doi:10.1093/jmcb/mjaa005 Journal of Molecular Cell Biology (2020), 12(7), 499–514 | 499 Published online March 12, 2020 Article Cyclophilins A and B oppositely regulate renal tubular epithelial cell phenotype Eduard Sarró 1, *, Mónica Durán1 , Ana Rico1 , Diana Bou-Teen2 , Vanesa Fernández-Majada3 , Anthony J. Croatt4 , Karl A. Nath4 , Maria Teresa Salcedo5 , Justin H. Gundelach6,7 , Daniel Batlle8 , Richard J. Bram6,9 , and Anna Meseguer1,10,11, * 1 Renal Physiopathology Group, CIBBIM-Nanomedicine, Vall d’Hebron Research Institute, 08035 Barcelona, Spain 2 Cardiovascular Diseases Group, Vall d’Hebron Research Institute, 08035 Barcelona, Spain 3 Biomimetic Systems for Cell Engineering Laboratory, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain 4 Division of Nephrology and Hypertension and Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA 5 Department of Pathology, Hospital Universitari Vall d’Hebron, 08035 Barcelona, Spain 6 Department of Pediatric and Adolescent Medicine, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA 7 Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA 8 Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA 9 Department of Immunology, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA 10 Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 11 Red de Investigación Renal (REDINREN), Instituto de Salud Carlos III-FEDER, 28040 Madrid, Spain * Correspondence to: Eduard Sarró, E-mail: eduard.sarro@vhir.org; Anna Meseguer, E-mail: ana.meseguer@vhir.org Edited by Xuebiao Yao Restoration of kidney tubular epithelium following sublethal injury sequentially involves partial epithelial–mesenchymal transition (pEMT), proliferation, and further redifferentiation into specialized tubule epithelial cells (TECs). Because the immunosuppressant cyclosporine-A produces pEMT in TECs and inhibits the peptidyl-prolyl isomerase (PPIase) activity of cyclophilin (Cyp) proteins, we hypothesized that cyclophilins could regulate TEC phenotype. Here we demonstrate that in cultured TECs, CypA silencing triggers loss of epithelial features and enhances transforming growth factor β (TGFβ)-induced EMT in association with upregulation of epithelial repressors Slug and Snail. This pro-epithelial action of CypA relies on its PPIase activity. By contrast, CypB emerges as an epithelial repressor, because CypB silencing promotes epithelial differentiation, prevents TGFβ-induced EMT, and induces tubular structures in 3D cultures. In addition, in the kidneys of CypB knockout mice subjected to unilateral ureteral obstruction, inflammatory and pro-fibrotic events were attenuated. CypB silencing/knockout leads to Slug, but not Snail, downregulation. CypB support of Slug expression depends on its endoplasmic reticulum location, where it interacts with calreticulin, a calcium- buffering chaperone related to Slug expression. As CypB silencing reduces ionomycin-induced calcium release and Slug upregu- lation, we suggest that Slug expression may rely on CypB modulation of calreticulin-dependent calcium signaling. In conclusion, this work uncovers new roles for CypA and CypB in modulating TEC plasticity and identifies CypB as a druggable target potentially relevant in promoting kidney repair. Keywords: cyclophilins, epithelial phenotype, Slug, TGFβ, UUO, fibrosis Introduction redifferentiation into functionally specialized TECs (Chang– Restoration of the renal tubular epithelium following sublethal Panesso and Humphreys, 2017). Failure of these processes kidney injury relies on the complex interplay of dedifferentiation can lead to pathological conditions such as fibrosis or carcino- of tubule epithelial cells (TECs), proliferation, and further genesis. Within the kidney, injured TECs play a major role in fibrosis by releasing inflammatory cytokines and pro-fibrotic Received January 28, 2019. Revised January 29, 2020. Accepted March 6, 2020. factors, in particular transforming growth factor β (TGFβ) (Liu © The Author(s) (2020). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. et al., 2018). TGFβ drives transient loss of epithelial features This is an Open Access article distributed under the terms of the Creative Com- and acquisition of mesenchymal features in TECs, the so- mons Attribution Non-Commercial License (http://creativecommons.org/licenses/ called partial epithelial–mesenchymal transition (pEMT). EMT- by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, activating transcription factors Snail, Slug, zinc finger E-box- please contact journals.permissions@oup.com binding homeobox 1 and 2 (ZEB1/2), and Twist1/2 negatively 500 | Sarró et al. regulate epithelial markers, including E-cadherin expression at Day 5 (Figure 1C). At this time point, CypB silencing upreg- (Moreno-Bueno et al., 2006), which is a major hallmark of ulated E-cadherin and occludin greatly and ZO-1 and keratin EMT. Unraveling the mechanisms modulating these epithelial to a less extent at both protein (Figure 1D) and mRNA levels repressors could provide therapeutic tools to mitigate kidney (Figure 1E). CypB silencing also intensified plasma membrane fibrosis after renal damage. (PM) E-cadherin staining (Figure 1F), suggesting a gain in Cyclosporine-A (CsA) is a potent immunosuppressant whose functionality, and increased proliferation indices (Figure 1G). clinical use is limited by its nephrotoxicity, including tubular On the other hand, CypA silencing reduced occludin, ZO-1, and atrophy and tubule–interstitial fibrosis (Bennett et al., 1996). keratin levels (Figure 1C). This downregulation correlated with In vitro, CsA induces loss of epithelial phenotype of TECs by reduced transepithelial electric resistance (TEER) (Figure 1H) and inducing Snail expression, TGFβ release, and pro-inflammatory increased fluorescein isothiocyanate (FITC)-dextran permeability cytokine production (Slattery et al., 2005; González-Guerrero (Figure 1I). et al., 2017). CsA binds to cyclophilins, a family of ubiquitous We next explored whether these phenotypic changes corre- highly conserved peptidyl-prolyl cis-trans isomerases (PPIase), lated with altered levels of epithelial repressors. Snail mRNA inhibiting their enzymatic activity. cyclophilins play prominent levels were slightly upregulated in both CypA and CypB-silenced roles as chaperones by mediating protein trafficking, protein– cells, whereas the expression of Slug was strongly increased in protein interactions, and assembly of macromolecular com- CypA-silenced cells but almost undetectable after CypB silencing plexes (Wang and Heitman, 