Francesca Bodega

Contribution of lymphatic drainage through stomata to albumin removal from pleural space.

The contribution of lymphatic drainage through the stomata of parietal mesothelium to the overall removal of labeled albumin from the pleural space was found 89% in sheep with very large hydrothoraces (10 ml/kg), a condition involving a approximately 20 times increase in lymphatic drainage [Broaddus et al., J. Appl. Physiol. 64 (1988) 384]. We determined this contribution in anesthetized rabbits with small (0.12 ml/kg) and large (2.4 ml/kg) hydrothoraces of Ringer-albumin with labeled albumin and labeled dextran-2000 kDa. This dextran was used as marker of liquid removal through the stomata because it should essentially leave the pleural space through the stomata only, owing to its size. The removal of labeled albumin by lymphatic drainage through the stomata was 39% of the overall removal in the small hydrothoraces, and 64% in the large ones. Hence, lymphatic drainage through the stomata does not contribute most of protein and liquid removal from the pleural space under physiological conditions, as it has been maintained. It markedly increases with the increase in pleural liquid volume.

Lubricating effect of sialomucin and hyaluronan on pleural mesothelium

Coefficient of kinetic friction (μ) between rabbit visceral and parietal pleura, sliding in vitro at physiological velocities and load, increases markedly after blotting mesothelial surface with filter paper; this increase is only partially reduced by wetting blotted mesothelium with Ringer solution. Given that mesothelial surface is covered by a thick coat with sialomucin and hyaluronan, we tested whether addition of sialomucin or hyaluronan solution after blotting lowers μ more than Ringer alone. Actually, these macromolecules lowered μ more than Ringer, so that μ was no longer significantly higher than its preblotting value. Moreover, Ringer addition, after washout of macromolecule solution, increased μ, in line with their dilution. These findings indicate that mesothelial blotting removes part of these molecules from the coat covering mesothelial surface, and their relevance for pleural lubrication. Transmission electron micrographs of pleural specimens after mesothelial blotting showed that microvilli were partially or largely removed from mesothelium, consistent with a substantial loss of macromolecules normally entrapped among them.

Evidence for Na+–glucose cotransporter in type I alveolar epithelium

Functional evidence of Na+–glucose cotransport in rat lung has been provided by Basset et al. (J. Physiol. 384:325–345, 1987). By autoradiography [3H]phloridzin binding has been found confined to alveolar epithelial type II cells in mouse and rabbit lungs (Boyd, J. Physiol. 422: 44P, 1990). In this research we checked by immunofluorescence whether Na+–glucose cotransporter (SGLT1) is also expressed in alveolar type I cells. Lungs of anesthetized rats and lambs were fixed by paraformaldehyde, perfused in pulmonary artery, or instilled into a bronchus, respectively. Tissue blocks embedded in paraffin or frozen were sectioned. Two specific anti-SGLT1 antibodies for rat recognizing aminoacid sequence 402–420, and 546–596 were used in both species. Bound primary antibody was detected by secondary antibody conjugated to fluorescein isothiocianate or Texas red, respectively. In some sections cellular nuclei were also stained. In rats alveolar type I cells were identified by fluorescent Erythrina cristagalli lectin. Sections were examined by confocal laser-scanning microscope. Both in rats and lambs alveolar epithelium was stained by either antibody; no labeling occurred in negative controls. Hence, SGLT1 appears to be also expressed in alveolar type I cells. This is functionally relevant because type I cells provide 95–97% of alveolar surface, and SGLT1, besides contributing to removal of lung liquid under some circumstances, keeps low glucose concentration in lining liquid, which is useful to prevent lung infection.

Expression of Na+–glucose cotransporter (SGLT1) in visceral and parietal mesothelium of rabbit pleura

Indirect evidence for a solute-coupled liquid absorption from rabbit pleural space indicated that it should be caused by a Na(+)/H(+)-Cl(-)/HCO(3)(-) double exchanger and a Na(+)-glucose cotransporter [Agostoni, E., Zocchi, L., 1998. Mechanical coupling and liquid exchanges in the pleural space. In: Antony, V.B. (Ed.), Clinics in Chest Medicine: Diseases of the Pleura, vol. 19. Saunders, Philadelphia, pp. 241-260]. In this research we tried to obtain molecular evidence for Na(+)-glucose cotransporter (SGLT1) in visceral and parietal mesothelium of rabbit pleura. To this end we performed immunoblot assays on total protein extracts of scraped visceral or parietal mesothelium of rabbits. These showed two bands: one at 72kDa (m.w. of SGLT1), and one at 55kDa (which should also provide Na(+)-glucose cotransport). Both bands disappeared in assays in which SGLT1 antibody was preadsorbed with specific antigen. Molecular evidence for Na(+)/K(+) ATPase (alpha1 subunit) was also provided. Immunoblot assays for SGLT1 on cultured mesothelial cells of rabbit pleura showed a band at 72kDa, and in some cases also at 55kDa, irrespectively of treatment with a differentiating agent. Solute-coupled liquid absorption hinders liquid filtration through parietal mesothelium caused by Starling forces, and favours liquid absorption through visceral mesothelium caused by these forces.

