Figure 6 |. Multiphoton microscopy can be used to measure changes in glomerular albumin leakage.

(a) Simultaneous measurement of Alexa 594 albumin florescence intensity in the Bowman’s space (BS) and within plasma-filled glomerular capillary loops (CL) allowed calculation of the glomerular sieving coefficient for albumin (GSCalb). Multiphoton images were selected for analysis provided the BS could be clearly defined and 2 sites with no capillary loops within 4.5 μm (X, Y, and Z plane) could be sampled, minimizing background florescence. The same glomeruli were imaged on day 0 and day 5, allowing calculation of the fold-change in GSCalb in each glomerulus before calculating the mean for each mouse. Bar = 50 μm. (b) The formula used to calculate the GSCalb for each glomerulus. Fluorescence intensity (FI) was measured at 2 points within the BS and at 3 points within the capillary lumens on each image, 4 images were analyzed for each glomerulus. The final GSCalb for each mouse was taken as the average result from all included glomeruli. (c) Young DBA2J mice developed significant albuminuria (elevated urine albumin creatinine ratio [uACR]) after 10 days of salt and aldosterone exposure (analysis of variance; P = 0.176; Tukey correction shown; n = 5). (d) Fold-change in the glomerular albumin sieving coefficients for young DBA2J mice after 5 days of salt and aldosterone (Mann-Whitney; P = 0.0079; n = 5). Day 10 measurements were attempted, but glomeruli were too deep for reliable assessment. Salt + aldosterone row: the checkmark indicates 1% NaCl in drinking water (ad libitum) and 0.6 μg/g/d aldosterone delivered subcutaneously via a minipump; the X indicates standard drinking water (ad libitum) and a vehicle (ethanol saline)-filled minipump (*P < 0.05; **P < 0.01). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.