2005). Cyclophilin A (CypA) and (Figure 1J). By contrast, Twist1 and Zeb1 levels were unaffected in cyclophilin B (CypB) are the best studied members of the either case (Figure 1J). At the protein level, Slug and Snail levels family. CypA is cytosolic, whereas CypB has an N-terminal progressively decreased from Day 2 to Day 10 (Figure 1K), corre- signal peptide targeting it to the endoplasmic reticulum (ER) lating with the increase in epithelial markers. At Day 5 postseed- (Wang and Heitman, 2005). CypB also contains a C-terminal ing, Slug protein levels were increased in CypA-silenced cells ER-retention sequence (VEKPFAIAKE) directing CypB to ER sub- compared to control and were almost undetectable after CypB compartments related to calcium signaling, where it colocalizes silencing (Figure 1L). Snail protein levels were increased further with the calcium-buffering chaperone calreticulin (CRT) (Arber than their mRNAs in both CypA and CypB knockdown conditions, et al., 1992). In addition, CypB interacts with the calcium suggesting additional post-transcriptional regulation (Figure 1L). signal-modulating cyclophilin ligand (CAML), potentially reg- The effects of cyclophilin silencing on E-cadherin, occludin, ulating intracellular calcium release (Bram and Crabtree, Slug, and Snail were also observed in the human telomerase 1994). CypB is secreted in the presence of CsA (Price et al., reverse transcriptase (hTERT) immortalized human renal proxi- 1994), and both CypA and CypB are released in response to mal tubular epithelial cell line (RPTEC/TERT1) (Supplementary inflammatory stimuli acting as pro-inflammatory mediators Figure S1A and B). In RPTEC/TERT1, E-cadherin upregulation was via their potent chemotactic effects on various immune cells less dramatic at the protein level, likely due to its higher basal (Bukrinsky, 2015). In the kidney, CypB interacts with kid- expression compared to HK-2 cells (Supplementary Figure S1C). ney androgen-regulated protein (KAP), a protein exclusively expressed in proximal TECs that protects against CsA toxicity CypA and CypB oppositely modulate TGFβ-induced phenotypic (Tornavaca et al., 2011). changes Considering the loss of epithelial phenotype triggered by CsA Considering cyclophilin modulation of TEC phenotype, we and its inhibitory action on cyclophilins, we hypothesized that next aimed to investigate the effects of cyclophilin silencing on these proteins could play a key role in the maintenance of the TGFβ-induced EMT. TGFβ treatment induced a time-dependent epithelial phenotype and that, consequently, their inhibition decrease in E-cadherin and occludin as well as an increase in would trigger the development of a pEMT and, eventually, kidney fibronectin, without affecting CypA or CypB levels (Figure 2A). fibrosis. These molecular changes were consistent with a morphological switch from a cobblestone-like monolayer to formation of cell aggregates containing a combination of poorly interconnected Results rounded cells and spindle-shaped cells with filopodia (Figure 2C, CypA and CypB differentially affect epithelial phenotype of TECs shCon), and to an increase in cell-to-substrate adhesiveness To investigate the involvement of cyclophilins in the regulation (shCon data of Figure 2D). CypB silencing partially prevented of the epithelial phenotype, we silenced CypA and CypB in TGFβ-induced EMT by maintaining E-cadherin and occludin human kidney-2 (HK-2) cells (Figure 1A) by short hairpin RNA levels closer to control and by preventing fibronectin expression (shRNA) and analyzed epithelial markers. Control cells reached (Figure 2B), TGFβ-induced morphological changes (Figure 2C), confluency at Day 2 postseeding, progressively increasing the and the increase in cell-to-substrate adhesiveness (Figure 2D). levels of E-cadherin, zonula occludens-1 (ZO-1), occludin, and By contrast, CypA silencing enhanced TGFβ-induced EMT by keratin (Figure 1B), and the activity of proximal tubule brush further decreasing E-cadherin and occludin and increasing border alkaline phosphatase (AP) (Figure 1C) within days in cul- fibronectin levels. Similar results were observed in RPTEC cells ture. AP activity was markedly enhanced in CypB-silenced cells (Supplementary Figure S2). and reduced in cyclophilin-silenced cells, with the greatest differ- Epithelial cells either migrate collectively as multicellular ences between control and cyclophilin-silenced cells observed sheets maintaining their intercellular junctions, or undergo CypA and CypB regulate epithelial phenotype | 501 Figure 1 CypA and CypB differentially affect epithelial phenotype of TECs (HK-2 cells). (A) Western blot analysis (WB) showing CypA and CypB expression after cyclophilin silencing. (B) Protein levels of E-cadherin, ZO-1, occludin, and keratin in control cells cultured for different days. (C) AP activity after 2, 5, and 10 days of culture. (D and E) WB (D) and quantitative polymerase chain reaction (qPCR; E) showing expression levels of the indicated epithelial markers in CypA- and CypB-silenced cells after Day 5 of culture. (F) Immunofluorescence staining of E- cadherin (green) in cells cultured for 5 days. Calnexin (red) is used to stain ER. Scale bar, 25 µm. (G) Cell proliferation as measured by CFSE labeling followed by flow cytometry. Values indicate the ratio FInd/MFI, where MFI is the median fluorescence intensity of all viable cells at collection and FInd is the peak fluorescence intensity of the viable non-divided cells. (H and I) To assess monolayer integrity, TEER (H) and FITC-labeled dextran permeability (I) were measured in cells cultured for 5 days. (J) The mRNA levels of the indicated transcriptional repressors were analyzed by qPCR. (K and L) Protein levels of Slug and Snail in control cells after 2, 5, and 10 days of culture (K) or in cyclophilin-silenced cells at Day 5 of culture (L). To simplify the graphs in C, only the statistically significant differences within each group are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. 