Proinflammatory cytokines contribute to renal injury, but the downstream effectors within kidney cells are not well understood. One candidate effector is Klotho, a protein expressed by renal cells that has antiaging properties; Klotho-deficient mice have an accelerated aging-like phenotype, including vascular injury and renal injury. Whether proinflammatory cytokines, such as TNF and TNF-like weak inducer of apoptosis (TWEAK), modulate Klotho is unknown. In mice, exogenous administration of TWEAK decreased expression of Klotho in the kidney. In the setting of acute kidney injury induced by folic acid, the blockade or absence of TWEAK abrogated the injury-related decrease in renal and plasma Klotho levels. TWEAK, TNF␣, and siRNA-mediated knockdown of IB␣ all activated NFB and reduced Klotho expression in the MCT tubular cell line. Furthermore, inhibition of NFB with parthenolide prevented TWEAK-or TNF␣-induced downregulation of Klotho. Inhibition of histone deacetylase reversed TWEAKinduced downregulation of Klotho, and chromatin immunoprecipitation showed that TWEAK promotes RelA binding to the Klotho promoter, inducing its deacetylation. In conclusion, inflammatory cytokines, such as TWEAK and TNF␣, downregulate Klotho expression through an NFB-dependent mechanism. These results may partially explain the relationship between inflammation and diseases characterized by accelerated aging of organs, including CKD.

No abstract

The longevity-promoting NAD + -dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells.coactivator-associated arginine methyltransferase 1 | HuR | neural differentiation | embryonic stem cells S irtuin 1 (SIRT1) is an NAD + -dependent lysine deacetylase involved in multiple cellular events, including chromatin remodeling, transcriptional silencing, mitosis, stress responses, DNA repair, apoptosis, cell cycle, genomic stability, insulin regulation, and control of lifespan (see ref. 1 for a review). In mammals, SIRT1 function is mediated by its deacetylating activity not only on histone tails (mainly K16-H4 and K9-H3 positions; refs. 2-4), but also on key transcription factors such as p53 (p53), forkhead transcription factors (FOXO), p300 histone acetyltransferase, the tumor protein p73 (p73), E2F transcription factor 1 (E2F1), the DNA repair factor Ku antigen, the 70-kDa subunit (Ku70), the nuclear factor κ-B (NF-κB), and the androgen receptor (AR) (see ref. 1 for a review).Recent studies in mouse models suggest the importance of Sirt1 in stem cell differentiation. Sirt1 influences the neural and glial specification of neural precursors (5), regulates differentiation of skeletal myoblast (6), and inhibits spermatogenesis (7). Independently generated Sirt1-deficient mice are reported to exhibit severe neural defects, including exencephaly and disturbed neuroretinal morphogenesis (8, 9). In contrast to mice, in man the role of SIRT1 in human embryonic stem cell (hESC) differentiation is poorly understood. Here, we report a pathway that down-regulates SIRT1 during stem cell differentiation. In addition, we demonstrate that SIRT1 regulates the expression of specific developmental genes in pluripotent hESC and, thus, that its down-regulation is necessary for correct establishment of specific differentiation programs during stem cell differentiation. Results SIRT1 Is Down-Regulated During hESC Differentiation.To study the putative role of SIRT1 in hESC differentiation, we first measured SIRT1 mRNA levels during the course of in vitro differentiation of the hESC lines Shef-1 and H-181. Withdrawal of basic fibroblast growth fac...

Primary/secondary hyperoxalurias involve nephrocalcinosis-related chronic kidney disease (CKD) leading to end-stage kidney disease. Mechanistically, intrarenal calcium oxalate crystal deposition is thought to elicit inflammation, tubular injury and atrophy, involving the NLRP3 inflammasome. Here, we found that mice deficient in NLRP3 and ASC adaptor protein failed to develop nephrocalcinosis, compromising conclusions on nephrocalcinosis-related CKD. In contrast, hyperoxaluric wild-type mice developed profound nephrocalcinosis. NLRP3 inhibition using the β-hydroxybutyrate precursor 1,3-butanediol protected such mice from nephrocalcinosis-related CKD. Interestingly, the IL-1 inhibitor anakinra had no such effect, suggesting IL-1-independent functions of NLRP3. NLRP3 inhibition using 1,3-butanediol treatment induced a shift of infiltrating renal macrophages from pro-inflammatory (CD45F4/80CD11bCX3CR1CD206) and pro-fibrotic (CD45F4/80CD11bCX3CR1CD206TGFβ) to an anti-inflammatory (CD45F4/80CD11bCD206TGFβ) phenotype, and prevented renal fibrosis. Finally, in vitro studies with primary murine fibroblasts confirmed the non-redundant role of NLRP3 in the TGF-β signaling pathway for fibroblast activation and proliferation independent of the NLRP3 inflammasome complex formation. Thus, nephrocalcinosis-related CKD involves NLRP3 but not necessarily via intrarenal IL-1 release but rather via other biological functions including TGFR signaling and macrophage polarization. Hence, NLRP3 may be a promising therapeutic target in hyperoxaluria and nephrocalcinosis.

