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Effect of colchicine treatment during the recovery phase on tubulin acetylation, cell proliferation, and cell cycle arrest, and progression of fibrosis following I/R injury. Mice were subjected to either 30 min of ischemia or sham operation (Sham). Mice were administered colchicine (Col, 100 μ g/kg BW) or 0.9% saline (Vehicle, Veh) beginning on one day following surgery, and continuing every other day until sacrifice. (A) Kidney samples were subjected to Western blot analysis using anti-ac-α-tubulin (ac-α-tub),-p21,-α-SMA or-GAPDH antibodies. (B–D) Band density was measured using the NIH Image J software. (E–G) Kidney sections were subjected to either picro-sirius red staining (red) (E), or immunohistochemical staining using anti-BrdU (brown) antibody (G). (G) Filled and blank arrowheads indicate BrdU-positive tubular and interstitial cells, respectively. Collagen-deposited area (%) (F) and BrdU-positive (BrdU + ) tubular (H) and interstitial (I) cell numbers were measured as described in the Materials & Methods. Results are expressed as mean ± SEM (n = 5). * p < 0.05 vs. respective sham-vehicle.
Source publication
The microtubule cytoskeleton is composed of α-tubulin and β-tubulin heterodimers, and it serves to regulate the shape, motility, and division of a cell. Post-translational modifications including acetylation are closely associated with the functional aspects of the microtubule, involving in a number of pathological diseases. However, the role of mi...
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Citations
... Ke et al. [21] reported that HDAC6 expression and activity were low in kidney tubule cells in ADPKD, a disease associated with defects in the primary cilia of kidney tubule cells. Furthermore, in a previous study, we found that inhibition of microtubule dynamics by taxol treatment during recovery delayed the restoration of kidney functional damage [40]. Therefore, we speculate that alteration of primary cilia length regulated by HDAC6 is associated with urine concentration. ...
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.
... In some cystic conditions, like PKD and NPH, cilia appear to lengthen (Garcia et al., 2022;Shao et al., 2020). Conversely, shortened cilia are observed with centrosome disruption, accumulation of ROS or, soon after, ischemic injury (Dionne et al., 2018;Han et al., 2016;Kim et al., 2013;Verghese et al., 2008). Detection of shed ciliary components, like acetylated α-tubulin, in urine has also been recently proposed as marker for kidney disease (Han et al., 2016;Kim et al., 2013;Park, 2018). ...
... Conversely, shortened cilia are observed with centrosome disruption, accumulation of ROS or, soon after, ischemic injury (Dionne et al., 2018;Han et al., 2016;Kim et al., 2013;Verghese et al., 2008). Detection of shed ciliary components, like acetylated α-tubulin, in urine has also been recently proposed as marker for kidney disease (Han et al., 2016;Kim et al., 2013;Park, 2018). Here, we provide new insights regarding the nexus of nephron and ciliary development, as esrrγa regulates prostaglandin signaling through cooperation with ppargc1a. ...
Cilia are essential for the ontogeny and function of many tissues, including the kidney. Here, we report that transcription factor ERRγ ortholog estrogen related receptor gamma a (Esrrγa) is essential for renal cell fate choice and ciliogenesis in zebrafish. esrrγa deficiency altered proximodistal nephron patterning, decreased the multiciliated cell populace and disrupted ciliogenesis in the nephron, Kupffer's vesicle and otic vesicle. These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued by PGE2 or the cyclooxygenase enzyme Ptgs1. Genetic interaction revealed that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (Ppargc1a), which acts upstream of Ptgs1-mediated prostaglandin synthesis, has a synergistic relationship with Esrrγa in the ciliogenic pathway. These ciliopathic phenotypes were also observed in mice lacking renal epithelial cell (REC) ERRγ, where significantly shorter cilia formed on proximal and distal tubule cells. Decreased cilia length preceded cyst formation in REC-ERRγ knockout mice, suggesting that ciliary changes occur early during pathogenesis. These data position Esrrγa as a novel link between ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with Ppargc1a.
... Внутри эпителиальных клеток находятся микротрубочки, которые определяют важное значение в поддержании цитоскелета, формы и подвижности клеток [31]. Микротрубочки состоят из гетеродимеров α-и β-тубулинов. ...