Pleural mesothelium lubrication after hyaluronidase, neuraminidase or pronase treatment

Coefficient of kinetic friction (μ) of pleural mesothelium has been found to increase markedly after mesothelial blotting and rewetting. This increase disappeared after addition of a solution with hyaluronan or sialomucin, though previous morphological studies showed that only sialomucin occurs in mesothelial glycocalyx. In this research we investigated whether μ of rabbit pleural mesothelium increased after hyaluronidase, neuraminidase or pronase treatment. Hyaluronidase and neuraminidase did not increase μ, though neuraminidase cleaved sialic acid from mesothelial glycocalyx of diaphragm specimens, and removed hystochemical stain of sialic acid from glycocalyx. Sialomucin treated with neuraminidase lowered μ of blotted mesothelium, though less than untreated sialomucin; this feature plus lubrication provided by other molecules could explain why μ did not increase after neuraminidase. Short pronase treatment (in order to affect only glycocalyx proteins) increased μ; this increase was removed by hyaluronan or sialomucin. After pronase treatment μ decreased with increase in sliding velocity, indicating a regime of mixed lubrication, as in blotted mesothelium.

Mixed lubrication after rewetting of blotted pleural mesothelium.

Coefficient of kinetic friction (μ) of pleural mesothelium blotted with filter paper, and rewetted with Ringer solution markedly increases; this increase is removed if a sufficient amount of sialomucin or hyaluronan is added to Ringer (Bodega et al., 2012. Respiratory Physiology and Neurobiology 180, 34-39). In this research we found that μ of pleural mesothelium blotted, rewetted, and sliding at physiological velocities and loads, decreased with increase of velocity, mainly at low velocities. Despite this decrease, μ at highest velocity was still double that before blotting. With small concentration of sialomucin or hyaluronan μ was markedly smaller at each velocity, decreased less with increase of velocity, and at highest velocity approached preblotting value. These findings indicate a regime of mixed lubrication in post-blotting Ringer, at variance with boundary lubrication occurring before blotting or postblotting with sufficient macromolecule addition. Greater roughness of mesothelial surface, caused by blotting, likely induces zones of elastohydrodynamic lubrication, which increase with velocity, while contact area decreases.

Pleural mesothelium lubrication after phospholipase treatment

Coefficient of kinetic friction (μ) of rabbit pleural mesothelium increased after short treatment of specimens with phospholipase C. This increase was removed by addition of a solution with hyaluronan or sialomucin, as previously shown in post-blotting Ringer or after short pronase treatment. After phospholipase μ decreased with increase in sliding velocity, but at highest velocity it was still greater than control; this difference was removed by addition of hyaluronan or sialomucin, as in post-blotting Ringer or after short pronase treatment. Hyaluronan placed on specimen before phospholipase treatment reduced increase in μ by protecting phospholipids from enzyme, as shown by others for alveolar and synovial phospholipids. Samples of parietal pleura stained with silver nitrate showed that mesothelial cells were not disrupted by short phospholipase treatment. Instead, they were disrupted if this treatment was preceded by a short pronase treatment; but even after this disruption addition of hyaluronan or sialomucin brought μ back to control.

Na+–glucose cotransporter is also expressed in mesothelium of species with thick visceral pleura

Molecular evidence for Na+-glucose cotransporter (SGLT1) in rabbit pleural mesothelium has been recently provided, confirming earlier functional findings on solute-coupled liquid absorption from rabbit pleural space. In this research we checked whether SGLT1 is also expressed in pleural mesothelium of species with thick visceral pleura, which receives blood from systemic circulation, but drains it into pulmonary veins. To this end immunoblot assays were performed on total protein extract of scraped visceral and parietal mesothelium of lambs and adult sheep, and of a human mesothelial cell line. All of them showed SGLT1 specific bands. Moreover, confocal immunofluorescence images of lamb pleural mesothelium showed that SGLT1 is located in apical membrane. Therefore, a solute-coupled liquid absorption should also occur from pleural space of species with thick visceral pleura. Because of this protein-free liquid entering interstitium between visceral mesothelium and capillaries, inherent Starling forces should be different than hitherto considered, and visceral pleura capillaries could absorb liquid even in these species.

Labeled albumin in plasma and removal paths from pleural space in control and increased ventilation.

Increased ventilation was shown to markedly increase lymphatic drainage and plasma content of labeled proteins injected into pleural space relative to control ventilation. These proteins reach plasma by lymphatic drainage: directly through parietal pleura stomata, and indirectly from pleural interstitium, reached by diffusion, convection and transcytosis. Increased drainage from interstitium should not involve a comparable increase in protein removal from pleural space by these transports, while increased drainage through stomata involves a comparable increase in protein removal. Hence, relative increase in labeled protein removal from pleural space caused by increased ventilation should be marked only if drainage through stomata contributed most of this removal, whereas relative increase of labeled proteins in plasma should be marked in either case. We injected 3 ml of albumin-Ringer with albumin-Texas red into the pleural space of three groups of anesthetized rabbits: control, CO2-, or muscle stimulation-increased ventilation. Increased ventilation for 3 h (78 and 61%, respectively) increased (P < 0.01) labeled albumin in plasma by 132 and 106%, respectively, but did not significantly increase its removal. Hence, lymphatic drainage through stomata should not contribute most of liquid and protein removal from pleural space.

Pleural liquid and kinetic friction coefficient of mesothelium after mechanical ventilation

Volume and protein concentration of pleural liquid in anesthetized rabbits after 1 or 3h of mechanical ventilation, with alveolar pressure equal to atmospheric at end expiration, were compared to those occurring after spontaneous breathing. Moreover, coefficient of kinetic friction between samples of visceral and parietal pleura, obtained after spontaneous or mechanical ventilation, sliding in vitro at physiological velocity under physiological load, was determined. Volume of pleural liquid after mechanical ventilation was similar to that previously found during spontaneous ventilation. This finding is contrary to expectation of Moriondo et al. (2005), based on measurement of lymphatic and interstitial pressure. Protein concentration of pleural liquid after mechanical ventilation was also similar to that occurring after spontaneous ventilation. Coefficient of kinetic friction after mechanical ventilation was 0.023±0.001, similar to that obtained after spontaneous breathing.