Studies of mitochondria-targeted nephroprotective agents suggest a key role of mitochondrial injury in AKI. Here we tested whether an improved perception of factors responsible for mitochondrial biogenesis may provide clues to novel therapeutic approaches to AKI. TWEAK is an inflammatory cytokine which is upregulated in AKI. Transcriptomic analysis of TWEAK-stimulated cultured murine tubular epithelial cells and folic acid-induced AKI in mice identified downregulation of peroxisome proliferator- activated receptor-γ coactivador-1α (PGC-1α) and its target genes (mitochondrial proteins Ndufs1, Sdha, and Tfam) as a shared feature. Neutralizing anti-TWEAK antibodies prevented the decrease in kidney PGC-1α and its targets during AKI. TWEAK stimulation decreased kidney PGC-1α expression in healthy mice and decreased expression of PGC-1α and its targets as well as mitochondrial membrane potential in cultured tubular cells. Adenoviral-mediated PGC-1α overexpression prevented TWEAK-induced downregulation of PGC-1α-dependent genes and the decrease in mitochondrial membrane potential. TWEAK promoted histone H3 deacetylation at the murine PGC-1α promoter. TWEAK-induced downregulation of PGC-1α was prevented by histone deacetylase or NF-κB inhibitors. Thus, TWEAK decreases PGC-1α and target gene expression in tubular cells in vivo and in vitro. Approaches that preserve mitochondrial function during kidney injury may be therapeutic for AKI.

No abstract

Renal inflammation has a key role in the onset and progression of immune- and nonimmune-mediated renal diseases. Therefore, the search for novel anti-inflammatory pharmacologic targets is of great interest in renal pathology. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) proteins, was previously found to preserve renal function in experimental polycystic kidney disease. We report here that JQ1-induced BET inhibition modulated the in vitro expression of genes involved in several biologic processes, including inflammation and immune responses. Gene silencing of BRD4, an important BET protein, and chromatin immunoprecipitation assays showed that JQ1 alters the direct association of BRD4 with acetylated histone-packaged promoters and reduces the transcription of proinflammatory genes (IL-6, CCL-2, and CCL-5). In vivo, JQ1 abrogated experimental renal inflammation in murine models of unilateral ureteral obstruction, antimembrane basal GN, and infusion of Angiotensin II. Notably, JQ1 downregulated the expression of several genes controlled by the NF-κB pathway, a key inflammatory signaling pathway. The RelA NF-κB subunit is activated by acetylation of lysine 310. In damaged kidneys and cytokine-stimulated renal cells, JQ1 reduced the nuclear levels of RelA NF-κB. Additionally, JQ1 dampened the activation of the Th17 immune response in experimental renal damage. Our results show that inhibition of BET proteins reduces renal inflammation by several mechanisms: chromatin remodeling in promoter regions of specific genes, blockade of NF-κB pathway activation, and modulation of the Th17 immune response. These results suggest that inhibitors of BET proteins could have important therapeutic applications in inflammatory renal diseases.

The role of MICA antibodies in acute heart allograft rejection was examined utilizing 190 pre-and posttransplant serum samples from 44 patients collected during the first year after transplantation. MICA antibodies were detected by CDC test on recombinant cell lines and by the newly developed Luminex MICA antibody detection assay. Additionally, MICA expression was analyzed by 'real time' RT-PCR and by immunohistochemistry in 10 endomyocardial biopsies. Only two subjects had HLA antibodies post-transplant. Nevertheless, MICA antibodies were found in a significant number of subjects. The prevalence of MICA antibodies was significantly higher among those with severe acute rejection (AR) than in those without rejection (60.7% vs. 14.3%, p = 0.0038 by CDC; 55.5% vs. 5.7%, p = 0.0020 by Luminex). In most cases, the appearance of MICA antibodies post-transplant precedes AR. Following transplantation, MICA up-regulation correlated with histological evidence of severe rejection. Monitoring for MICA antibodies post-transplant may be useful to establish new risk factors for acute rejection.