Using 2-photon microscopy to understand albuminuria

Abstract

Intravital 2-photon microscopy, along with the development of fluorescent probes and innovative software, has rapidly advanced the study of intracellular and intercellular processes at the organ level. Researchers can quantify the distribution, behavior, and dynamic interactions of up to four labeled chemical probes and proteins simultaneously and repeatedly in four dimensions (3D + time) with subcellular resolution in real time. Transgenic fluorescently labeled proteins, delivery of plasmids, and photo-activatable probes enhance these possibilities. Thus, multi-photon microscopy has greatly extended our ability to understand cell biology intra-vitally at cellular and subcellular levels. For example, evaluation of rat surface glomeruli and accompanying proximal tubules has shown the long held paradigm regarding limited albumin filtration under physiologic conditions is to be questioned. Furthermore, the role of proximal tubules in determining albuminuria under physiologic and disease conditions was supported by direct visualization and quantitative analysis.

Fig. 1

Texas Red Rat Serum Albumin uptake by proximal tubule cells. A 12-micron volume of a superficial glomerulus given a single bolus of labeled albumin (red) ∼20 minutes post-infusion shows avid uptake in the early S1 segment and other proximal tubules (PT). Note the absence of labeled albumin in distal tubules (DT), corroborating the high capacity of PTs to internalize and transcytose filtered albumin. (Abbreviations: mv, microvasculature; Glom cap, glomerular capillary loops; BowSp, Bowman's space; Bar = 20 μm.)

Fig. 2

Transcytosis of albumin across proximal tubule cells in vivo using 2-photon microscopy. (A) Two-photon intra-vital time image taken in a Simonsen Munich Wistar rat given 2 mg of Alexa 568-RSA intravenously 24 hours before imaging shows vesicular and tubular structures containing albumin. A single frame with a large accumulation of albumin is indicated by the arrow; note the orientation of the apical membrane is opposite the arrow. The formation of a tubular structure extending from an intracellular compartment toward the basal pole of the PTC is shown at the end of the arrow. (B) Schematic of albumin entering into a PTC either via unbound in a fluid phase vesicle/endosome (FPE) or bound to megalin-cubulin as a receptor mediated endosome (RME) at the apical surface via a clatherin coated pit. With acidification to a pH of less than or equal to 6, the megalin-cubulin binding of albumin diminishes while that of FcRn increases dramatically. As such, there is an exchange of albumin from megalin-cubulin to binding to FcRn and this carrier then mediates transcytosis. When the transcytotic vesicle unites with the basolateral membrane, the increase in pH of the interstitial compartment releases albumin to diffuse into the interstitium and be transcytosed across endothelial cells again using FcRn as the carrier. FcRn then recycles to the apical membrane area via the recycling vesicle (RV). L stands for lysosome.