Pharmacologic inhibition of RGD-binding integrins ameliorates fibrosis and improves function following kidney injury

Fibrosis is a final common pathway for many causes of progressive chronic kidney disease (CKD). Arginine-glycine-aspartic acid (RGD)-binding integrins are important mediators of the pro-fibrotic response by activating latent TGF-β at sites of injury and by providing myofibroblasts information about the composition and stiffness of the extracellular matrix. Therefore, blockade of RGD-binding integrins may have therapeutic potential for CKD. To test this idea, we used small-molecule peptidomimetics that potently inhibit a subset of RGD-binding integrins in a murine model of kidney fibrosis. Acute kidney injury leading to fibrosis was induced by administration of aristolochic acid. Continuous subcutaneous administration of CWHM-12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM-12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM-680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small-molecule antagonists is a promising therapeutic approach in fibrotic kidney disease

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

Whole mount expression pattern of <i>Six2</i>-cre in E12.5 kidney and stomach.

<p>(A) <i>Six2</i>-cre (GFP, green) is expressed in the cap metanephric mesenchyme (mm) surrounding the ureteric bud tips. Wnt9b expression and canonical Wnt signaling is highest in the ureteric bud which is labeled with cytokeratin (red). (B) In the stomach, <i>Six2</i>-cre is expressed in the hindstomach and pyloric region. Canonical Wnt signaling activity is downregulated in this region <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043098#pone.0043098-Kim1" target="_blank">[13]</a>.</p

Lef1 expression exhibits dose-dependent effects in the <i>Wnt9b</i> and stabilized β-catenin alleles.

<p>(A–C) Lef1 expression (green) is low in the self-renewing cap metanephric mesenchyme cells (mm) and upregulated in the differentiating renal vesicle structures (rv). NCAM expression is also increased as the cap mm differentiates, but is not expressed in the ureteric bud (ub). (D–I) <i>Wnt9b</i> transgenic kidneys have increased Lef1 staining which is further upregulated in the ectopic vesicles seen in the stabilized β-catenin allele. Notably, not all ectopic vesicles in the stabilized β-catenin allele are NCAM positive, suggesting abnormal specification of these differentiated structures.</p

Duodenogastric reflux and pyloric sphincter abnormalities in <i>Six2</i>-cre^tg/+, <i>Wnt9b</i>^tg/+ double heterozygotes.

<p>(A, B) Yellow amniotic fluid refluxed into the stomach of E18.5 double heterozygotes, but was properly retained in the intestine in control animals. A lack of constriction (arrow) and an increased diameter (inset) suggests that the reflux is due to a malformed pyloric sphincter. (C, D) Whole mount staining for <i>Nephrocan</i> mRNA delineates a thin band demarking the sphincter at E16.5 that is expanded in the double heterozygotes. (E, F) Smooth muscle actin staining in sections from control adult stomach demonstrates a thick muscular layer that leads to the sphincter. Double heterozygotes exhibit a thickened muscular layer; however it does not coalesce at the point of highest constriction. Vertical lines mark the transition from antral stomach (left side) to duodenum (right side) in E–H. (G, H) Alcian blue stains mucosal cells in the antral stomach in control sections and these cells are absent in the double heterozygotes. Mucosal cells in the intestine are stained normally. (I, J) Staining for the H⁺/K⁺ ATPase detects parietal cells in the forestomach that are excluded from the distal stomach. These cells are found throughout the distal stomach and pylorus in double heterozygotes. Sections depicted in panels I and J highlight the transition between forestomach and distal stomach (diagonal line) and do not include the duodenum.</p

Kidney cysts in double transgenics can be found throughout all segments of the nephron.

<p>(A–D) H&E staining of adult kidneys demonstrates numerous cystic dilatations in the cortex and outer medulla in <i>Six2-cre^tg/+</i>, <i>Wnt9b^tg/+</i> double transgenics and a normal kidney architecture in <i>Wnt9b^tg/+</i> controls. (E–H) Cysts were derived from proximal tubules (LTL-positive) and distal loop of Henle (THP-positive). DBA-positive and Aquaporin 2- positive collecting duct cysts were also identified in smaller number. Since <i>Six2</i>-cre is not expressed in the collecting duct, these cysts are produced through signaling from neighboring cells or secondary to dilatation of more proximal segments.</p

Pyloric gene expression exhibits dose-dependent effects in the <i>Wnt9b</i> and stabilized β-catenin alleles.

<p>Whole mount in situ analysis of <i>Grem1</i>, <i>Nkx2.5</i>, and <i>Gata3</i> expression in E12.5 stomachs. All three genes are restricted to the pyloric sphincter in wild-type stomachs (<i>Six2</i>-cre^tg/+). <i>Grem1</i> and <i>Gata3</i> show decreased expression in <i>Wnt9b</i> transgenics (<i>Six2-cre^tg/+</i>, <i>Wnt9b^tg/+</i>) that is further downregulated in the stabilized β-catenin allele (<i>Six2-cre^tg/+</i>, <i>Ctnnb1^ex3/+</i>). <i>Nkx2.5</i> expression is expanded into the stomach in <i>Wnt9b</i> transgenics and further upregulated in the stabilized β-catenin allele. <i>Nkx2.5</i> expression that can be seen at the upper right of the stomach is found in the spleen on the opposite side.</p