Remote sensing and signaling in kidney proximal tubules stimulates gut microbiome-derived organic anion secretion

Abstract

Membrane transporters and receptors are responsible for balancing nutrient and metabolite levels to aid body homeostasis. Here, we report that proximal tubule cells in kidneys sense elevated endogenous, gut microbiome-derived, metabolite levels through EGF receptors and downstream signaling to induce their secretion by up-regulating the organic anion transporter-1 (OAT1). Remote metabolite sensing and signaling was observed in kidneys from healthy volunteers and rats in vivo, leading to induced OAT1 expression and increased removal of indoxyl sulfate, a prototypical microbiome-derived metabolite and uremic toxin. Using 2D and 3D human proximal tubule cell models, we show that indoxyl sulfate induces OAT1 via AhR and EGFR signaling, controlled by miR-223. Concomitantly produced reactive oxygen species (ROS) control OAT1 activity and are balanced by the glutathione pathway, as confirmed by cellular metabolomic profiling. Collectively, we demonstrate remote metabolite sensing and signaling as an effective OAT1 regulation mechanism to maintain plasma metabolite levels by controlling their secretion.

Keywords: indoxyl sulfate; kidney proximal tubule; organic anion transporter 1; remote sensing and signaling.

Fig. 1.

Human (h) and rat (r) kidneys sense and signal elevated gut-derived metabolite plasma levels and induce their secretion. (A) Schematic diagram of the human study design. (B) IS excretion before and after high-protein diet intervention. (C) Relative mRNA hOAT1 expression before and after high-protein diet intervention in human kidney cells. (D) Schematic diagram of the second human study (n = 36 volunteers) using protein concentrates extracted from corn, whey, and bovine plasma in a randomized manner. (E) IS and (F) p-cresyl sulfate excretion following corn, whey, and bovine plasma protein concentrate intervention. (G) Schematic diagram of the animal study design. (H) IS clearance over time in vehicle (triangle) and IS (square)-treated CKD rats. (I) Relative mRNA rOAT1 expression in vehicle (triangle) and IS (IS, square)-treated rat kidney cells. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2.

IS induces OAT1 expression and function in vitro via the AhR and EGFR axis under the control of miR-223. (A) Schematic diagram of the experimental in vitro design using renal proximal tubule cells. (B) Relative mRNA hOAT1 gene expression in control and IS treated ciPTEC cells in the presence or absence of alpha-naphtoflavone (aNF) and (C) in primary kidney cells. (D) Relative OAT1 protein expression corrected for loading control using Na,K-ATPase and normalized to control. Control and treated cells in the presence or absence of CH-223191 (CH) and cetuximab (CTX) are shown. (E) OAT1 activity monitored using fluorescein transport in control and treated cells in the presence or absence of CH and CTX and normalized to control. (F) Fluorescein transport in control and treated cells in the presence or absence of EGF. (G) Relative mRNA expression EGF signaling in IS-treated cells compared with control. White boxes are not measured. For complete result list of genes tested, see SI Appendix, Table S3. (H) Fluorescein transport in control and treated cells in the presence or absence of bisindolylmaleimide (BIM; protein kinase C inhibitor), MEK (U-0126; MEK inhibitor), and LY-294002 (LY; phosphoinositide 3-kinase inhibitor) and normalized to control. (I) Fluorescein transport in control and treated cells in the presence or absence of SN50 trifluoroacetate salt (SN50; NF-κB inhibitor) and normalized to control. (J and K) Nuclear ARNT expression in control and treated cells in the presence or absence of CTX. Representative images are shown in K. (L) Fluorescein transport in control and treated cells in the presence or absence of scrambled antagomiR-223 (Scr miR223) or antagomiR-223 (a-miR223) and normalized to control. (M) Nuclear ARNT expression in control and treated cells in the presence or absence of Scr miR223 or a-miR223 and normalized to control. Representative images are shown in SI Appendix, Fig. S4. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. (Scale bar: K, 10 µm.)

Fig. 3.

Ameliorated transepithelial transport using IS exposure in a 3D kidney model. (A) Schematic showing the experimental design of transepithelial transport across a 3D bioengineered kidney tubule. (B) IS clearance in control and IS-treated bioengineered kidney tubules. Data are presented as mean ± SEM. *P < 0.05.

Fig. 4.

Reactive oxygen species aid remote metabolite sensing and signaling. (A) ROS production in control and treated cells in the presence or absence of trolox. Data were normalized to control. (B) Fluorescein transport in control and treated cells in the presence or absence of N-acetyl-l-cysteine (AcCyst) and trolox. Data were normalized to control and are presented as mean ± SEM. **P < 0.01 and ***P < 0.001.

Fig. 5.

Induced glutathione and reduced beta-alanine metabolism during IS sensing and signaling in response to oxidative stress. (A) Metabolomic analysis of IS-treated cells compared with control. Relative metabolite abundance of the glutathione metabolism, TCA cycle, urea cycle, oxidate pentose phosphate pathway (PPP), and glycosis are plotted. Color code: orange, P < 0.05; yellow, 0.10 > P > 0.05; white, P > 0.10; gray, not analyzed. (B) Pathway analysis based on impact and P value showing that glutathione metabolism is enhanced and beta-alanine is reduced in IS-treated cells compared with control. Larger circles farther from the y axis and orange-red color show higher impact of pathway. P-CoA, pantothenate and co-A biosynthesis; P, propanoate metabolism; CC, citrate cycle; GST, glycine, serine, and threonine metabolism. *P < 0.05 and **P < 0.01.