Quantitative morphology of renal cortical structures during compensatory hypertrophy

The compensatory hypertrophy in different renal cortical structures was studied in rats 10 and 21 days after unilateral nephrectomy (UNX). Quantitative morphological/stereological analysis revealed significant increases in total renal cortical volume - 33% on day 10 and 48% on day 21 - after UNX. These changes were paralleled by significant increments in the volumes of proximal convoluted tubule (PCT, 55%), distal convoluted tubule (DCT, 114%), and cortical collecting duct (CCD, 106%) segments on day 10. The corresponding changes on day 21 were 76, 122, and 212%, respectively. These alterations were accompanied by increases in segment length; 3% PCT, 23% DCT, and 50% CCD on day 10 and 9% PCT, 30% DCT, and 142% CCD on day 21 after UNX. The total luminal and basolateral cell membrane surface areas also exhibited a time-dependent increase after UNX. The increments in both luminal and basolateral membrane domains in PCT and DCT after 10 days were not significant, but reached significance after 21 days (PCT: luminal membrane 21%, basolateral membrane 63%; DCT: luminal membrane 98%, basolateral membrane 63%). In contrast, CCD membrane areas had increased substantially already 10 days after UNX (luminal membrane 92%, basolateral membrane 71%). It declined subsequently by day 21 (luminal membrane 57%, basolateral membrane 32%). The cell rubidium concentration after a 30-second rubidium infusion, an index of Na-K-ATPase activity, as well as sodium concentrations were unaltered in cells of all nephron segments investigated. Altogether the stereological analysis shows that the compensatory increase in organ volume can be attributed primarily to an increase in nephron epithelial volume. The PCT responds with `radial' hypertrophy (thickening of the tubular epithelial wall), while the DCT undergoes `length' hypertrophy (increase of tubular length without thickening of the tubular wall and without an increase in number of cells). This type of hypertrophy is especially prominent on day 21 after UNX for the CCD which doubles in length. Only on day 10 does the CCD seem to respond with hyperplasia. Adaptive changes in response to UNX develop gradually. Only a few of the morphological parameters studied had completed their change by 10 days, the majority required longer

A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse

The increasing number of available genetically manipulated mice makes it necessary to develop tools and techniques for examining the phenotypes of these animals. We have developed a straightforward and rapid method for the isolation of large quantities of single tubule fragments from the mouse kidney. Immunohistochemistry, electron microscopy, and fluorescence microscopy were used to evaluate the viability, functional characteristics, and morphology of proximal tubules (PT), and collecting ducts from cortex (CCD) and inner stripe of the outer medulla (ISOMCD). Tubules were isolated using a modified collagenase digestion technique, and selected under light microscopy for experimentation. Electron microscopy and trypan blue exclusion showed that a large portion of unselected proximal tubules were damaged by the digestion procedure. The selected tubules, however, all excluded trypan blue, indicating that the plasma membrane had remained intact. Immunocytochemistry on isolated CCD showed normal distribution of H+-ATPase, pendrin, and anion exchanger-1 (AE-1) staining. The pH-sensitive dye 2′,7′-bis(2-carboxylethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure Na+-dependent and -independent intracellular pH (pHi) recovery rates in PT, and in single intercalated cells of CCD and ISOMCD fragments. Na+-dependent pHi-recovery was 0.144±0.008 (PT), 0.182±0.013 (CCD), and 0.112±0.010pH units/min. (ISOMCD). Na+-independent pHi recovery was found in all three segments (PT: 0.021±0.002, CCD: 0.037±0.002, ISOMCD: 0.033±0.002pH units/min) and was sensitive to concanamycin. In summary, we have developed a new technique for rapid and straightforward preparation of large quantities of defined tubule fragments from mouse kidney. Using this technique, the first measurements of plasma membrane vacuolar H+-ATPase activities in mouse PT and collecting duct were made. This technique will facilitate further characterization of kidney function in normal and genetically manipulated animal