Volumetric Imaging Approaches Reveal Unique Spatial Interactions Between Cysts and Vasculature in Polycystic Mouse Kidneys

Abstract

A significant part of the polycystic kidney disease (PKD) pathology occurs in the renal vasculature. Some vascular pathology may be caused by direct mechanical pressures exerted by expanding cysts, while others caused by other factors. One such factor is compensatory remodeling consequent to nephron dropout, often manifesting as glomerular hypertrophy and glomerular afferent arteriole dilation. Determination of direct, mechanical cyst effects upon the vasculature all but requires assessment of the spatial relationship between the vasculature and the cysts, difficult to assess using conventional two-dimensional (e.g. histology) approaches. The primary objective of this project is to establish 3D imaging approaches of the PKD kidney cysts and vasculature thereby providing valuable insight into the spatial interactions between them. Methods. PKD mouse renal vascular casts were made and the tissue, but not cast, made optically transparent through ethanol, methyl salicylate chemical clearing. This enabled dissection microscope visualization of both the vascular cast and cysts. The vascular cast has excellent intrinsic radiocontrast, and therefore we micro-CT scanned one PKD kidney vascular cast. We applied the CLARITY technique to additional kidneys. This technique renders tissue optically transparent but retains protein integrity thus enabling 3D imaging by conventional immunostaining. Through application of this technique, we were able to visualize cysts, arteries, arterioles, and glomeruli. Results. Dissection microscope imaging of the vascular cast, tissue-cleared kidneys revealed numerous examples of vessels, often ~20 µm in diameter, circumferentially following cyst boundaries. On micro-CT imaging, many interlobar vessels, diameters typically 50-100 µm, follow an irregular, wave-like path, where path undulations often spatially coincided with cyst boundaries. The CLARITY technique similarly revealed relatively large diameter (45 µm) vessels circumferentially following cyst boundaries. In an n=1 study, PKD glomerular size and glomerular afferent arteriole size were larger than that in a non-PKD kidney (glomerular diameter: 66.3 ± 2.4 vs. 79.6 ± 4.1 µm; glomerular afferent arteriole diameter: 9.9 ± 0.7 vs. 15.5 ± 1.1, µm). However, the glomerular diameter to afferent arteriole diameter ratio was reduced in PKD (6.9 ± 0.5 vs. 5.3 ± 0.4, µm/µm). Within the PKD kidney, glomerular diameter and afferent arteriole diameter were nearly identical, on average, for glomeruli located immediately adjacent to cysts versus those not. Conclusions. These imaging approaches enable visualization of cysts and all levels of the renal vasculature in 3D and may be used to find structural correlates of known PKD functional deficits such as autoregulation. This limited n, proof-of-concept study revealed abnormalities in PKD renal vasculature that are likely caused, at least in part, by direct mechanical cyst forces, while other possible abnormalities may be caused by other factors.