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

Sang Jun Han,Jee In Kim,Joshua H. Lipschutz,Kwon Moo Park 대한생리학회-대한약리학회 2021 The Korean Journal of Physiology & Pharmacology Vol.25 No.6

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 H2S 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

The role of the exocyst in renal ciliogenesis, cystogenesis, tubulogenesis, and development

Joshua H. Lipschutz 대한신장학회 2019 Kidney Research and Clinical Practice Vol.38 No.3

The exocyst is a highly conserved eight-subunit protein complex (EXOC1-8) involved in the targeting and docking of exocytic vesicles translocating from the trans-Golgi network to various sites in renal cells. EXOC5 is a central exocyst component because it connects EXOC6, bound to the vesicles exiting the trans-Golgi network via the small GTPase RAB8, to the rest of the exocyst complex at the plasma membrane. In the kidney, the exocyst complex is involved in primary ciliognesis, cystogenesis, and tubulogenesis. The exocyst, and its regulators, have also been found in urinary extracellular vesicles, and may be centrally involved in urocrine signaling and repair following acute kidney injury. The exocyst is centrally involved in the development of other organs, including the eye, ear, and heart. The exocyst is regulated by many different small GTPases of the RHO, RAL, RAB, and ARF families. The small GTPases, and their guanine nucleotide exchange factors and GTPase-activating proteins, likely give the exocyst specificity of function. The recent development of a floxed Exoc5 mouse line will aid researchers in studying the role of the exocyst in multiple cells and organ types by allowing for tissue-specific knockout, in conjunction with Cre-driver mouse lines.

Shortening of primary cilia length is associated with urine concentration in the kidneys

( Min Jung Kong ),( Sang Jun Han ),( Sung Young Seu ),( Ki-hwan Han ),( Joshua H. Lipschutz ),( Kwon Moo Park ) 대한신장학회 2023 Kidney Research and Clinical Practice Vol.42 No.3

Background: The primary cilium, a microtubule-based cellular organelle present in certain kidney cells, functions as a mechano-sensor to monitor fluid flow in addition to various other biological functions. In kidneys, the primary cilia protrude into the tubular lumen and are directly exposed to pro-urine flow and components. However, their effects on urine concentration remain to be defined. Here, we investigated the association between primary cilia and urine concentration. Methods: Mice either had free access to water (normal water intake, NWI) or were not allowed access to water (water deprivation, WD). Some mice received tubastatin, an inhibitor of histone deacetylase 6 (HDAC6), which regulates the acetylation of α-tubulin, a core protein of microtubules. Results: WD decreased urine output and increased urine osmolality, concomitant with apical plasma membrane localization of aquaporin 2 (AQP2) in the kidney. After WD, compared with after NWI, the lengths of primary cilia in renal tubular epithelial cells were shortened and HDAC6 activity increased. WD induced deacetylation of α-tubulin without altering α-tubulin levels in the kidney. Tubastatin prevented the shortening of cilia through increasing HDAC6 activity and consequently increasing acetylated α-tubulin expression. Furthermore, tubastatin prevented the WD-induced reduction of urine output, urine osmolality increase, and apical plasma membrane localization of AQP2. Conclusions: WD shortens primary cilia length through HDAC6 activation and α-tubulin deacetylation, while HDAC6 inhibition blocks the WD-induced changes in cilia length and urine output. This suggests that cilia length alterations are involved, at least in part, in the regulation of body water balance and urine concentration.

Downregulation of exocyst Sec10 accelerates kidney tubule cell recovery through enhanced cell migration

Noh, Mi Ra,Jang, Hee-Seong,Song, Dae-Kyu,Lee, Seong-Ryong,Lipschutz, Joshua H.,Park, Kwon Moo,Kim, Jee In Elsevier 2018 Biochemical and biophysical research communication Vol. No.