Currently, there is a high scientific interest in studying the features of the structure and functions of the tubules of the kidneys. The relevance of the topic is due to the potential possibility of identifying various markers of tubular dysfunction and using them for early diagnosis of not only tubulopathies, but also glomerular disorders. In clinical practice, markers of tubular dysfunction are used insufficiently. The article presents information about the anatomical and functional features of the proximal and distal parts of the tubular apparatus, outlines highly organized mechanisms of intermolecular interaction, presents the main biologically active substances, the change in the concentration of which is a consequence of damage to the tubules. The presented manuscript is the product of a deep analysis and systematization of the available data in Russian and foreign information and analytical portals.
... Quantification of positive staining for Masson's trichrome, α-SMA, 4-HNE or NOX-4 was analyzed using the IMT i-Solution software (IMT i-Solution, Inc., Coquitlam, BC, Canada) in 10 random cortical fields (×400) per sample according to the manufacturer's instructions. This computer-assisted automated image analyzer has been widely used to analyze positively stained areas [25][26][27]. The number of F4/80-postive cells was counted in 10 random cortical fields (×600) per sample. ...
Renal fibrosis is a common feature of chronic kidney disease and is a promising therapeutic target. However, there is still limited treatment for renal fibrosis, so the development of new anti-fibrotic agents is urgently needed. Accumulating evidence suggest that oxidative stress and endoplasmic reticulum (ER) stress play a critical role in renal fibrosis. Carnosol (CS) is a bioactive diterpene compound present in rosemary plants and has potent antioxidant and anti-inflammatory properties. In this study, we investigated the potential effects of CS on renal injury and fibrosis in a murine model of unilateral ureteral obstruction (UUO). Male C57BL/6J mice underwent sham or UUO surgery and received intraperitoneal injections of CS (50 mg/kg) daily for 8 consecutive days. CS improved renal function and ameliorated renal tubular injury and interstitial fibrosis in UUO mice. It suppressed oxidative injury by inhibiting pro-oxidant enzymes and activating antioxidant enzymes. Activation of ER stress was also attenuated by CS. In addition, CS inhibited apoptotic and necroptotic cell death in kidneys of UUO mice. Furthermore, cytokine production and immune cell infiltration were alleviated by CS. Taken together, these findings indicate that CS can attenuate renal injury and fibrosis in the UUO model.
... Colchicine not only failed to attenuate the epithelial-mesenchymal transition (EMT) in the UUO model, but also hindered the repair of tubular epithelial cells injured by ischemiareperfusion injury (IRI). 5,6,8 Thus, we speculate that the antifibrotic effect of colchicine is primarily attributed to its effect on fibroblasts. Since colchicine works by disruption of microtubule polymerization, the biological action of colchicine is closely related to the inhibition of cellular microtubule dynamics. ...
The transition of fibroblasts into myofibroblasts is a crucial step in kidney fibrosis. However, the biological processes involved in this transdifferentiation are incompletely understood. In this study, we discovered that the midbody plays a role in the fibroblast‐myofibroblast transition by mediating TGF‐β/Smad signaling. Combining bulk RNA‐seq, histology, and the western blot of unilateral ureteral obstruction kidneys, we demonstrated that the pathway related to microtubules is implicated in kidney fibrosis, and the blocking of microtubule dynamics by colchicine improved kidney fibrosis. Subsequently, to explore microtubule‐based organelles in detail, we cultured NRK‐49F (rat kidney fibroblast cell line) and HKC‐8 (human proximal tubule cell line) under transforming growth factor‐β1 (TGF‐β1) stimulation, which caused deciliation in both cell lines during epithelial‐mesenchymal and fibroblast‐myofibroblast transition. We identified another microtubule‐based organelle, the midbody, whose formation is promoted by TGF‐β1 in fibroblasts as a result of proliferation in contrast to tubular cells. Notably, TGF‐β receptors were present in the midbody of both cell lines. In TGF‐β1‐treated fibroblasts, colchicine or Hedgehog pathway inhibitor 4 impaired the midbody formation, and attenuated the upregulation of canonical TGF‐β/Smad signaling and α‐SMA expression. These findings offer novel insight into the midbody as an active organelle involved in fibroblast‐myofibroblast transition by mediating TGF‐β/Smad signaling, which could be a potential therapeutic target.
... In clinical practice, IR may cause renal interstitial fibrosis in the later stages and consequently, may affect the prognosis of AKI. A similar situation was discovered in animal models (24). In the present study, IR resulted in significant fibrosis of the renal interstitium in mice two weeks after IR, which was assessed using the ratio of Masson staining in high resolution fields (Fig. 6). ...