502 | Sarró et al. Figure 2 CypA and CypB oppositely modulate TGFβ-induced phenotypic changes. (A) HK-2 cells were treated with 1.5 ng/ml TGFβ for different times and the expression levels of the indicated proteins were analyzed by WB. (B–D) CypA- and CypB-silenced HK-2 cells were treated with TGFβ for 24 h and analyzed as above (B), or treated for 48 h and visualized under a light microscope (C; scale bar, 100 µm) or assayed for cell adhesion (D). (E) Wound healing assay of CypA- and CypB-silenced HK-2 cells treated or not with TGFβ. The rate of gap closure was measured. Scale bar, 250 µm. (F) HK-2 cells were seeded on MatrigelTM -coated transwells and treated with TGFβ for 24 h. The number of cells that migrated through the transwell was monitored. Scale bar, 100 µm. Values are expressed as fold or percentage of controls. Only statistically significant differences within control or TGFβ-treated groups are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. a pEMT and migrate as individual mesenchymal-like cells, stimulated control cells migrated at rates similar to untreated although these features combine in variable degrees (Campbell CypB-silenced cells, whereas CypA silencing left cells unre- and Casanova, 2016). We analyzed collective cell migration by sponsive to TGFβ’s pro-migratory effects. In contrast, in Boyden means of a wound healing assay and individual migration by a chamber assays, CypA-silenced cells showed higher rates of Boyden chamber assay. Our results show that in scratch assays cell migration in both basal and TGFβ conditions, whereas and in basal conditions, CypB-silenced cells exhibited faster CypB silencing partially prevented TGFβ-induced individual cell gap closure than control or CypA-silenced cells (Figure 2E). TGFβ- migration (Figure 2F). CypA and CypB regulate epithelial phenotype | 503 Cyclophilin modulation of TGFβ-induced phenotypic effects whereas reintroduction of PPI-CypA failed to do so (Figure 4C). coincides with Slug and SMAD6/7 expression patterns but not These results were also observed in the presence of TGFβ SMAD2/3 activation (Figure 4D). On the other hand, reintroduction of wt-CypB, but We next explored whether cyclophilin modulation of TGFβ- not of ER-CypB, rescued Slug expression in CypB-silenced induced EMT correlated with Slug and Snail levels and the cells (Figure 4G). Remarkably, PPI-CypB increased Slug levels activation of SMAD2/3, which canonically transduce TGFβ even further than those achieved by wt-CypB (Figure 4G). As signals. TGFβ treatment of HK-2 cells induced a time-dependent before, these effects were reproduced in the presence of TGFβ expression of Slug and Snail that was preceded by SMAD2/3 (Figure 4H). activation (Figure 3A). Both CypA and CypB silencing enhanced TGFβ-induced Snail expression. CypA silencing also potenti- Slug modulation by CypB is independent of the CD147 receptor ated TGFβ-induced Slug expression, whereas CypB silencing and extracellular CypB prevented Slug induction by TGFβ (Figure 3B). These effects Extracellular CypA and CypB are important mediators of inter- were also observed at the mRNA level (Figure 3B right, bottom). cellular communications, both in physiological and pathological Cyclophilin modulation of TGFβ-induced Slug and Snail levels processes (Yurchenko et al., 2010; Bukrinsky, 2015). Accord- occurred without changes in SMAD4 levels or TGFβ-induced ingly, we explored whether cyclophilins could regulate Slug and SMAD2/3 phosphorylation (Figure 3B) and nuclear translocation Snail in an autocrine/paracrine manner. Analysis of conditioned (Figure 3C). Treatment with the proteasome inhibitor MG132 media (CM) showed that CypB and CypA were progressively inhibited Slug degradation, thereby increasing Slug protein secreted into the extracellular milieu and that their secretion levels (Figure 3D). This increase was diminished in CypB- was unaffected by TGFβ treatment (Figure 5A). Pretreatment with silenced cells, confirming that CypB silencing reduced de brefeldin-A (BFA), a secretion inhibitor that impedes ER–Golgi novo Slug expression. Simultaneous treatment with TGFβ and protein transport, blocked both basal and CsA-induced CypB MG132 did not increase Slug levels above those observed secretion but had no effect on that of CypA (Figure 5B). This, with MG132 alone. By contrast, CypB silencing downregulated together with the lack of CsA effect on CypA release, confirms that Slug levels more effectively when TGFβ and MG132 were added CypA and CypB secretion involves different mechanisms. BFA together compared to MG132 alone. Because TGFβ limits its own prevented basal and TGFβ-induced Slug expression (Figure 5C) signaling by initiating an autoinhibitory feedback loop (Yan and but, unlike CypB silencing, also reduced Snail expression and Chen, 2011), these results suggest that CypB silencing might SMAD2/3 activation. We next silenced CD147, the only known overactivate TGFβ-induced termination signaling. receptor for extracellular CypB and CypA (Yurchenko et al., 2010). We analyzed inhibitory SMADs (I-SMADs) SMAD6/7 and CD147 silencing increased rather than decreased Slug and Snail Ski-like (SKIL) which mediate a TGFβ autoregulatory feed- levels, which was reminiscent of CypA silencing (Figure 5D). back loop (Yan and Chen, 2011). In addition to upregula- Finally, exogenously added recombinant human CypB (rCypB) tion of these genes by TGFβ, we observed that CypB silenc- was unable to restore Slug or downregulate Snail to levels found ing further increased the levels of SMAD6/7 and SKIL in in non-silenced cells (Figure 5E). ERK1/2 phosphorylation was basal and TGFβ-treated cells, whereas CypA silencing slightly used as a control for CypB activity (Yurchenko et al., 2001). Taken reduced them (Figure 3E). We also analyzed the levels of together these results argue against an autocrine loop in Slug bone morphogenic protein (BMPs) 2 and 7, which coun- modulation by CypB but hint that CypA–CD147 interaction could teract TGFβ-induced EMT in a SMAD7-dependent way (Yang be key in Slug and Snail regulation. et al., 2009). BMP2 mRNA was found to be upregulated in CypB- silenced cells (Figure 3E), whereas we were unable to detect CypB reduces ionomycin- and thapsigargin-induced calcium BMP7 in HK-2 cells (data not shown). release and prevents ionomycin-induced Slug expression CypB and CypA participate in ER calcium regulation in Disruption of the PPIase activity of CypA and CypB induces Slug lymphocytes (Bram and Crabtree, 1994) and platelets (Rosado expression, whereas mislocalization of CypB out of the ER et al., 2010), respectively. Since Slug expression is modulated abrogates Slug expression by calcium (Hayashida et al., 2006), we investigated whether To further investigate the mechanisms underlying Slug and Slug regulation by cyclophilins involved calcium signaling. Snail modulation by cyclophilins, we reintroduced either wild- Control and cyclophilin-silenced cells were loaded with a type (wt) or mutant CypA and CypB lacking PPIase activity (PPI) fluorescent Ca2+ indicator and