<P><B>Abstract</B></P> <P>Migration of surviving kidney tubule cells after sub-lethal injury, for example ischemia/reperfusion (I/R), plays a critical role in recovery. Exocytosis is known to be involved in cell migration, and a key component in exocytosis is the highly-conserved eight-protein exocyst complex. We investigated the expression of a central exocyst complex member, Sec10, in kidneys following I/R injury, as well as the role of Sec10 in wound healing following scratch injury of cultured Madin-Darby canine kidney (MDCK) cells. Sec10 overexpression and knockdown (KD) in MDCK cells were used to investigate the speed of wound healing and the mechanisms underlying recovery. In mice, Sec10 decreased after I/R injury, and increased during the recovery period. In cell culture, Sec10 OE inhibited ruffle formation and wound healing, while Sec10 KD accelerated it. Sec10 OE cells had higher amounts of diacylglycerol kinase (DGK) gamma at the leading edge than did control cells. A DGK inhibitor reversed the inhibition of wound healing and ruffle formation in Sec10 OE cells. Conclusively, downregulation of Sec10 following I/R injury appears to accelerate recovery of kidney tubule cells through activated ruffle formation and enhanced cell migration.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Exocyst Sec10 decreases by I/R injury and restores with functional recovery. </LI> <LI> Sec10 inversely regulates kidney tubule cell wound healing after scratch injury. </LI> <LI> Sec10 inversely regulates ruffle formation at the leading edge during recovery. </LI> <LI> Upregulation of Sec10 increases DGKγ expression at the leading edge during recovery. </LI> <LI> DGK inhibitor reversed Sec10-mediated ruffle formation and wound healing defect. </LI> </UL> </P>

Novel MAPK-dependent and -independent tubulogenes identified via microarray analysis of 3D-cultured Madin-Darby canine kidney cells

Chacon-Heszele, Maria F.,Zuo, Xiaofeng,Hellman, Nathan E.,McKenna, Sarah,Choi, Soo Young,Huang, Liwei,Tobias, John W.,Park, Kwon Moo,Lipschutz, Joshua H. American Physiological Society 2014 American Journal of Physiology Vol.306 No.9

<P>Cystogenesis and tubulogenesis are basic building blocks for many epithelial organs, including the kidney. Most researchers have used two-dimensional (2D) cell culture to investigate signaling pathways downstream of hepatocyte growth factor (HGF). We hypothesize that three-dimensional (3D) collagen-grown Madin-Darby canine kidney (MDCK) cells, which form cysts and then tubulate in response to HGF, are a much more in vivo-like system for the identification of novel tubulogenes. With the use of a canine microarray containing over 20,000 genes, 2,417 genes were identified as potential tubulogenes that were differentially regulated, exclusively in 3D-grown MDCK cells. Among these, 840 were dependent on MAPK signaling. Importantly, this work shows that many putative tubulogenes, previously identified via microarray analysis of 2D cultures, including by us, do not change in 3D culture and vice versa. The use of a 3D-culture system allowed for the identification of novel MAPK-dependent and -independent genes that regulate early renal tubulogenesis in vitro, e.g., matrix metalloproteinase 1 (MMP1). Knockdown of MMP1 led to defects in cystogenesis and tubulogenesis in 3D-grown MDCK cells, most likely due to problems establishing normal polarity. We suggest that data obtained from 2D cultures, even those using MDCK cells treated with HGF, should not be automatically extrapolated to factors important for cystogenesis and tubulogenesis. Instead, 3D culture, which more closely replicates the biological environment and is therefore a more accurate model for identifying tubulogenes, is preferred. Results from the present analysis will be used to build a more accurate model of the signaling pathways that control cystogenesis and tubulogenesis.</P>

Exocyst Complex Member EXOC5 Is Required for Survival of Hair Cells and Spiral Ganglion Neurons and Maintenance of Hearing

Lee, Byeonghyeon,Baek, Jeong-In,Min, Hyehyun,Bae, Seung-Hyun,Moon, Kyeonghye,Kim, Min-A,Kim, Ye-Ri,Fogelgren, Ben,Lipschutz, Joshua H.,Lee, Kyu-Yup,Bok, Jinwoong,Kim, Un-Kyung Springer-Verlag 2018 Molecular neurobiology Vol.55 No.8