CKD development in sickle mice after repeated hemolytic events. (A) Experimental scheme showing the regimen of repeated low-dose hemin injections, mimicking multiple hemolytic events in SS mice. (B) Kidney function represented by GFR, 1 week after the last hemin challenge in the younger SS mice (4-6 weeks) compared with the vehicle-injected mice (n = 8; M, 4; F, 4). (C-E) Increased albuminuria (represented by uACR), plasma Cys C, and urinary KIM-1 in multiheme-challenged SS mice showing CKD development in younger SS mice. (F) Evans blue extravasation data showing elevated renal endothelial barrier disruption in younger SS mice injected with the multiheme regimen (n = 5-6; M, 2-3; F, 3). (G) Vehicle- and heme-injected SS mice were subjected to SRU imaging 1 week after the last hemin challenge. The reconstructed 2-dimensional, cross-sectional, SRU images of the long-axis mouse kidney showing vessel distribution and the velocity map for the vehicle and hemin-challenged mice (n = 3; M, 2; F, 1). The velocity values were separated into positive (displayed as red in the color map) and negative values (displayed as blue in the color map), representing the blood flow direction toward and away from the US probe to separate the arteries and veins, respectively. Quantitation of (H-I) vessel densities and (J-K) renal blood flow calculated from the SRU data showing loss of vessels and reduced blood flow in the SS mouse kidney after multiheme-induced CKD development. ∗P < .05; ∗∗∗P < .001 (unpaired Student t test).