subsequently treated with the or CypB’s ER-directing signal peptide (ER), in the corresponding calcium ionophore ionomycin or the sarcoplasmic/endoplasmic silenced cells. Our results show that all constructs were correctly reticulum calcium ATPase (SERCA) inhibitor thapsigargin. expressed (Figure 4A, B, and E) and that wt-CypB and PPI-CypB Ionomycin induced a stronger Ca2+ increase that rapidly peaked were properly located in the ER (Figure 4F) and secreted to the and gradually declined to a steady-state above baseline extracellular medium (Figure 4E, CM blot), whereas ER-CypB (Figure 6A), whereas thapsigargin triggered a weaker and more was not. flattened response (Figure 6B). CypB silencing reduced the Reintroduction of wt-CypA in CypA-silenced cells downreg- calcium rise elicited by both compounds. Interestingly, Slug ulated Slug and Snail to the levels found in control cells, expression was either transiently increased or reduced after 504 | Sarró et al. Figure 3 Cyclophilin modulation of TGFβ-induced phenotypic effects coincides with Slug and I-SMADs expression patterns but not SMAD2/3 activation. (A) HK-2 cells were treated with 1.5 ng/ml TGFβ for the indicated times and the expression levels of Slug, Snail, and total and phosphorylated SMAD3 and SMAD2 were analyzed by WB. (B) CypA- and CypB-silenced HK-2 cells were treated with TGFβ for 4 or 24 h. Protein levels were analyzed by WB (left) and quantified for Slug/actin and Snail/actin ratios, expressed as percentage of that of control shRNA cells exposed to TGFβ for 4 h (right, upper), and the mRNA levels of Slug and Snail were analyzed by qPCR after 24 h of TGFβ treatment (right, bottom). (C) Control and CypB-silenced cells were treated with TGFβ for 24 h and Slug levels were analyzed by WB. When indicated, 5 µM of the proteasome inhibitor MG132 was added to cells for the last 16 h of TGFβ treatment. (D) Nuclear translocation of SMAD3 and SMAD2 (green) after treatment with TGFβ or vehicle for 4 h. Nuclei were stained with Hoechst (blue). Scale bar, 25 µm. (E) The mRNA levels of of the indicated genes were analyzed by qPCR after treatment with TGFβ for 24 h. Unless otherwise indicated values are expressed as fold to controls. Only statistically significant differences within control or TGFβ-treated groups are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. ionomycin (Figure 6C) or thapsigargin (Figure 6D) treatment, expression in the absence of CypB (Figure 6E) or when CypB respectively. These striking differences may be attributable was not localized within the ER (Figure 6F). By contrast, CypB to the distinct intensities and kinetics in calcium release PPIase activity was unnecessary for ionomycin-induced Slug of each compound. Ionomycin was unable to induce Slug expression. Conversely, the reduction in Slug levels after CypA and CypB regulate epithelial phenotype | 505 Figure 4 CypA PPIase activity is required to downregulate Snail and Slug, whereas ER location of CypB is mandatory to sustain Slug expression. (A) Scheme summarizing the mutations introduced in CypA and CypB expression vectors to generate HA-tagged shRNA-resistant wt, PPIase- defective (PPI), and signal peptide-defective (ER) cyclophilins. (B) The expression levels of wt-CypA and PPI-CypA forms reintroduced into CypA-silenced cells were determined by WB. (C and D) Slug and Snail levels in CypA-silenced cells carrying wt-CypA or PPI-CypA, either untreated (C) or treated with TGFβ for 4 h (D). (E) The expression levels of wt-CypB and mutant forms reintroduced into CypB-silenced cells were analyzed by WB in cell extracts and CM. (F) Immunofluorescence staining showing that wt-CypB and PPI-CypB colocalized with the ER marker calnexin (CNX), whereas ER-CypB did not. Scale bar, 25 µm (left) and 90 µm (right, magnifications). (G and H) WB of Slug and Snail levels after reintroduction of wt-CypB, PPI-CypB, or ER-CypB, either in untreated cells (G) or in cells treated with TGFβ for 4 h (H). In all cases, Ø corresponds to the empty expression vector. In D and H, the statistically significant differences with shCon+ Ø are not shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. 506 | Sarró et al. Figure 5 Slug modulation by CypB is independent of the CD147 receptor and extracellular CypB. (A) HK-2 cells were treated with 1.5 ng/ml TGFβ for the indicated times, and the presence of CypA and CypB in the CM was analyzed by WB. Culture medium was used as a negative control. (B) HK-2 cells were treated with 1 µM BFA for 30 min before addition of either 1.5 ng/ml TGFβ or 0.5 µM CsA for 4 h and CM were analyzed as above. (C) HK-2 cells were treated with 1 µM BFA for 30 min before addition of either 1.5 ng/ml TGFβ or not for 4 h. The levels of Slug, Snail, and total and phosphorylated SMAD3 and SMAD2 were analyzed by WB. (D) HK-2 cells were stably silenced for CD147 and treated or not with TGFβ for 4 h before WB for the indicated protein levels. (E) Control and CypB-silenced cells were treated with increasing doses of rCypB for 4 h and the levels of Slug, Snail, and total and phosphorylated ERK1/2 were analyzed by WB. thapsigargin treatment was not inhibited but instead increased tilayered cell sheet-like structure with higher cell density in CypB-silenced cells (Supplementary Figure S3), indicating that and multicellular protrusions in the edges of the cell sheet. both CypB silencing and thapsigargin effects are not mutually Interestingly, the sheets of CypB-silenced cells formed longer exclusive. and more branched multicellular protrusions than sheets of The ER calcium-buffering chaperone calreticulin (CRT) has control or CypA-silenced cells, resembling tubular-like structures been shown to be involved in Slug upregulation and EMT (Figure 7B). In addition, and as in 2D, 3D-cultured CypB-silenced promotion (Hayashida et al., 2006). Coimmunoprecipitation cells exhibited higher levels of epithelial markers and lower (coIP) assays with reintroduced wt or mutant CypB showed that levels of Slug than control or CypA-silenced cells (Figure 7C CRT interacted with wt-CypB and to a higher extent with PPI- and D). CypB, but not with ER-CypB (Figure 6G). We next explored whether CypB could be modulating CRT by altering its subcellular CypB deletion ameliorates inflammation and early events localization. Figure 6H shows that CRT localization was not of renal fibrosis after unilateral ureteral obstruction (UUO) affected by either CypA or CypB silencing. To expand on our in vitro results, we investigated the contribution of CypB in the development of