Sustained hyperosmolarity increses TGF-beta1 and Egr-1 expression in the rat renal medulla.

BACKGROUND: Although TGF-ss and the transcription factor Egr-1 play an important role in both kidney fibrosis and in response to acute changes of renal medullary osmolarity, their role under sustained hypo- or hyperosmolar conditions has not been elucidated. We investigated the effects of chronic hypertonicity and hypotonicity on the renal medullary TGF-ss and Egr-1 expression. METHODS: Male adult Sprague Dawley rats (n = 6/group) were treated with 15 mg/day furosemide, or the rats were water restricted to 15 ml/200 g body weight per day. Control rats had free access to water and rodent chow. Kidneys were harvested after 5 days of treament. In cultured inner medullary collecting duct (IMCD) cells, osmolarity was increased from 330 mOsm to 900 mOsm over 6 days. Analyses were performed at 330, 600 and 900 mOsm. RESULTS: Urine osmolarity has not changed due to furosemide treatment but increased 2-fold after water restriction (p < 0.05). Gene expression of TGF-ss and Egr-1 increased by 1.9-fold and 7-fold in the hypertonic medulla, respectively (p < 0.05), accompanied by 6-fold and 2-fold increased c-Fos and TIMP-1 expression, respectively (p < 0.05) and positive immunostaining for TGF-ss and Egr-1 (p < 0.05). Similarly, hyperosmolarity led to overexpression of TGF-ss and Egr-1 mRNA in IMCD cells (2.5-fold and 3.5-fold increase from 330 to 900 mOsm, respectively (p < 0.05)) accompanied by significant c-Fos and c-Jun overexpressions (p < 0.01), and increased Col3a1 and Col4a1 mRNA expression. CONCLUSION: We conclude that both TGF-ss and Egr-1 are upregulated by sustained hyperosmolarity in the rat renal medulla, and it favors the expression of extracellular matrix components

Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease

Background: Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. Methods: Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGFβ1) ± apyrase, or ATPγS for 48h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43+/- mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. Results: Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGFβ1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43+/- mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. Conclusion: Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD

Mediator of DNA Damage Checkpoint 1 (MDC1) Contributes to High NaCl-Induced Activation of the Osmoprotective Transcription Factor TonEBP/OREBP

Background: Hypertonicity, such as induced by high NaCl, increases the activity of the transcription factor TonEBP/OREBP whose target genes increase osmoprotective organic osmolytes and heat shock proteins. Methodology: We used mass spectrometry to analyze proteins that coimmunoprecipitate with TonEBP/OREBP in order to identify ones that might contribute to its high NaCl-induced activation. Principal Findings: We identified 20 unique peptides from Mediator of DNA Damage Checkpoint 1 (MDC1) with high probability. The identification was confirmed by Western analysis. We used small interfering RNA knockdown of MDC1 to characterize its osmotic function. Knocking down MDC1 reduces high NaCl-induced increases in TonEBP/OREBP transcriptional and transactivating activity, but has no significant effect on its nuclear localization. We confirm six previously known phosphorylation sites in MDC1, but do not find evidence that high NaCl increases phosphorylation of MDC1. It is suggestive that MDC1 acts as a DNA damage response protein since hypertonicity reversibly increases DNA breaks, and other DNA damage response proteins, like ATM, also associate with TonEBP/OREBP and contribute to its activation by hypertonicity. Conclusions/Significance: MDC1 associates with TonEBP/OREBP and contributes to high NaCl-induced increase of tha

CD98 Increases Renal Epithelial Cell Proliferation by Activating MAPKs

CD98 heavy chain (CD98hc) is a multifunctional transmembrane spanning scaffolding protein whose extracellular domain binds with light chain amino acid transporters (Lats) to form the heterodimeric amino acid transporters (HATs). It also interacts with β1 and β3 integrins by its transmembrane and cytoplasmic domains. This interaction is proposed to be the mechanism whereby CD98 mediates cell survival and growth via currently undefined signaling pathways. In this study, we determined whether the critical function of CD98-dependent amino acid transport also plays a role in cell proliferation and defined the signaling pathways that mediate CD98-dependent proliferation of murine renal inner medullary collecting duct (IMCD) cells. We demonstrate that downregulating CD98hc expression resulted in IMCD cell death. Utilizing overexpression studies of CD98hc mutants that either lacked a cytoplasmic tail or were unable to bind to Lats we showed that CD98 increases serum-dependent cell proliferation by a mechanism that requires the CD98hc cytoplasmic tail. We further demonstrated that CD98-dependent amino acid transport increased renal tubular epithelial cell proliferation by a mechanism that does not require the CD98hc cytoplasmic tail. Both these mechanisms of increased renal tubular epithelial cell proliferation are mediated by Erk and p38 MAPK signaling. Although increased amino transport markedly activated mTor signaling, this pathway did not alter cell proliferation. Thus, these studies demonstrate that in IMCD cells, the cytoplasmic and extracellular domains of CD98hc regulate cell proliferation by distinct mechanisms that are mediated by common MAPK signaling pathways

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

A conserved 12-amino acid motif in Sall1 recruits the nucleosome remodeling and deacetylase corepressor complex.

Sall1 is a multi-zinc finger transcription factor that represses gene expression and regulates organogenesis. In this report, we further characterize the domain of Sall1 necessary for repression. We show that endogenous Sall1 binds to the nucleosome remodeling and deacetylase corepressor complex (NuRD) and confirm the functionality of the Sall1-associating macromolecular complex by showing that the complex possesses HDAC activity. NuRD is involved in global transcriptional repression and regulation of specific developmental processes. The mechanism by which sequence-specific DNA-binding proteins associate with NuRD is not well understood. We have identified a highly conserved 12-amino acid motif in the transcription factor Sall1 that is sufficient for the recruitment of NuRD. Single amino acid substitutions defined the critical amino acid peptide motif as RRKQXK-PXXF. This motif probably exhibits a more general role in regulating gene expression, since other proteins containing this domain, including all Sall family members and an unrelated zinc finger protein Ebfaz, mediate transcriptional repression and associate with NuRD. These results also have important implications for the pathogenesis of Townes-Brocks, a syndrome caused by SALL1 mutations

A conserved 12-amino acid motif in Sall1 recruits the nucleosome remodeling and deacetylase corepressor complex.

Sall1 is a multi-zinc finger transcription factor that represses gene expression and regulates organogenesis. In this report, we further characterize the domain of Sall1 necessary for repression. We show that endogenous Sall1 binds to the nucleosome remodeling and deacetylase corepressor complex (NuRD) and confirm the functionality of the Sall1-associating macromolecular complex by showing that the complex possesses HDAC activity. NuRD is involved in global transcriptional repression and regulation of specific developmental processes. The mechanism by which sequence-specific DNA-binding proteins associate with NuRD is not well understood. We have identified a highly conserved 12-amino acid motif in the transcription factor Sall1 that is sufficient for the recruitment of NuRD. Single amino acid substitutions defined the critical amino acid peptide motif as RRKQXK-PXXF. This motif probably exhibits a more general role in regulating gene expression, since other proteins containing this domain, including all Sall family members and an unrelated zinc finger protein Ebfaz, mediate transcriptional repression and associate with NuRD. These results also have important implications for the pathogenesis of Townes-Brocks, a syndrome caused by SALL1 mutations