Korean J Physiol Pharmacol.  2021 Nov;25(6):593-601. 10.4196/kjpp.2021.25.6.593.

Hydrogen sulfide, a gaseous signaling molecule, elongates primary cilia on kidney tubular epithelial cells by activating extracellular signal-regulated kinase

• Han SJ
• Kim JI
• Lipschutz JH
• Park KM

• KMID: 2521485
• DOI: http://doi.org/10.4196/kjpp.2021.25.6.593

Abstract

Primary cilia on kidney tubular cells play crucial roles in maintaining structure and physiological function. Emerging evidence indicates that the absence of primary cilia, and their length, are associated with kidney diseases. The length of primary cilia in kidney tubular epithelial cells depends, at least in part, on oxidative stress and extracellular signal-regulated kinase 1/2 (ERK) activation. Hydrogen sulfide (H₂S) is involved in antioxidant systems and the ERK signaling pathway. Therefore, in this study, we investigated the role of H₂S in primary cilia elongation and the downstream pathway. In cultured Madin-Darby Canine Kidney cells, the length of primary cilia gradually increased up to 4 days after the cells were grown to confluent monolayers. In addition, the expression of H₂S-producing enzyme increased concomitantly with primary cilia length. Treatment with NaHS, an exogenous H₂S donor, accelerated the elongation of primary cilia whereas DL-propargylglycine (a cystathionine γ-lyase inhibitor) and hydroxylamine (a cystathionine-β-synthase inhibitor) delayed their elongation. NaHS treatment increased ERK activation and Sec10 and Arl13b protein expression, both of which are involved in cilia formation and elongation. Treatment with U0126, an ERK inhibitor, delayed elongation of primary cilia and blocked the effect of NaHS-mediated primary cilia elongation and Sec10 and Arl13b upregulation. Finally, we also found that H ₂ S accelerated primary cilia elongation after ischemic kidney injury. These results indicate that H₂S lengthens primary cilia through ERK activation and a consequent increase in Sec10 and Arl13b expression, suggesting that H₂S and its downstream targets could be novel molecular targets for regulating primary cilia.

Figure

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  Fig. 1 Relationship between expression of H2S-producing enzyme and primary cilium length in MDCK cells. MDCK cells grown on the coverslip were fixed and processed for immunofluorescence staining using anti-acetylated-α-tubulin antibody (green) at the indicated day following growth of the cells to confluent monolayers. DAPI (blue) indicates nuclear staining. (A) Representative images and length of primary cilia are shown at the indicated times. (B) The percentage of primary cilia in each length range are shown at the indicated times. (C) Representative immunoblot image of CBS. GAPDH was used as loading control. The results are expressed as the mean ± SEM (n = 3–4). H2S, hydrogen sulfide; MDCK, Madin-Darby Canine Kidney; CBS, cystathionine β-synthase. *p < 0.05 vs. 0 day.

• 

  Fig. 2 Hydrogen sulfide elongates primary cilia in cultured MDCK cells. MDCK cells grown on coverslips were treated with vehicle, 10 μM NaHS, 0.5 mM PAG, 0.1 mM HA or combination of PAG and HA daily after cells were grown to confluency. After 4 days, cells were fixed and processed for immunofluorescence staining using anti-acetylated-tubulin antibody (green). DAPI (blue) indicates nuclear staining. (A) Representative images of primary cilia and the length of primary cilia are shown. (B) The percentage of primary cilia in each length range is shown. The results are expressed as the mean ± SEM (n = 4). MDCK, Madin-Darby Canine Kidney; HA, hydroxylamine; PAG, propargylglycine; ns, non-significant. *p < 0.05 vs. vehicle. **p < 0.01 vs. vehicle.

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  Fig. 3 Hydrogen sulfide increases expressions of phospho-ERK and Sec10. MDCK cells were treated with vehicle or 1, 10, and 50 μM of NaHS after cells reached confluence. (A) Ten minutes after treatment, cell lysates were subjected to Western blot analysis using antibodies against Sec10, Arl13b, phosphorylated ERK (p-ERK), and total-ERK (t-ERK). GAPDH was used as loading control. (B–D) Band densities were measured using ImageJ software. (E, F) After 4 days, cells were fixed and processed for immunofluorescence staining using anti-acetylated-tubulin antibody (green). DAPI (blue) indicates nuclear staining. The results are expressed as the mean ± SEM (n = 4). ERK, extracellular signal-regulated kinase. *p < 0.05 vs. vehicle.

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  Fig. 4 Inhibition of ERK activation prevents hydrogen sulfide-induced elongation of primary cilia. MDCK cells were treated daily with either vehicle or 10 μM NaHS 30 min after treatment with either vehicle (DMSO) or 10 μM U0126. Cell lysates were subjected to Western blot analysis, which was performed for phospho-ERK (p-ERK, A), total-ERK (t-ERK, A), Arl13b and Sec10 (B) 10 min after treatment. (C–E) Band densities were measured using ImageJ software. (F) Four days after treatment, cells were fixed and processed for immunofluorescence staining using anti-acetylated-α-tubulin antibody (green). DAPI (blue) indicates nuclear staining. The length of primary cilia (G) and the percentage of primary cilia in each length range (H) were measured. The results are expressed as the mean ± SEM (n = 4). ERK, extracellular signal-regulated kinase; MDCK, Madin-Darby Canine Kidney; ns, non-significant. *p < 0.05 vs. vehicle.

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  Fig. 5 Effect of H2S on the primary cilia elongation and recovery of kidney after ischemia/reperfusion injury. Mice were subjected to either 30 min of bilateral renal ischemia or sham surgery. Mice were treated daily with NaHS (500 μg/kg BW), PAG (50 mg/kg BW) or vehicle (0.9% saline) beginning 2 days after surgery. (A) Eight days after ischemia, kidney sections were subjected to immunofluorescent staining using anti-acetylated α-tubulin (green) and aquaporin 1 (AQP-1, red) antibodies. Pictures were obtained from the outer medulla. Average length of primary cilia (B), frequency of primary cilia length (C), and the ratio of primary cilia to proximal tubular cell nuclei (D) were measured as described in the Methods. Results were expressed as the means ± SEM (n = 6). H2S, hydrogen sulfide; BW, body weight; PAG, propargylglycine. *p < 0.05 vs. their respective vehicle-treated sham on the same day. †p < 0.05 vs. vehicle-treated ischemia on the same day.

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  Fig. 6 Schematic of the proposed mechanisms for H2S-mediated primary cilia elongation. The biological production of H2S in the kidney is mediated by the transsulfuration pathway. CSE and CBS generate H2S by using L-cysteine and homocysteine as substrates. PAG and CBS are inhibitor of CSE and CBS respectively, and NaHS is the exogenous H2S donor. H2S elongates primary cilia by activating ERK with concomitant upregulation of Sec10 and Arl13b. H2S, hydrogen sulfide; CBS cystathionine β-synthase; CSE, cystathionine γ-lyase; HA, hydroxylamine; PAG, propargylglycine.

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