fibrosis using CypB and CypA differentially affect epithelial phenotype of TECs global CypB knockout (KO) mice and wt littermates subjected in 3D cultures to UUO (Figure 8A). To study early events in renal fibrosis, mice Three-dimensional (3D) cultures increase cell differentiation underwent ureteral ligation of the left kidney for 1 week and and partially recapitulate tissue-specific morphology (Secker were compared with contralateral (CL) non-ligated right kidneys et al., 2018). To validate two-dimensional (2D) results, we (Figure 8B). In non-obstructed kidneys, there were no apparent performed 3D ‘sandwich’ experiments where control and histological differences between wt and CypB KO mice. By cyclophilin-silenced cells were embedded in between two contrast, kidneys from CypB KO mice were partially protected layers of MatrigelTM and allowed to differentiate for 14 days from the UUO-induced tubular distension and inflammation (Figure 7A). Our results show that HK-2 cells formed a mul- (Figure 8C). In these conditions, no overt fibrosis was observed CypA and CypB regulate epithelial phenotype | 507 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 Figure 6 Effect of CypB silencing on ionomycin and thapsigargin-induced cytosolic calcium increase and Slug expression in HK-2 cells. (A) Left: cytosolic calcium increase in ionomycin-treated cyclophilin-silenced cells, expressed as the ratio between maximal fluo-4 fluores- cence achieved after the addition of 1 µM ionomycin (Fmax) and the fluorescence before treatment (F0). Dotted line indicates fluorescent value before treatment. Right: cytosolic calcium kinetics in two representative cells. The arrow points the time when ionomycin was added. (B) Same as A after addition of 5 µM thapsigargin. Data represent mean ± SEM of 5–10 replicates per group. *corresponds to significant statistical difference (P < 0.05) respect to shCon group. (C and D) HK-2 cells were treated with 1 µM ionomycin (C) or 5 µM thapsigargin (D) for the indicated times and Slug expression was analyzed by WB. (E and F) Slug levels were analyzed after 4-h treatment with 1 µM ionomycin in CypA- and CypB-silenced cells (E) or in CypB-silenced cells carrying wt or mutated CypB forms (F). (G) HA-tagged wt-CypB, PPI-CypB, and ER- CypB were immunoprecipitated with an anti-HA affinity matrix, followed by immunoblotting with CRT and HA. (H) Immunofluorescence staining of CRT (ER luminal and cytosolic; in red) and CNX (ER transmembrane; in green). Scale bar, 25 µm (left) and 90 µm (right, magnifications). using Masson’s trichrome staining (data not shown), likely due reduced CypA, E-cadherin, CD147, and BMP7 whereas increased to the short-term obstruction. We next analyzed the expression fibronectin, collagen-Ia, matrix metalloproteinase 9 (MMP-9), of relevant pro-fibrotic and pEMT-related markers at the mRNA TGFβ, Snail, Slug, and BMP6 levels. Genetic deletion of CypB level. In wt kidneys, UUO did not have significant effects on significantly prevented the decrease of CypA, E-cadherin, CD147, CypB, BMP2, SMAD7, and SMAD6 mRNA levels, but significantly and BMP7 and the increase of fibronectin, MMP9, and Slug 508 | Sarró et al. Figure 7 CypA and CypB differentially affect epithelial phenotype of TECs in 3D cultures. (A) Control or CypA- or CypB-silenced HK-2 cells were embedded between two layers of MatrigelTM and allowed to differentiate for 14 days. (B) Bright-field images showing HK-2 cells after 14 days. Scale bar, 500 µm (for 2.5× magnification) and 125 µm (for 10× magnification). (C and D) WB (C) and qPCR (D) of cell extracts obtained from HK-2 cells embedded in MatrigelTM . *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. produced by UUO. CypB knockdown further increased the UUO- Discussion induced expression of Snail and BMP-6 levels. Finally, there This paper shows that CypA and CypB are opposite regulators were no significant differences in collagen-Ia, TGFβ, or BMP2 of TEC plasticity since silencing of CypA hampered, whereas levels between obstructed kidneys of wt and CypB KO mice. silencing of CypB promoted the epithelial phenotype of kidney SMAD6/7 and Slug were the only genes that were upregulated cells (HK-2 cells). Accordingly, we postulate that CypA favors kid- or downregulated, respectively, in non-ligated kidneys of CypB ney epithelial differentiation, whereas CypB acts as an epithelial KO mice. Regarding pro-inflammatory cytokines, UUO-induced repressor. We also propose that, based on our in vivo results tumor necrosis factor α (TNFα), macrophage chemoattracting in a model of fibrosis caused by UUO, CypB may participate in protein 1 (MCP1), and the pan-macrophage marker CD68 were the development of kidney fibrosis and inflammation in the early all significantly reduced in CypB KO mice (Figure 8E). To unravel stages. whether cytokine reduction underlies a downregulation of pro- CypA silencing results in loss of epithelial characteristics, inflammatory signaling in TECs, we analyzed the activity of including downregulation of tight junction proteins, decreased nuclear factor kappa B (NFκB) in HK-2 cells. Results in Figure 8F TEER and increased dextran permeability, reduced AP activity, show that NFκB promoter activity was strongly increased after and increased invasion and fibronectin expression. Although CypA silencing and considerably decreased in CypB-silenced some of these effects were previously reported in CypA-silenced cells. human primary TECs (Pallet et al., 2008), we now describe for the CypA and CypB regulate epithelial phenotype | 509 Figure 8 CypB depletion ameliorates inflammation and early events of renal fibrosis after UUO. (A) WB showing CypB levels in kidneys from CypB KO (CypB−/− ) mice and control wt littermates. (B) Experimental scheme: CypB KO mice and control littermates were subjected to UUO as a model of renal fibrosis. Mice were sacrificed 7 days after obstruction and total RNA was extracted from the right CL kidneys and left obstructed kidneys (UUO). (C) Left: hematoxylin and eosin (H&E) and mouse macrophage marker F4/80 staining of kidney sections. Scale bar, 100 µm. Right: semi-quantitative immunohistochemistry analysis for tubular dilatation and inflammation; each parameter was assessed independently by two pathologists and was scored on a scale of 0–4, ranging from none (0), minimum (1), mild (2), moderate (3), to severe (4). Mann–Whitney U test was used to detect differences. (D and E) The mRNA levels of the indicated genes were detected by qPCR. Dots represent individual values for either CL or UUO kidneys, with eight mice in each group. (F) The NFκB promoter activity in HK-2 cells transfected with a control empty plasmid pCMV-HA or the p65 subunit of NFκB was analyzed with a luciferase reporter assay. Only statistically significant differences within empty vector or p65-transfected groups are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. 