Regional metabolic analysis of structurally preserved kidney slices by ex vivo respirometry

A comprehensive spatial analysis of kidney metabolism is essential for advancing knowledge of both normal kidney physiology and pathophysiology. The kidney exhibits marked regional differences in bioenergetic demands and substrate utilization, reflecting the distinct functional profiles of each nephron segment. To complement existing approaches with freshly isolated tubules or primary cell cultures, we established and validated an ex vivo respirometry method using structurally preserved kidney slices on a Seahorse XFe24 platform. This protocol avoids tissue disruption or enzymatic digestion and enables simultaneous, region-specific measurements of metabolic fluxes in the cortex, outer medulla, and inner medulla. It provides an integrated readout of the metabolic properties of the cell types present within each anatomical region. We demonstrate the utility of this approach through proof-of-principle studies that profile region-specific metabolic fluxes under hyperglycemic conditions in a mouse model of obesity and type 2 diabetes, as well as the metabolic alterations that accompany the transition from acute ischemic injury to chronic kidney disease. Furthermore, to highlight its relevance for therapeutic discovery, we applied this method to assess the impact of pharmacological hypoxia-inducible factor activation on regional kidney bioenergetics. In summary, this protocol advances the study of kidney metabolism by providing a robust platform for region-specific analysis of kidney respiration and bioenergetics and holds promise for accelerating the development of novel therapies targeting metabolic pathways in kidney disease.NEW & NOTEWORTHY Assessment of regional metabolism in kidney tissue is crucial for understanding normal physiology and disease. We have developed a robust ex vivo method to measure respiration in structurally preserved kidney slices using a metabolic flux analyzer. This approach enables analysis of metabolic fluxes and substrate utilization in the kidney cortex, outer medulla, and inner medulla while maintaining tissue architecture, providing region-specific insights into kidney metabolism with broad applications in disease modeling and therapeutic discovery. Keywords: acute kidney injury; diabetic nephropathy; hypoxia-inducible factor; mitochondria; respirometry.