Oxidative stress, caused by renal ischemia reperfusion (IR)/hypoperfusion, is one of the main causes of acute kidney injury (AKI). Previous studies have demonstrated that sevoflurane (SEV) protects organs from the damage caused by oxidative stress. In the present study, mice were randomly assigned to a sham operation group (Sham), IR-vehicle group (IR+ vehicle), IR + SEV low-dose preconditioning group and an IR + SEV high-dose preconditioning group. The effect of SEV on nuclear factor E2-related factor 2 (Nrf2), a key regulatory protein of the endogenous antioxidant defense system and, consequently oxidative stress, inflammation and apoptosis-related factors, were all quantified using commercial kits or by western blotting. SEV preconditioning was demonstrated to ameliorate kidney injury as a result of decreased blood urine nitrogen and serum creatinine levels, activated Nrf2 expression in the kidney and decreased oxidative stress and inflammatory index levels an AKI mouse model. SEV preconditioning also protected injured kidney via the downregulation of caspase-3 protein expression levels. In addition, using the Nrf2 inhibitor, Brusatol, significantly abolished the SEV preconditioning renal protective effect. Using an in vitro HK-2 cell model of hypoxia/reoxygenation, it was also demonstrated that Nrf2 pathway activation was necessary for SEV to exert its beneficial effect for tubular cell injury caused by hypoxia/reoxygenation. These results indicated that SEV may protect against renal injury caused by IR via Nrf2 upregulation.
... Primary cilia play an essential role in sensing environmental cues (e.g. mechanotransduction), planar cell polarity of epithelial cells 43 , lumen formation, and EMT/fibrosis after acute kidney injury 44,45,46 , in which many factors (such as, chemical or physical cues) can modulate the ciliary length and frequency. Therefore, we stained for primary cilia (acetylated α-tubulin (aTUB) and ADP Ribosylation Factor Like GTPase 13B (ARL13B)) to investigate whether ciliary frequency and length vary in the different hydrogels. ...
Pluripotent stem cell-derived kidney organoids offer a promising solution to renal failure, yet current organoid protocols often lead to off-target cells and phenotypic alterations, preventing maturity. Here, we created various dynamic hydrogel architectures, conferring a controlled and biomimetic environment for organoid encapsulation. We investigated how hydrogel stiffness and stress relaxation affect renal phenotype and undesired fibrotic markers. We observed stiff hydrogel encapsulation led to an absence of certain renal cell types and signs of an epithelial-mesenchymal transition (EMT), whereas encapsulation in soft-stress-relaxing hydrogels led to all major renal segments, fewer fibrosis/EMT associated proteins, apical proximal tubule enrichment, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids. Our findings show that engineering hydrogel mechanics and dynamics have a decided benefit for organoid culture. These structure-property-function relationships can enable rational design of materials, bringing us closer to functional engraftments and disease-modelling applications.
... 7,8,13,28,29 This laboratory was able to sort the HRTPT cells isolated from a renal following ischaemia/reperfusion. 31 The only gene grouping that did not appear to be linked to microtubules and cytoskeleton was the neuroblastoma breakpoint genes that have not been highly studied, but do have a role in kidney development. 35 Overall, the analysis of global gene expression allowed the definition of genes and pathways associated with a CD133 and CD24 co-expressing cell line, known to have features associated with renal progenitor cells. ...
Damage to proximal tubules due to exposure to toxicants can lead to conditions such as acute kidney injury (AKI), chronic kidney disease (CKD) and ultimately end‐stage renal failure (ESRF). Studies have shown that kidney proximal epithelial cells can regenerate particularly after acute injury. In the previous study, we utilized an immortalized in vitro model of human renal proximal tubule epithelial cells, RPTEC/TERT1, to isolate HRTPT cell line that co‐expresses stem cell markers CD133 and CD24, and HREC24T cell line that expresses only CD24. HRTPT cells showed most of the key characteristics of stem/progenitor cells; however, HREC24T cells did not show any of these characteristics. The goal of this study was to further characterize and understand the global gene expression differences, upregulated pathways and gene interaction using scRNA‐seq in HRTPT cells. Affymetrix microarray analysis identified common gene sets and pathways specific to HRTPT and HREC24T cells analysed using DAVID, Reactome and Ingenuity software. Gene sets of HRTPT cells, in comparison with publicly available data set for CD133+ infant kidney, urine‐derived renal progenitor cells and human kidney‐derived epithelial proximal tubule cells showed substantial similarity in organization and interactions of the apical membrane. Single‐cell analysis of HRTPT cells identified unique gene clusters associated with CD133 and the 92 common gene sets from three data sets. In conclusion, the gene expression analysis identified a unique gene set for HRTPT cells and narrowed the co‐expressed gene set compared with other human renal–derived cell lines expressing CD133, which may provide deeper understanding in their role as progenitor/stem cells that participate in renal repair.