510 | Sarró et al. first time that CypB silencing promotes epithelial differentiation I-SMADs expression is, in turn, regulated by BMPs, upregulation and prevents TGFβ-induced EMT-like phenotypic changes. of BMP2 in CypB-silenced HK-2 cells, as well as BMP6/7 in Those included downregulation of epithelial markers, altered CypB KO mice further supports this concept. Curiously, BMP2 cell morphology, and increased cell-to-substrate adhesion upregulation was not observed in CypB KO mice. As BMPs and invasion. There are, however, some similarities between have both redundant and isoform-specific functions (Yuasa CypB silencing and TGFβ in promoting collective cell migration and Fukuda, 2008), we hypothesize that BMP2-mediated CypB- and cell-to-substrate adhesion that might involve different silencing effects in HK-2 cells would be carried out by BMP6 or mechanisms of action. Faster collective migration of CypB- BMP7 in kidneys of CypB KO mice. silenced cells would result from upregulation of adherens and In this work we also demonstrate that CypA PPIase activity is tight junction proteins. This ought to facilitate transmission of necessary to repress Slug and Snail expression. Interestingly, the traction force between leader and follower cells and between while reintroducing PPI-CypA reproduced CypA-silencing follower cells, as described (Campbell and Casanova, 2016; effects on Slug and Snail, reintroduction of PPI-CypB did not Friedl and Mayor, 2017). Conversely, TGFβ treatment acts by mimic CypB silencing. In contrast, PPI-CypB further increased enhancing the migratory phenotype of leader cells at the edge Slug levels above those observed with wt-CypB, suggesting that of the gap, accelerating wound closure. The small increase in mutation of CypB PPIase activity may represent a gain of function cell-to-substrate adhesion in CypB-silenced cells may result over CypB silencing. Thus, while CypA and CypB exert almost from upregulation of hemi-desmosomal protein keratin-8, as opposite actions in regulating epithelial phenotype of TECs, previously described (Liu et al., 2008). The stronger TGFβ pro- mutation to block either of their PPIase activities resulted in Slug adhesive effect may relate to acquisition of a rear–front polarity, upregulation. As CsA inhibits the PPIase activities of both CypA which, in turn, is inhibited by CypB silencing. and CypB, our results suggest that CsA pro-fibrotic action could Cells cultured in 3D more accurately reproduce in vivo behavior be mediated, at least in part, by Slug upregulation consequent (Secker et al., 2018). In a 3D environment, HK-2 cells formed to the inhibition of CypA and CypB activity. multicellular protrusions, which resembled the collective sprout- Accumulating evidence points to an important role for extra- ing underlying the branching morphogenesis of the kidney (Gray cellular CypA and CypB in mediating intercellular communica- et al., 2010; Friedl and Mayor, 2017). The growing sprout moves tions through receptor-mediated autocrine and paracrine sig- collectively with a row of front–rear polarized leader cells form- naling pathways (Bukrinsky, 2015). However, no evidence was ing a terminal end bud, coupled to lumen-enclosing apicobasal found to support an extracellular role of CypB in modulating polarized follower cells by adherens, desmosomal, and tight Slug expression in HK-2 cells because: (i) no differences in CypB junctions (Shamir and Ewald, 2015; Huebner et al., 2016). In 3D- secretion were observed after TGFβ treatment; (ii) silencing of cultured HK-2, extension of multicellular protrusions was much CD147, the only known receptor for extracellular cyclophilins, longer and branched in CypB-silenced cells. We hypothesize did not prevent but rather increased Slug expression; and (iii) that, while the EMT-like processes in the tip of sprouting strands addition of recombinant CypB in the extracellular milieu of CypB- could be occurring in a similar way in all cell lines, the higher silenced cells was unable to restore Slug levels. Because CD147 proliferation and collective migration rates of follower cells with silencing upregulated Slug and Snail levels as in CypA-silenced silenced CypB could boost sprout extension. cells, we hypothesize that interaction between extracellular CypA The strikingly opposite actions of CypB and CypA on HK-2 and CD147 could account for Slug and Snail repression in differ- epithelial phenotype revealed differential regulation of the entiated TECs. epithelial repressors Snail and Slug. In HK-2 cells, CypA acts Slug modulation by CypB might involve calcium signaling as a repressor of Slug and Snail, whereas CypB represses Snail because CypB silencing prevents ionomycin-induced calcium but is mandatory for Slug expression. This raises the question of release and Slug expression. Although the SERCA inhibitor whether Slug and Snail function in redundant, specific, or even thapsigargin also increased calcium release, it reduced Slug antagonistic manners. Interestingly, there are examples of both levels. This disparity reflects the fact that calcium signals cooperativity and antagonism depending on the context (Nieto, differing in amplitude, spatial, and temporal properties trigger 2002; Moreno-Bueno et al., 2006). Snail upregulation in CypB- different transcriptional responses (Carrasco et al., 2004). silenced cells could also involve a compensatory mechanism Ionomycin catalyzes calcium transport not only across the PM to counterbalance the decrease in Slug levels, as previously but also out of intracellular calcium stores (Kao et al., 2010). described (Ganesan et al., 2016). Regardless, our results Because both ionomycin and thapsigargin commonly target the indicate that, at least in CypB-silenced cells, Slug deficiency, ER calcium store, reduced calcium release in CypB-silenced cells rather than augmented Snail levels, determines cell phenotype. could reflect lower ER calcium levels or buffering capacity. This This predominant role of Slug is particularly relevant because, was further supported by CypB interaction with the calcium- so far, Snail has been considered as sufficient and necessary buffering chaperone