Lymphatic Microvessels in the Rat Remnant Kidney Model of Renal Fibrosis

Abstract

Abstract. Rat remnant kidney is an established model of renal tubulointerstitial fibrosis and progression to end-stage renal failure. The morphologic lesions comprise nephron loss and regeneratory tubular hypertrophy, interstitial infiltration, predominately by macrophages, and progressive fibrosis. A critical role in this complex pathology was assigned to tubulointerstitial blood microvessels that regulate the supply of oxygen and nutrients of tubuli. Whereas some investigations reported a rarefaction of the vascular network in association with the degenerative cortical changes, others observed an increase in vascularization. Here these discrepant findings are addressed by reinvestigation of the vascularization of rat remnant kidneys by the use of two novel endothelial lineage specific, discriminatory markers, i.e., the membrane mucoprotein podoplanin with specificity for lymphatic endothelia, and the glycosylphosphatidylinositol (GPI)-anchored membrane enzyme aminopeptidase P that is recognized by a monoclonal antibody designated JG12 and that is specifically expressed by endothelial cells of blood vessels only. The results obtained confirm a regional rarefaction of aminopeptidase P-positive blood microvessels; they also establish major changes in the renal lymphatic vasculature. Massive proliferation of lymphatic vessels was observed in fibrotic tubulointerstitial regions, whereas in kidneys of sham-operated rats, only a few lymphatic vessels were found adjoined with arteries. The lymphatic vessels frequently contained mononuclear cells that were also encountered in the interstitial spaces and expressed relative large amounts of vascular endothelial growth factor-C mRNA by in situ hybridization. Collectively, these results indicate that a large proportion of the microvessels encountered in the cortex of remnant kidneys are of lymphatic origin and cannot be discriminated by common endothelial markers, such as CD34, that are expressed by both lymphatic and blood endothelia cells. As lymphatic endothelial cells secrete chemokines that attract dendritic cells, it is possible that the increase in lymphatic vascularization could enhance the immunologic surveillance of remnant kidneys.

Comparison of the diagnostic accuracy of lymphatic endothelium markers: Bayesian approach

“…Up to date, experts' opinions favored the antibody against podoplanin as the most reliable and specific marker at hand. 1,5,9,14,15,17,21 Nevertheless, scientists emphasize the necessity for a quantitative evaluation of the accuracy of the so-called 'specific lymphatic endothelium markers'. 1,5,[19][20][21][22] To our knowledge, our work is the first report that pursues direct comparisons between two lymphatic endothelium markers and provides quantitative estimates for their specificity, sensitivity and positive predictive value.…”

“…However, the used priors are based on the available knowledge and a comprehensive literature search. 1,5,9,14,15,17,21,26 Finally, we considered stained vessels with intraluminal red blood cells as potential 'false-positives'. Intraluminal erythrocytes might be a histologic artifact resulting from pressure-induced leakage of a neighboring blood vessel in a true lymphatic vessel.…”

“…These monoclonal antibodies are considered to be highly specific. 1,5,9,14,15,17,21,26 Therefore, we assigned to both antibodies high specificities. Also, prior beliefs regarded the antibody against podoplanin as the most specific lymphatic endothelium marker; 1,5,9,14,15,17,21 hence, we set priors between 90 and 100% for the specificity of the antibody against podoplanin, and between 80 and 100% for D2-40 ( Figure 1).…”

“…1,5 It has been postulated that the density of the lymphatic vessels is a predictor of clinically meaningful outcomes in a number of malignancies. [6][7][8][9][10][11][12][13] One of the most critical steps in the assessment of lymphatic vessel density is the selection of the optimal lymphatic endothelium marker, 1,2,5,[12][13][14][15][16][17][18][19][20][21][22][23] in order to avoid spurious findings arising from the low accuracy of the staining. However, given the absence of a gold standard, the evaluation of the sensitivity and the specificity of the lymphatic endothelium markers is a daunting task.…”

“…1,5,9,14,15,17,21 Such claims are not based on formal quantitative comparisons between different lymphatic endothelium markers in the same samples, despite the numerous relative calls in the literature. 3,12,16,[20][21][22] We set out to compare the diagnostic accuracy of podoplanin and D2-40 in the same samples, employing a Bayesian approach to account for the absence of a gold standard.…”