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Purpose: The increasing incidence of kidney diseases is a global concern, and current biomarkers and treatments are inadequate. Changes in renal tubule luminal volume fraction (TVF) serve as a rapid biomarker for kidney disease and improve understanding of renal (patho)physiology. This study uses the amplitude of the long T 2 component as a surrogate for TVF in rats, by applying multiexponential analysis of the T 2 -driven signal decay to examine micromorphological changes in renal tissue., Methods: Simulations were conducted to identify a low mean absolute error (MAE) protocol and an accelerated protocol customized for the in vivo study of T 2 mapping of the rat kidney at 9.4 T. We then validated our bi-exponential approach in a phantom mimicking the relaxation properties of renal tissue. This was followed by a proof-of-principle demonstration using in vivo data obtained during a transient increase of renal pelvis and tubular pressure., Results: Using the low MAE protocol, our approach achieved an accuracy of MAE < 1% on the mechanical phantom. The T 2 mapping protocol customized for in vivo study achieved an accuracy of MAE < 3%. Transiently increasing pressure in the renal pelvis and tubules led to significant changes in TVF in renal compartments: ΔTVF cortex  = 4.9%, ΔTVF outer&#95;medulla  = 4.5%, and ΔTVF inner&#95;medulla  = -14.6%., Conclusion: These results demonstrate that our approach is promising for research into quantitative assessment of renal TVF in in vivo applications. Ultimately, these investigations have the potential to help reveal mechanism in acute renal injury that may lead to chronic kidney disease, which will support research into renal disorders., (© 2024 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)