CRT. CypB–CRT interaction only occurred to induce EMT and fibrosis in the mouse kidney (Grande et al., when CypB was located in the ER, and was stronger when 2015). CypB lacked its PPIase activity. Because this interaction pattern Our results indicate that cyclophilin-mediated regulation of paralleled that of Slug expression, we speculate that CRT–CypB Slug and Snail might be exerted through I-SMADs. Because interaction might be crucial for Slug expression. In agreement CypA and CypB regulate epithelial phenotype | 511 with this, CRT has been previously involved in Slug upregulation were starved in medium A supplemented with 0.1% fetal bovine and EMT induction (Hayashida et al., 2006). Although CypB serum (FBS) for 16 h. mislocalization to the cytosol prevented ionomycin-induced Slug expression, CypB modulation of calcium signaling could Gene silencing and rescue experiments involve other cellular compartments apart from the ER. Recent CypA, CypB, and CD147 silencing and cyclophilin reintroduc- publications highlight the importance of ER–PM contacts in tion were performed using lentiviral particles as described in calcium signaling (Wang et al., 2018). Interestingly, CypB Supplementary material. All shRNA sequences are provided in associates with the calcium channel, transient receptor potential Supplementary Table S2. Briefly, for shRNA rescue experiments, cation channel subfamily V member 6 (TRPV6), regulating we first generated a shRNA-resistant wt-CypB by introducing calcium uptake (Stumpf et al., 2008). TRPV6 is part of the store- silent mutations into the shRNA-targeted sequence to prevent operated calcium entry (SOCE) mechanism and translocates RNA-induced silencing complex (RISC)-mediated degradation. to the PM via Orai1 (Raphaël et al., 2014). CypB, through ShRNA against CypA was directed to the 3 UTR, and thus reintro- its chaperone activity, could be involved in the formation of duction of wt-CypA did not require further modification. Within multiprotein complexes containing TRPV6–Orai1 in the ER–PM these constructs, we mutated critical residues to inactivate PPI- junctions, thereby modulating Ca2+ influx and downstream Slug ase activity (PPI) of CypA (c.161_162delinsGC; p.Arg55Ala) expression. and CypB (c.259_260delinsGC; p.Arg62Ala) or to eliminate CypB In vivo, we observed that kidneys of CypB KO mice exhib- ER-directing signal peptide (ER) (c.4_75del; p.2_25del). All ited downregulated and upregulated Slug and SMAD6/7 constructs were stably transduced into cyclophilin-silenced cells levels, respectively, largely recapitulating the results observed using lentiviral particles as for shRNA infection. in cultured TECs. In addition, kidneys from CypB KO mice were protected from the UUO-induced tubular distension and Immunoblotting, immunocytochemistry, and coIP inflammation after 1-week UUO. No overt fibrosis was observed A detailed description of immunoblotting, immunocytochem- at this time point but the upregulation of pro-fibrotic factors istry, and coIP protocols is provided in Supplementary material. indicated that early events of renal fibrosis were taking place. For WB of extracellular cyclophilins, CM were collected and cen- Considering our in vitro results showing a pro-epithelial role of trifuged 5 min at 1500× g. Supernatants were mixed with 5× CypA, the reduction of CypA levels seen in the UUO model might sample buffer, and 50 µl of the resulting mix was resolved via gel also be considered to possibly exert a pro-fibrotic action. As CypB electrophoresis. A list of all antibodies used in this work is shown knockdown not only partially prevents UUO-induced fibronectin, in Supplementary Table S3. MMP9 and Slug upregulation but also CypA and E-cadherin downregulation, we postulate that CypB may participate in RNA extraction and qPCR kidney fibrosis. In addition, CypB also mediates UUO-induced RNA extraction and qPCR were performed as indicated in Sup- inflammation because CypB knockdown reduced the expression plementary material. All probes used in this work are listed in of pro-inflammatory markers. Moreover, our results showing Supplementary Table S4. Analysis of relative gene expression reduced activity of the pro-inflammatory transcription factor was performed using the 2–Ct method normalizing to TATA- NFκB in CypB-silenced HK-2 cells support a central role of TEC’s binding protein (TBP) expression. in the development of interstitial inflammation (Liu et al., 2018). Altogether, our results demonstrate key roles for CypA and CypB in kidney epithelial cell plasticity and have, moreover, pin- Cell proliferation pointed Slug as a key regulator of EMT. We suggest a role of CypB Cells were trypsinized, washed with phosphate buffered saline at the crossroads between the inflammatory and the fibrogenic (PBS), and incubated with carboxyfluorescein diacetate succin- processes by regulating the inflammatory response of TECs. imidyl ester (CFSE; Sigma-Aldrich) at 2.5 µM final concentration for 10 min at 37◦ C in a cell culture incubator. An aliquot of cells was left unlabeled to set background fluorescence. CFSE was Materials and methods then quenched by washing cells twice with complete medium, Materials and a portion of cells was taken to measure fluorescence at the The source of all materials used in this work is described in beginning of the experiment. The rest of labeled cells were plated Supplementary Table S1. and incubated at 37◦ C for 5 days. Fluorescence was measured Cell culture with a flow cytometer (Becton Dickinson) and analyzed with Cell Human kidney proximal tubule cells HK-2 (ATCC) and RPTEC/ Quest software (Becton Dickinson). TERT1 (ATCC), which retain morphological and functional attributes of normal adult human proximal tubular epithelium TEER and FITC-dextran permeability (Ryan et al., 1994; Wieser et al., 2008), were cultured as indi- Cells were seeded on 24-well transwell plates with 0.4-µm cated in Supplementary material. Unless otherwise indicated, pore polyester membrane inserts (Sigma-Aldrich) and measure- cells were seeded at 2.25 × 104 cells/cm2 and experiments ments were performed 5 days later. TEER was measured using were performed at Day 5 postseeding. Before treatment, cells an epithelial voltmeter (Millipore) with STX100C electrode (World 512 | Sarró et al. Precision Instruments) according to manufacturer’s instructions. with the lower surface facing up. Nuclei were visualized with For permeability assays, 40 kDa FITC-dextran (Sigma-Aldrich) an Olympus IX71 fluorescence microscope and counted using in a