Preparation and Composition of Mouse Tubular and Glomerular Basement Membranes

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Abstract
Methods were developed to obtain tubules and glomeruli and their respective basement membranes from mouse kidneys. While the procedures are especially useful for preparing tubules, one method can be used to simultaneously prepare both tubules and glomeruli. Tubules can be obtained from single animals, while a minimum of five animals are required in order to prepare glomeruli. Either minced whole kidneys or dissected cortex tissue is dispersed using a polytron and the desired fractions are obtained by sucrose density gradient centrifugation. Discontinuous gradients consisting of 49.5, 53, 57, and 60% sucrose are used in the first method. Following centrifugation, tubules are collected on the 49.5% sucrose layer and glomeruli are pelleted on the bottom of the tube. In the second method, disrupted tissue is mixed with 57% sucrose and after centrifugation a layer of pure tubules is obtained from the top of the sucrose solution. Basement membranes are then obtained by a sonication method and the average yields per kidney are 0.1 mg and 0.02 mg for tubular and glomerular basement membranes respectively. Their chemical compositions are similar to the respective basement membranes from other species.