E-NTPDases in human airways: Regulation and relevance for chronic lung diseases

Chronic obstructive lung diseases are characterized by the inability to prevent bacterial infection and a gradual loss of lung function caused by recurrent inflammatory responses. In the past decade, numerous studies have demonstrated the importance of nucleotide-mediated bacterial clearance. Their interaction with P2 receptors on airway epithelia provides a rapid ‘on-and-off’ signal stimulating mucus secretion, cilia beating activity and surface hydration. On the other hand, abnormally high ATP levels resulting from damaged epithelia and bacterial lysis may cause lung edema and exacerbate inflammatory responses. Airway ATP concentrations are regulated by ecto nucleoside triphosphate diphosphohydrolases (E-NTPDases) which are expressed on the mucosal surface and catalyze the sequential dephosphorylation of nucleoside triphosphates to nucleoside monophosphates (ATP → ADP → AMP). The common bacterial product, Pseudomonas aeruginosa lipopolysaccharide (LPS), induces an acute reduction in azide-sensitive E-NTPDase activities, followed by a sustained increase in activity as well as NTPDase 1 and NTPDase 3 expression. Accordingly, chronic lung diseases, including cystic fibrosis (CF) and primary ciliary dyskinesia, are characterized by higher rates of nucleotide elimination, azide-sensitive E-NTPDase activities and expression. This review integrates the biphasic regulation of airway E-NTPDases with the function of purine signaling in lung diseases. During acute insults, a transient reduction in E-NTPDase activities may be beneficial to stimulate ATP-mediated bacterial clearance. In chronic lung diseases, elevating E-NTPDase activities may represent an attempt to prevent P2 receptor desensitization and nucleotide-mediated lung damage

Protease activity of plasma hemopexin

Background. Previous studies into the relevance of a putative circulating factor in the pathogenesis of minimal change nephrotic syndrome have opened the possibility that plasma hemopexin might be an important effector molecule in this disorder. Thus, intra renal infusion of isolated plasma hemopexin into rats induced minimal change like glomerular lesions and proteinuria. Both, in vivo and in vitro effects of the active isoform of hemopexin could be attributed to protease activity of this molecule. However, the question remained whether hemopexin per se rather than some contaminating plasma factor is responsible for the potential enzymatic activity of this molecule. Methods Recombinant hemopexin was prepared according to standard methods in Pichia pastoris and compared for its identity and protease activity with plasma hemopexin using Western blotting and various in vitro assays. Unilateral renal perfusion in anesthetized rats was used to test the proteinuria inducing capacity of recombinant hemopexin versus heat-inactivated recombinant hemopexin. Results. The blotting results show identical 85 kD bands in both native as well as recombinant hemopexin. Incubation of kidney tissue with recombinant hemopexin resulted in loss of of glomerular ectoapyrase and sialoglycoproteins, as shown by immunohistochemistry, which effect can be inhibited with the serine protease inhibitor phenylmethanesulfonyl fluoride. Artificial substrates for serine proteases, like kallikrein or thrombin, are hydrolysed by recombinant hemopexin in vitro, and not by heat-inactivated recombinant hemopexin or saline. Unilateral kidney perfusion of recombinant hemopexin, in contrast to control Pichia transfection products or heat-inactivated recombinant hemopexin, followed by a protein marker showed significantly enhanced urinary protein leakage 5.0, 10.0, and 15.0 minutes after perfusion. Conclusion. It is concluded that the hemopexin molecule as such can potentially act as a toxic protease, leading in the rat to proteinuria and glomerular alterations characteristic for minimal change nephrotic syndrome

Altered activity of plasma hemopexin in patients with minimal change disease in relapse

Since an active isoform of plasma hemopexin (Hx) has been proposed to be a potential effector molecule in minimal change disease (MCD), we tested plasma and urine samples from subjects with MCD in relapse (n =18) or in remission (n =23) (after treatment with prednisolone) for presence or activity of Hx. For comparison, plasma or urine from proteinuric subjects with focal and segmental glomerulosclerosis (FSGS, n =11), membranoproliferative glomerulonephritis (MPGN, n =9), IgA nephropathy (n =5) or healthy control donors (n =10), were incorporated into the study. Electrophoresis and Western blotting methods were used for evaluation of the Hx status, whereas protease activity of Hx was tested upon kidney tissue in vitro according to standard methods. The results show (1) a decreased mean titer of plasma Hx exclusively in MCD relapse subjects as compared with MCD in remission (0.21 +/- 0.14 mg/ml vs 0.44 +/- 0.06 mg/ml; p <0.01). Mean Hx titers in other proteinuric subjects ranged from 0.38 +/- 0.05 mg/ml to 0.40 +/- 0.06 mg/ml, whereas, the mean titer of healthy controls was 0.59 +/- 0.03 mg Hx/ml; (2) an increased Hx activity (expressed in arbitrary units) exclusively in plasma from MCD relapse subjects (3.3 +/- 0.72 vs 1.16 +/- 0.56, MCD remission; p <0.01); (3) different Western blot patterns in MCD relapse vs remission plasma; (4) reduced stainability or virtual absence of the 80-kD Hx band in blots of urine from MCD relapse in contrast to urine samples from other proteinuric subjects with FSGS, MPGN, or IgA nephropathy. It is concluded that Hx in MCD relapse subjects may exist in an altered isoform, showing enhanced protease activity as compared with subjects in remission, subjects with other forms of primary glomerulopathy, or healthy control individuals