... Ischaemia has been associated with disruption of the cytoskeleton, leading to loss of tissue integrity [48][49]. However, the effects of brain death specifically on renal tissue integrity are still unknown. ...
Assessment of donor kidney quality is based on clinical scores or requires biopsies for histological assessment. Noninvasive strategies to identify and predict graft outcome at an early stage are, therefore, needed. We evaluated the perfusate of donation after brain death (DBD) kidneys during nonoxygenated hypothermic machine perfusion (HMP). In particular, we compared perfusate protein profiles of good outcome (GO) and suboptimal outcome (SO) 1‐year post‐transplantation. Samples taken 15 min after the start HMP (T1) and before the termination of HMP (T2) were analysed using quantitative liquid chromatography–tandem mass spectrometry (LC‐MS/MS). Hierarchical clustering of the 100 most abundant proteins showed discrimination between grafts with a GO and SO at T1. Elevated levels of proteins involved in classical complement cascades at both T1 and T2 and a reduced abundance of lipid metabolism at T1 and of cytoskeletal proteins at T2 in GO versus SO was observed. ATP‐citrate synthase and fatty acid‐binding protein 5 (T1) and immunoglobulin heavy variable 2‐26 and desmoplakin (T2) showed 91% and 86% predictive values, respectively, for transplant outcome. Taken together, DBD kidney HMP perfusate profiles can distinguish between outcome 1‐year post‐transplantation. Furthermore, it provides insights into mechanisms that could play a role in post‐transplant outcomes. Experimental design, workflow and results. Elevated complement, cytoskeleton and lipid metabolism proteins were identified.
... Water restriction deacetylates a-tubulin along with the increase of HDAC6 activity in the kidney. The disassembly of the microtubule is associated with the shortening of the primary cilium; this microtubule disassembly is regulated by the deacetylation of α-tubulins [15,16]. Therefore, we determined the levels of ac-α-tubulin. ...
Background: The primary cilium, a microtubule-based cellular organelle, acts as a mechano-sensor for monitoring the fluid flow in cells. In kidneys, the primary cilia protrude into the tubular lumen from the tubular cells and therefore, directly contact pro-urine flow and components. However, it remains to be defined how the cilia are associated with kidney function and diseases. Here, we investigated whether water access restriction affects the cilia length in the renal tubular cells, whether cilia length changes are associated with kidney functions, and how cilia lengths are regulated.
Methods: C57BL/6 mice were provided free access to water (control), but water supply was stopped for some mice for 24 to 48 h (water restriction). Among each group, some mice were administered with tubastatin A (10 mg/kg BW), a specific inhibitor of histone deacetylase 6 (HDAC6), daily from 2 days before water restriction. Cultured tubular epithelial cells were treated with either 10 or 20 mM NaCl or 20 mM mannitol with or without tubastatin A. Primary cilia were determined by immunofluorescence staining using acetylated-α-tubulin antibody or scanning electron microscope.
Results: Water restriction shortened the primary cilia of kidney tubular epithelial cells along with increasing urine osmolality. Water restriction increased the activity of HDAC6 with increased the deacetylation of α-tubulin, a substrate of HDAC6 and a major comprising protein of microtubule of primary cilia. HDAC6 inhibitor blocked water restriction-induced primary cilia shortening along with the inhibition of α-tubulin deacetylation. In addition, HDAC6 inhibitor blocked the increase in water restriction-induced urine osmolality. Increases of NaCl or mannitol concentration in the medium for Madin-Darby canine kidney tubule cell culture shortened the cilia length and increased HDAC6 activity and α-tubulin deacetylation. HDAC6 inhibitor blocked those NaCl and mannitol-induced effects.
Conclusions: Our data have demonstrated that water restriction shortened the primary cilia of kidney tubular cells via HDAC6 activation and α-tubulin deacetylation along with increasing urine osmolality, suggesting that the alteration of primary cilia length is an adaptive response to the water intake to maintain body water balance and that the primary cilia length regulation may be a therapeutic strategy of kidney diseases related to the body water and electrolyte imbalances.
