians 1.75. and urine Žmedians 15.41. of patients was significantly increased when compared to control plasma Žmedians 0.55, p - 0.001. or control urine Ž0.74, p - 0.001.. These results are shown in Fig. 3. Kininase II activity in patient urine was significantly higher than in patient plasma ŽTable 2.. No signifi-

levels Žmedians 1.83. for patients were significantly increased compared to controls Žmedians 0.49, p 0.001.. Similarly, LKg levels in plasma of patients Žmedians 4.24. were significantly increased in relation to controls Žmedians 0.78, p - 0.001.. 3.2. Plasma kallikrein Plasma kallikrein levels were significantly increased in patients Žmedians 3.58. when compared to controls Žmedians 1.68, p - 0.001.. Table 1 and Fig. 2 illustrate these results. No significant correlation was observed when the levels of plasma kallikrein were compared to the levels of its natural substrate ŽHKg.. 3.3. Tissue kallikrein in plasma and urine The tissue kallikrein-like activity in plasma of lupus nephritis patients Žmedians 1.95. was significantly increased in relation to that observed in controls Žmedians 1.58, p - 0.001, Fig. 2.. Similarly, the amidase activity of urinary kallikrein seen

Fig. 3. Kininase II activity in plasma of patients with systemic lupus erythematosus presenting with nephritis and of controls. Data are expressed as m molrml plasma. Horizontal bars indicate median values.

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cant correlations were seen when kininase II activity in plasma was compared to kininogen or plasma kallikrein levels or activity of tissue kallikreins or urinary kallikrein.

4. Discussion Little attention has been paid to the role of the kinin system in systemic lupus erythematosus. Overall, the patients of this series presented increased plasma concentrations of kinin precursors Žkininogens. associated with increased activities of kininogenases Žkallikreins. and kininases Žkinin inactivators. in plasma and urine. Regarding the plasma results, increased activities of both kallikreins and kininase II would be compatible with an increased kinin production and kininogen consumption; however, we did not observe decreased levels of kininogens. On the contrary, kininogen concentrations were increased. Two major points should be considered with respect to these findings. First, the regulatory mechanisms, which induce kininogen synthesis or promote kininogen consumption, have not been completely elucidated. Second, kininogens are multifunctional molecules, possessing in their structure several domains, six for HKg ŽD 1 – D6 . and five for LKg ŽD1 –D5 .. The D4 domain of both kininogens contains the amino acid sequence of bradykinin, a pro-inflammatory peptide. On the other hand, the D 2 and D 3 domains are cysteine proteinase inhibitors presenting antiinflammatory properties w23x. Then, the amount of kininogens in plasma may depend on the balance between the inflammatory and antiinflammatory stimuli and may also depend on the rate of kininogen synthesisrconsumption. In addition, the half-life of kinins is extremely short Žapproximately 30 s., making it difficult for the direct detection of the released kinin. The quantification of more stable kinin metabolite Arg 1-Pro-Pro-Gly-Phe 5 would help to clarify these points w24x. There are no previous studies evaluating the levels of plasma kallikrein in systemic lupus erythematosus. Plasma kallikrein has been reported to have chemoattractant activity for neutrophils w25x. Thus, increased plasma kallikrein levels, in addition to kinin generation, may also contribute to the inflam-

matory process observed in lupus nephritis. On the other hand, increased plasma kallikrein levels have been reported in synovial fluid of patients presenting with arthritis due to several autoimmune disorders w26x. Since the method used in this study to detect tissue kallikrein evaluated only the active form of the enzyme, and since the activity of urinary kallikrein in lupus nephritis was at least seven times higher than that seen in the plasma, we may conclude that there is a large amount of active urinary kallikrein in lupus nephritis. Urinary kallikrein is reported to be synthesized in connecting tubule cells and secreted into the interstitial space and into the circulation w27x. Additional tissue kallikrein may be present in the urine of lupus nephritis patients originating from the plasma by increased permeability of the damaged glomeruli. Kininase II has been considered to be one of the major kinin inactivators in human kidneys, and has been localized in the brush border of proximal tubules, endothelial cells and urine w27x. In this series, the activities of both urinary and plasma kininase II were increased in patients in relation to controls. In addition, the activity of the enzyme in patient urine was at least seven times higher than that observed in the plasma. Increased activity of kininase I and II in plasma of lupus patients has been reported, irrespective of clinical manifestations w28x. The results reported here confirm the increased kininase II activity in plasma of SLE patients and further show an increased kininase II activity in the urine of patients presenting with lupus nephritis. Kininogens, kallikrein, kininase II and bradykinin B 2 receptors have been localized in human nephrons. Once produced, kinins may have autocrine and paracrine functions, regulating glomerular filtration, sodium excretion and renal hemodynamics w1,27x. The results reported here for lupus nephritis indicate the participation of the kinin system at the systemic and renal levels. Since lupus nephritis has been considered an immunecomplex disease, the kinin system may contribute to its pathogenesis by facilitating immunecomplex deposition and inducing the release of other pro-inflammatory mediators including cytokines. Future investigations evaluating the localization and the amount of kinin system components in lupus nephrons may help in the understand-

R. Dellalibera-JoÕiliano et al.r International Immunopharmacology 1 (2001) 1889–1896

ing of the role of kinin system in this disease. Recent studies have reported the presence of almost all kinin system components on the membrane and in the granules of neutrophils w29–32x, also justifying future investigations of kinin system components in neutrophils of patients presenting with lupus nephritis.

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