TRPV1 Hyperfunction Involved in Uremic Toxin Indoxyl Sulfate-Mediated Renal Tubular Damage

Abstract

Indoxyl sulfate (IS) is accumulated during severe renal insufficiency and known for its nephrotoxic properties. Transient receptor potential vanilloid 1 (TRPV1) is present in the kidney and acts as a renal sensor. However, the mechanism underlying IS-mediated renal tubular damage in view of TRPV1 is lacking. Here, we demonstrated that TRPV1 was expressed in tubular cells of Lilly Laboratories cell-porcine kidney 1 (LLC-PK₁) and Madin-Darby canine kidney cells (MDCK). IS treatment in both cells exhibited tubular damage with increased LDH release and reduced cell viability in dose- and time-dependent manners. MDCK, however, was more vulnerable to IS. We, therefore, investigated MDCK cells to explore a more detailed mechanism. Interestingly, IS-induced tubular damage was markedly attenuated in the presence of selective TRPV1 blockers. IS showed no effect on TRPV1 expression but significantly increased arachidonate 12-lipoxygenase (ALOX12) protein, mRNA expression, and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) amounts in a dose-dependent manner, indicating that the ALOX12/12(S)-HETE pathway induced TRPV1 hyperfunction in IS-mediated tubulotoxicity. Blockade of ALOX12 by cinnamyl-3,4-dihydroxy-α-cyanocinnamate or baicalein attenuated the effects of IS. Since aryl hydrocarbon receptor (AhR) activation after IS binding is crucial in mediating cell death, here, we found that the AhR blockade not only ameliorated tubular damage but also attenuated ALOX12 expression and 12(S)-HETE production caused by IS. The uremic toxic adsorbent AST-120, however, showed little effect on ALOX12 and 12(S)-HETE, as well as IS-induced cell damage. These results clearly indicated that IS activated AhR and then upregulated ALOX12, and this induced endovanilloid 12(S)-HETE synthesis and contributed to TRPV1 hyperfunction in IS-treated tubular cells. Further study on TRPV1 may attenuate kidney susceptibility to the functional loss of end-stage kidney disease via IS.

Keywords: 12(S)-hydroxyeicosatetraenoic acid; arachidonate 12-lipoxygenase; aryl hydrocarbon receptor; indoxyl sulfate; renal tubular damage; transient receptor potential vanilloid 1.

Figure 1

Indoxyl sulfate induces cytotoxicity in tubular cells. (A,C) Lilly Laboratories cell-porcine kidney 1 (LLC-PK₁) and Madin-Darby canine kidney cells (MDCK) were treated with phosphate-buffered saline (PBS) or indoxyl sulfate (IS, 2.5, 5.0, and 10.0 mM) for 24, 48, and 72 h. Lactate dehydrogenase (LDH) release was measured as a marker of cell injury; (B,D) Both cell lines were cultured for 72 h in the presence of PBS or IS, and the cell viability was monitored in an MTT assay. n = 6 of experiments performed at each time point and dose. * p < 0.05 versus the control group (C) at the same time point.

Figure 2

Transient receptor potential vanilloid type-1 (TRPV1) expression in tubular cell lines. (A) Representative immunoblots show TRPV1 expression in rat dorsal root ganglia (DRG) tissues, rat kidney (RK), LLC-PK₁ cells (LLC), and MDCK cells using 20 μg of the total protein in each lane. The lower bar graph shows densitometrically quantified results by the ratio of TRPV1 to β-actin; (B) The expression of TRPV1 mRNA in rat tissues and tubular cells was examined by real-time quantitative RT-PCR. n = 4 of experiments performed in each group. * p < 0.05 versus DRG.

Figure 3

Blockade of TRPV1 attenuates indoxyl sulfate-induced tubular cell damage. MDCK cells were treated with a selective TRPV1 blockers capsazepine (Capz, 10 μM), SB-366791 (SB, 5 μM), or indoxyl sulfate (IS, 5 mM) alone or in combination for 24, 48, and 72 h. (A,C) Lactate dehydrogenase (LDH) was determined as a marker of cell injury; (B,D) Cells were cultured for 72 h in the presence of the indicated agents, and the viability was measured in an MTT assay. n = 6 of experiments performed in each time point of the group. * p < 0.05 versus the control group (C), ^@ p < 0.05, Capz (or SB) + IS versus Capz (or SB) group, ^# p < 0.05, Capz (or SB) + IS versus IS group.

Figure 4

Effects of indoxyl sulfate on TRPV1 and arachidonate 12-lipoxygenase (ALOX12) expression. (A) Representative blots show the protein expression of TRPV1 and ALOX12 in MDCK cells with or without treatment of indoxyl sulfate (IS) using 20 μg of total protein in each lane. The right bar graphs show the ratio of TRPV1 or ALOX12 to β-actin (n = 6 per group). Note that IS treatment for 72 h did not affect TRPV1 expression but increased ALOX12 expression in a dose-dependent manner; (B) Changes in TRPV1 and ALOX12 mRNA were examined by real-time quantitative RT-PCR. Note that ALOX12 mRNA demonstrated a similar trend as its protein increased, caused by IS; (C) Changes in 12(S)-HETE levels in cells were determined by an enzyme-linked immunosorbent assay. Note that the level of 12(S)-HETE significantly increased after IS treatment in a dose-dependent manner; (D) Treatment of IS cells with selective ALOX12 inhibitors cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) or baicalein (Bai) reduced LDH release and enhanced cell viability by an MTT assay. n = 6 of experiments performed in each group. * p < 0.05 versus the control group (C), ^# p < 0.05 versus IS group.

Figure 5

Effects of uremic toxin adsorbent (AST-120) and aryl hydrocarbon receptor (AhR) inhibitor on IS-treated cells. (A) Representative blots show the protein expression of ALOX12 in MDCK cells treated with IS (5 mM), uremic toxin adsorbent AST-120 (AST, 2 g/mL), AhR inhibitor CH-223191 (CH, 10 μM), and a combination of these drugs using 20 μg of the total protein in each lane. The lower bar graphs show the ratio of ALOX12 to β-actin (n = 6 per group); (B) Changes in 12(S)-HETE levels in cells were determined by an enzyme-linked immunosorbent assay; (C) Lactate dehydrogenase (LDH) was determined as a marker of cell injury; (D) Cells were cultured for 72 h in the presence of the indicated agents, and the viability was measured in an MTT assay. n = 6 of experiments performed in each group. * p < 0.05 versus the control group (C), ^# p < 0.05 versus IS group.

Figure 6

Schematic diagram showing how indoxyl sulfate induced cytotoxicity in tubular cells. After the uptake of indoxyl sulfate (IS), IS then induced tubular cell damage via AhR activation, and this effect could be abrogated by the AhR inhibitor CH-223191. AhR upregulated ALOX12 expression and cellular 12(S)-HETE synthesis. The blockade of ALOX12 also attenuated IS-mediated tubular damage. Excess formation of 12(S)-HETE overstimulated TRPV1 to induce cell damage, and this effect could be attenuated after TRPV1 inhibition by capsazepine and SB-366791.