Multiphoton imaging reveals axial differences in metabolic autofluorescence signals along the kidney proximal tubule

Abstract

Kidney proximal tubules (PTs) are densely packed with mitochondria, and defects in mitochondrial function are implicated in many kidney diseases. However, little is known about intrinsic mitochondrial function within PT cells. Here, using intravital multiphoton microscopy and live slices of mouse kidney cortex, we show that auto-fluorescence signals provide important functional readouts of redox state and substrate metabolism, and that there are striking axial differences in signals along the PT. Mitochondrial NAD(P)H intensity was similar in both S1 and S2 PT segments, and was sensitive to changes in respiratory chain (RC) redox state, while cytosolic NAD(P)H intensity was significantly higher in S2. Mitochondrial NAD(P)H increased in response to lactate and butyrate, but decreased in response to glutamine and glutamate. Cytosolic NAD(P)H was sensitive to lactate and pyruvate, and decreased dramatically in S2 in response to inhibition of glucose metabolism. Mitochondrial flavoprotein (FP) intensity was markedly higher in S2 than in S1, but was insensitive to changes in RC redox state. Mitochondrial FP signal increased in response to palmitate, but decreased in response to glutamine and glutamate. Fluorescence lifetime decays were similar in both S1 and S2, suggesting that intensity differences are explained by differences in abundance of the same molecular species. Expression levels of known fluorescent mitochondrial FPs were higher in S2 than S1. In summary, substantial metabolic information can be obtained in kidney tissue using a label free live imaging approach, and our findings suggest that metabolism is tailored to the specialized functions of S1 and S2 PT segments.

Autophagy and Inflammation Regulation in Acute Kidney Injury

“…Moreover, rodents exhibit different results upon AKI injury based on their sex and strains, and we expect differences in the responses between humans and rodents owing to the expression of different immune cell markers and different lymphocyte expression ratios. Autophagy functions may be different in different proximal tubule segments (Milica et al, 2018). Compared to rodents, the kidney structure of a pig is more similar to that of a human.…”

Albumin uptake and processing by the proximal tubule: physiological, pathological, and therapeutic implications

“…The change in these cellular processes is substantial as one goes from the S2 to S3 PT cells ( 9 , 20 , 26 , 27 ). The different segments also have differing metabolic, autofluorescence, and frequency-domain fluorescence lifetime imaging microscopy signatures allowing for identification in in vivo studies ( 28 , 29 ). In these studies, the S1 segment, compared with the S2 and S3 segments, was shown to have the greater mitochondrial activity consistent with histological studies.…”

NAD+ homeostasis in renal health and disease

“…For example, the hypoxic environment of the inner medulla is matched by a near absence of mitochondria within those tubular cells. Even in the welloxygenated cortex, different segments of the proximal tubule preferentially utilize different fuel substrates 37 . Fatty acids may be a chief fuel source for the S1 and S2 segments of the proximal tubule, whereas the less welloxygenated straight or S3 segment may also utilize glucose 38 .…”