Robust capability of renal tubule fatty acid uptake from apical and basolateral membranes in physiology and disease

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Excess lipid accumulation is associated with obesity-related chronic kidney disease, but the mechanisms of fatty acid (FA) uptake have been poorly understood. To this end, we investigated how FAs are taken up by tubular epithelial cells (TECs) in mice by using in vivo FA tracing and histological methods. Immunohistochemistry showed that CD36, which is a well-known FA transporter, was abundantly expressed on the basolateral side of proximal TECs (PTECs). The uptake of 125 I-BMIPP (a radiolabeled FA tracer) was significantly reduced in CD36-knockout kidneys at 1 min after injection. In vivo imaging with multiphoton microscopy revealed that BODIPY-C 12 (a fluorescence-labeled FA tracer) accumulated on both the basolateral and apical sides of PTECs. Numerous lipid droplets accumulated in PTECs after accelerated lipolysis. Furthermore, PTEC-specific injury via diphtheria toxin (DT) injection in transgenic mice expressing the DT receptor resulted in a compensatory increase in lipid accumulation in downstream TECs. Importantly, urinary FAs were undetectable, even in mice and humans with remarkable albuminuria. Our data demonstrate that renal TECs take up FAs from blood (CD36-dependent) and primary urine (CD36-independent) and can store excess FAs as neutral lipids. The results further show that renal tubules have hitherto largely unappreciated mechanisms by which the excretion of FAs into the urine is avoided.

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  1. eLife assessment

    This manuscript presents valuable and significant data on how lipids may accumulate in the tubulointerstitial compartment of the diseased kidney, but the work is largely descriptive, using methods that are inadequate for quantification (colorimetric assays versus mass spec), thus rendering data interpretation not very convincing. Therefore, while a major strength is the presentation of innovative ideas, additional experiments would be needed to support the main conclusions.

  2. Reviewer #1 (Public Review):

    Kidneys have very high energy needs and they preferentially use lipids as their energy source. Lipid metabolism however poorly understood in the kidney. Lipids accumulate in diseased kidneys, however the mechanism of lipid accumulation is not well understood. The team has studied lipid metabolism in the kidney.

    1. The team has first performed some basic lipid accumulation studies in kidneys of healthy mice. Lipid uptake show very significant feeding and fasting differences. It is unclear what stage of the feeding cycle the experiments were performed and whether the team has standardized it.

    2. All measurements have been performed using the colorimetric kit, the team should also use mass spec to validate results.

    3. The team did not measure lipolysis, fatty acid oxidation or fatty acid synthesis so statements so statements made around these pathways appear to be only speculative. They could measure it or adjust the text.

    4. Figure2 is probably the most valuable information in the paper. Lipid accumulation is only measured by staining. The team should also perform Plin stain or other methods to support their statement.

    5. The apical vs basal lipid update also seems speculative. I am not sure that we could see these differences. In addition, no quantification is presented to support the findings.

    6. For fatty acid uptake the team has analyzed the cd36 KO but not the fatp2 KO mice.

    7. The team has analyzed a PT injury model but most proteinuria is the result of glomerular injury. Unclear whether the data is relevant for glomerular disease

    8. The human urinary fatty acid quantification would need positive control samples. Clinically we often see lipid droplets in the urine, which is inconsistent with the presented data.

    9. No clear conclusion can be drawn from the data

    Overall while the project has some interesting elements and the presented data is relatively weak and no clear conclusion can be drawn and the overall message is unclear.

  3. Reviewer #2 (Public Review):

    Dr. Kawakami and colleagues investigate that FA is taken up via both apical and basolateral sides of tubular epithelial cells. CD36 is known to be expressed in tubular cells, so it is expected and well known that FA was taken up via CD36 at the basolateral side. However, FA is also taken up at the apical side (primary urinary side) independent of CD36 activity, and albumin reabsorption is an interesting new finding, although the specific mechanism involved in this process is not shown but discussed possible mechanism in a discussion section in the manuscript. Authors provide the evidence of CD36 expression in the basolateral side of tubules, TG contents in kidney tissue, and FA levels in serum and urine utilizing CD36KO mice, PTi (PT specific injury), and megalin KO mice to support the author's hypothesis.

    Although it is an interesting study, this study is overall descriptive by performing staining and testing FA levels in serum or urine rather than conducting functional studies in tubule cells. Moreover, the authors exclude the possibility that TG content is associated with TG lipolysis. Cell stores uptake FA as TG in lipid droplets and lipase activity is required to use FA as their energy source especially in tubular cells that are known to use FA as their energy source. Thus, there is a high probability that the balance between FA uptake and TG lipolysis determines TG contents. To exclude the TG lipolysis and ensure the TG contents are highly and solely associated with serum FA, the author should provide lipase activity, level of lipolysis, and TG content in tubular cells in vitro.

  4. Reviewer #3 (Public Review):

    The authors of this study were trying to determine the mechanisms of of fatty acid uptake and accumulation in the kidney. Their work identified clear evidence for both basolateral (CD36-dependent) and apical uptake of fatty acids in the kidney. The apical uptake of fatty acids is independent of megalin. Interestingly there is absence of fatty acids in the urine even in subjects with significant proteinuria indicating that fatty acids in the urine are completely taken up by the renal tubules.