A Comparative study of Permeabilized Proximal tubules and Cortical Mitochondrial Respiratory Functions in Kidneys

Background: The kidneys have the second highest O 2 consumption in mammals next to the heart, which is closely linked to tubular Na + transport functions. Proximal tubules (PTs) are responsible for reabsorbing a majority of Na + (~65%) from the glomerular filtrate in the cortex (Cx). This is highly energy demanding and requires a huge dependence on the PT mitochondrial bioenergetics. Mitochondrial dysfunction in the kidney is implicated in the pathophysiology of various diseases, including salt-sensitive hypertension, but studies of the PT mitochondrial bioenergetics have been hampered by the limited yield of PT mitochondria during isolation. Researchers therefore utilize Cx mitochondria as surrogates of PT mitochondria given the high density of PTs in the kidney Cx. However, the Cx has different cell types besides PTs, which may influence the study. To address this limitation, we have developed methods to permeabilize isolated PTs to study its mitochondrial bioenergetics and determined its reliability by comparing against Cx mitochondrial bioenergetics. Methods: PTs were bulk isolated from one kidney of normal Sprague-Dawley rats (6-8 wks) by collagenase perfusion, digestion, and sieving, and Cx mitochondria (Cx m ) were isolated from the other kidney of the same rats using differential centrifugation method. The isolated PTs were permeabilized (PT p ) using mechanical syringe strokes at 3 mg/ml and 5 mg/ml concentrations and compared to determine optimal condition for mitochondrial bioenergetic studies. The PT p and Cx m were studied for mitochondrial respiration (JO 2 ) and membrane potential (dPsi) changes to reveal their functional differences/similarity. Dynamic changes in JO 2 and dPsi were measured in PT p and Cx m using an Oroboros Oxygraph-2k respirometer with the addition of the complex I-linked substrate pyruvate+malate (PM) and complex II-linked substrate succinate (state 2; S2) followed by ADP (state 3; S3) and the uncoupler FCCP (state 5; S5). The JO 2 during S2 and S3 were used to determine the respiratory control index (RCI = S3/S2 JO 2 ) and P/O ratio (ADP consumed/O 2 consumed). Results: The total yield of PT p was estimated to be about twice of Cx m . Hence, dynamic changes in JO 2 of 0.2 mg/ml PT p were compared against 0.1 mg/ml Cx m . Based on preliminary studies, PTs permeabilized at 3 mg/ml was chosen for further study, considering its significant higher RCI than PTs permeabilized at 5 mg/ml. The JO 2 traces of PT p closely resembled to that of Cx m , when energized with PM or succinate. Cx m and PT p did not exhibit a significant difference in states 2 and 3 JO 2 magnitudes and state 3 JO 2 duration, confirming similar mitochondrial efficiency and coupling of oxidative phosphorylation (OxPhos) in the PT p and Cx m . The RCI and P/O ratio were ~6.5 and ~2.7 with PM and ~3.5 and ~1.6 with succinate, respectively, depicting close to theoretical values, confirming the efficient coupling of PT p mitochondria similar to Cx m . In addition, dPsi of PT p and Cx m were also close, confirming the mitochondrial membrane integrity. Conclusion: Our results show that PT p have mitochondrial bioenergetics similar to that of Cx m , in terms of OxPhos and coupling efficiency and mitochondrial membrane integrity. These pivotal preliminary studies provide the foundational basis for employing isolated permeabilized PTs, as opposed to isolated Cx mitochondria, to investigate PT-specific mitochondrial bioenergetics in pathological conditions. Funding: NIH R01 HL151587. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.