Abstract 5266: A Novel Technique for Assessing the Renal Microvasculature in Chronic Kidney Disease

Abstract

A variety of factors have been identified as playing an important role in the progression of chronic kidney disease (CKD) to end-stage renal failure. Microvascular loss plays a critical role, being pathologically linked to the development of glomerular sclerosis and tubulointerstitial fibrosis. Thus, we have developed a new and novel post-mortem fixation technique, fluorescent microangiography (FMA), for assessing the microvasculature in animal models of CKD. Methods: 8 week old make Fischer 344 rats underwent subtotal nephrectomy (n=10) (SNX), or sham surgery (n=10). Systolic blood pressure (SBP), serum creatinine, and proteinuria were assessed at baseline and 8 weeks follwing surgery. At sacrifice rats were subjected to FMA. (10% solution of 0.02 μ m fluorescent microspheres) Thick (200 μ m) sections of fixed tissue can then be visualized under confocal microscopy. Stacks of images are collected and 3D reconstruction and visualization carried out in order to quantify the density, size and shape of blood vessels. An automated tracing algorithm was used to connect the tubular microvessels in 3 dimensions, and a solid 3D model was created (Neurolucida). Results : SNX animals showed increased in SBP (192±36 versus 123±4 mm Hg in controls; p < 0.0001), serum creatinine (65±12 versus 32±4 μ M in controls; p < 0.001) and urinary protein (84±26 versus 13±1 mg/d in controls; p < 0.05). FMA analysis demonstrated glomerular hypertrophy (average glomerular diameter [185±7 versus 136±4 μ M in controls; p < 0.01]) with microvascular loss in SNX animals (3.02E-03±2.20E-04 versus 4.45E-03±2.29E-04 μ M in controls; p < 0.01, see figure below). SNX increased total vessel length (9809±627 versus 5879±393 μ M in controls; p < 0.05), average vessel diameter (5.59±0.15 versus 5.24±0.18 μ M in controls; p < 0.05). Conclusion: Fluorescent microangiography (FMA), with a spatial resolution of less than 1 μ M is a practical and quantifiable method for visualizing the microvasculature of the kidney that allows for direct measurements of volume, diameter, length and vascular density. In addition FMA provides visualization of the phenotypic changes in the renal microvasculature associated with CKD.