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Fluorescence microangiography for quantitative assessment of peritubular capillary changes after AKI in mice.

ABSTRACT: AKI predicts the future development of CKD, and one proposed mechanism for this epidemiologic link is loss of peritubular capillaries triggering chronic hypoxia. A precise definition of changes in peritubular perfusion would help test this hypothesis by more accurately correlating these changes with future loss of kidney function. Here, we have adapted and validated a fluorescence microangiography approach for use with mice to visualize, analyze, and quantitate peritubular capillary dynamics after AKI. A novel software-based approach enabled rapid and automated quantitation of capillary number, individual area, and perimeter. After validating perfusion in mice with genetically labeled endothelia, we compared peritubular capillary number and size after moderate AKI, characterized by complete renal recovery, and after severe AKI, characterized by development of interstitial fibrosis and CKD. Eight weeks after severe AKI, we measured a 40%±7.4% reduction in peritubular capillary number (P<0.05) and a 36%±4% decrease in individual capillary cross-sectional area (P<0.001) for a 62%±2.2% reduction in total peritubular perfusion (P<0.01). Whereas total peritubular perfusion and number of capillaries did not change, we detected a significant change of single capillary size following moderate AKI. The loss of peritubular capillary density and caliber at week 8 closely correlated with severity of kidney injury at day 1, suggesting irreparable microvascular damage. These findings emphasize a direct link between severity of acute injury and future loss of peritubular perfusion, demonstrate that reduced capillary caliber is an unappreciated long-term consequence of AKI, and offer a new quantitative imaging tool for understanding how AKI leads to future CKD in mouse models.

SUBMITTER: Kramann R

PROVIDER: S-EPMC4147987 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Vascular endothelial growth factor (VEGF) and its cognate receptor (VEGFR2) system are crucial for cell functions associated with angiogenesis and vasculogenesis. Klotho contributes to vascular health maintenance in the kidney and other organs in mammals, but it is unknown whether renoprotection by Klotho is dependent on VEGF/VEGFR2 signaling. We used heterozygous VEGFR2-haploinsufficient (VEGFR2+/-) mice resulting from heterozygous knockin of green fluorescent protein in the locus of fetal liver kinase 1 encoding VEGFR2 to test the interplay of Klotho, phosphate, and VEGFR2 in kidney function, the vasculature, and fibrosis. VEGFR2+/- mice displayed downregulated VEGF/VEGFR2 signaling in the kidney, lower density of peritubular capillaries, and accelerated kidney fibrosis, all of which were also found in the homozygous Klotho hypomorphic mice. High dietary phosphate induced higher plasma phosphate, greater peritubular capillary rarefaction, and more kidney fibrosis in VEGFR2+/- mice compared with wild-type mice. Genetic overexpression of Klotho significantly attenuated the elevated plasma phosphate, kidney dysfunction, peritubular capillary rarefaction, and kidney fibrosis induced by a high-phosphate diet in wild-type mice but only modestly ameliorated these changes in the VEGFR2+/- background. In cultured endothelial cells, VEGFR2 inhibition reduced free VEGFR2 but enhanced its costaining of an endothelial marker (CD31) and exacerbated phosphotoxicity. Klotho protein maintained VEGFR2 expression and attenuated high phosphate-induced cell injury, which was reduced by VEGFR2 inhibition. In conclusion, normal VEGFR2 function is required for vascular integrity and for Klotho to exert vascular protective and antifibrotic actions in the kidney partially through the regulation of VEGFR2 function.NEW & NOTEWORTHY This research paper studied the interplay of vascular endothelial growth factor receptor type 2 (VEGFR2), high dietary phosphate, and Klotho, an antiaging protein, in peritubular structure and kidney fibrosis. Klotho protein was shown to maintain VEGFR2 expression in the kidney and reduce high phosphate-induced cell injury. However, Klotho cytoprotection was attenuated by VEGFR2 inhibition. Thus, normal VEGFR2 function is required for vascular integrity and Klotho to exert vascular protective and antifibrotic actions in the kidney.

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Fluorescence microangiography for quantitative assessment of peritubular capillary changes after AKI in mice.

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