final concentration of 100 µg/ml was added to the apical Image J. compartment of the cells. Three hours later, samples were collected from the basolateral compartment and absorbance (λex /λem = 485/528 nm) was measured using a microplate Cytosolic calcium measurement reader (Molecular Devices). Attached HK-2 cells were loaded with 5 µM fluo-4 (ThermoFischer) in control buffer (containing 140 mM NaCl, AP activity 3.6 mM KCl, 1.2 mM MgSO4 , 1 mM CaCl2 , 20 mM HEPES, pH 7.4) AP activity was analyzed as described in Supplementary for 1 h at 37◦ C. Time-dependent changes in cell fluorescence material. (Ex/Em 488/528 nm) secondary to the addition of ionomycin (1 µM) or thapsigargin (5 µM) were monitored every 5 sec using a multimode microplate fluorometer (Molecular Devices). Cell adhesion assay Cells were trypsinized and washed twice with culture medium to completely eliminate trypsin. Cells were counted and 2 × 104 3D cultures cells/well were seeded onto two duplicated 96-well plates and Three-dimensional cell cultures were performed in 24-well allowed to adhere at 37◦ C for 30 min. After the incubation plates as indicated in (Secker et al., 2018), with the following period, unattached cells from one of the plates were removed modifications: each well was first coated with 210 µl MatrigelTM by washing twice with PBS. The respective amounts of remaining (BD Biosciences) and allowed to polymerize at 37◦ C for 1 h. attached cells (from the washed plate) and total of cells (from Thereafter, 0.15 × 106 HK-2 cells/cm2 were seeded onto the the unwashed plate) were determined using the tetrazolium salt MatrigelTM layer and left to attach for 16 h at 37◦ C. Subsequently, (XTT) assay. Results were expressed as the ratio of XTT values medium was removed and cells were overlaid with 150 µl/well between washed and unwashed plate. MatrigelTM . After polymerization of the new layer of MatrigelTM , 400 µl/well medium was added and cultures were maintained for Wound migration assay 14 days at 37◦ C, with medium replacement every 48 h. Images For wound migration assay, 7.5 × 104 cells were seeded on were obtained using a Leica DM IRBE inverted microscope. each of the two compartments (growth area: 0.22 cm2 /compartm For protein or RNA isolation from cells embedded in MatrigelTM , -ent) of silicone culture inserts (Ibidi GmbH), grown for 5 days, cell cultures were washed with cold PBS followed by addition and starved for 16 h. At the onset of the experiment, the silicone of 500 µl 0.5 mM EDTA/PBS per well and incubated on ice for insert was removed leaving a gap free of cells of 500 ± 100 µm 15 min. Cell/MatrigelTM mixture was dislodged from the dish width (according to manufacturer). Cells were gently washed with with a p1000 pipet with the tip cutoff and transferred to a serum-free media to remove cell debris and treated as indicated. microcentrifuge tube. Cells were then pelleted by centrifugation Digital images were obtained with an Olympus CellR microscope and resuspended in either radioimmunoprecipitation assay and area measurements were performed using ImageJ software. (RIPA) buffer or Trizol for protein or RNA extraction, respectively. The rate of wound closure was measured by subtracting the area of the wound at 24 h from the area at 0 h. UUO procedure Transwell migration assay UUO was performed as previously described (Kie et al., 2008). For transwell migration assays, polycarbonate cell cul- Briefly, mice were anesthetized by intraperitoneal injection of ture inserts with 8-µm pore size and 0.47 cm2 growth area pentobarbital (50 mg/kg). Through a 2-cm midline abdominal (ThermoFischer) were used. The inserts were coated with 50 µl incision, the left kidney was exposed by retraction of the growth factor reduced (GFR)-MatrigelTM matrix (BD Biosciences) intestines using a self-retaining microdissection retractor and diluted 10 times in serum-free media and incubated at 37◦ C the left ureter was carefully dissected from surrounding tissue. for 1.5 h. A total of 200 µl serum-free media containing The ureter was then doubly ligated at the midpoint between the 5 × 104 serum-starved HK-2 cells were placed in the top well kidney and the bladder using sterile 6-0 silk suture. The surgical chamber, whereas the bottom chamber was filled with 500 µl incision was then closed and the mouse was allowed to recover complete medium enriched with up to 10% FBS. Media in the from anesthesia; postoperative analgesia (buprenorphine, top and bottom chambers were supplemented with vehicle 0.1 mg/kg, SQ) was administered. Sham-operated control mice or TGFβ. After 24 h of treatment, cells were washed in PBS, underwent a similar surgical procedure without ligation of the fixed with 4% paraformaldehyde, and permeabilized with ureter. 0.1% Triton X-100. Cell nuclei were then stained with Hoechst (ThermoFischer). Cells from the top surface of the membrane were gently removed with a cotton swab. Membranes were then NF κB activity cut out from the inserts with a scalpel blade and mounted on NFκB promoter activity was measured as indicated in Supple- microscope slides using antifade mountant (ThermoFischer), mentary material. CypA and CypB regulate epithelial phenotype | 513 Statistics Chang-Panesso, M., and Humphreys, B.D. (2017). Cellular plasticity in kidney All statistical analyses were performed using GraphPad Prism injury and repair. Nat. Rev. Nephrol. 13, 39–46. Friedl, P., and Mayor, R. (2017). Tuning collective cell migration by cell–cell 6.0 (GraphPad Software). In all charts, dots represent individual junction regulation. Cold Spring Harb. Perspect. Biol. 9, pii: a029199. values from at least three independent experiments and the Ganesan, R., Mallets, E., and Gomez-Cambronero, J. (2016). The transcription horizontal and vertical bars represent the mean and SEM, factors Slug (SNAI2) and Snail (SNAI1) regulate phospholipase D (PLD) respectively. Statistical differences were determined by one-way promoter in opposite ways towards cancer cell invasion. Mol. Oncol. 10, analysis of variance (ANOVA) (Figures 1D, 1F–H, 1J, 4C, 4D, 4G, 663–676. González-Guerrero, C., Cannata-Ortiz, P., Guerri, C., et al. (2017). TLR4- 4H, 6A, 6B, and 7D) or two-way ANOVA (Figures 1I, 2D–F, 3B, 3E, mediated inflammation is a key pathogenic event leading to kidney damage 8C–F) followed by Tukey’s multiple comparison test. and fibrosis in cyclosporine nephrotoxicity. Arch. Toxicol. 91, 1925–1939. Grande, M.T., Sánchez-Laorden, B., López-Blau, C., et al. (2015). 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