At present, there are no effective therapies to ameliorate injury, accelerate recovery, or prevent postinjury fibrosis after AKI. Here, we sought to identify candidate compounds that accelerate recovery after AKI by screening for small molecules that increase proliferation of renal progenitor cells in zebrafish embryos. One compound identified from this screen was the histone deacetylase inhibitor methyl-4-(phenylthio) butanoate, which we subsequently administered to zebrafish larvae and mice 24-48 hours after inducing AKI. In zebrafish, treatment with the compound increased larval survival and proliferation of renal tubular epithelial cells. In mice, treatment accelerated recovery, reduced postinjury tubular atrophy and interstitial fibrosis, and increased the regenerative capacity of actively cycling renal tubular cells by decreasing the number of cells in G2/M arrest. These data suggest that accelerating recovery may be a viable approach to treating AKI and provide proof of concept that a screen in zebrafish embryos can identify therapeutic candidates for kidney injury.

BackgroundThe primary site of damage during AKI, proximal tubular epithelial cells, are highly metabolically active, relying on fatty acids to meet their energy demands. These cells are rich in mitochondria and peroxisomes, the two organelles that mediate fatty acid oxidation. Emerging evidence shows that both fatty acid pathways are regulated by reversible posttranslational modifications, particularly by lysine acylation. Sirtuin 5 (Sirt5), which localizes to both mitochondria and peroxisomes, reverses post-translational lysine acylation on several enzymes involved in fatty acid oxidation. However, the role of the Sirt5 in regulating kidney energy metabolism has yet to be determined.MethodsWe subjected male Sirt5-deficient mice (either +/− or −/−) and wild-type controls, as well as isolated proximal tubule cells, to two different AKI models (ischemia-induced or cisplatin-induced AKI). We assessed kidney function and injury with standard techniques and measured fatty acid oxidation by the catabolism of 14C-labeled palmitate to 14CO2.ResultsSirt5 was highly expressed in proximal tubular epithelial cells. At baseline, Sirt5 knockout (Sirt5−/−) mice had modestly decreased mitochondrial function but significantly increased fatty acid oxidation, which was localized to the peroxisome. Although no overt kidney phenotype was observed in Sirt5−/− mice, Sirt5−/− mice had significantly improved kidney function and less tissue damage compared with controls after either ischemia-induced or cisplatin-induced AKI. This coincided with higher peroxisomal fatty acid oxidation compared with mitochondria fatty acid oxidation in the Sirt5−/− proximal tubular epithelial cells.ConclusionsOur findings indicate that Sirt5 regulates the balance of mitochondrial versus peroxisomal fatty acid oxidation in proximal tubular epithelial cells to protect against injury in AKI. This novel mechanism might be leveraged for developing AKI therapies.

Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.

Retinoic acid (RA) has been used therapeutically to reduce injury and fibrosis in models of AKI, but little is known about the regulation of this pathway and what role it has in regulating injury and repair after AKI. In these studies, we show that RA signaling is activated in mouse and zebrafish models of AKI, and that these responses limit the extent of injury and promote normal repair. These effects were mediated through a novel mechanism by which RA signaling coordinated the dynamic equilibrium of inflammatory M1 spectrum versus alternatively activated M2 spectrum macrophages. Our data suggest that locally synthesized RA represses proinflammatory macrophages, thereby reducing macrophage-dependent injury post-AKI, and activates RA signaling in injured tubular epithelium, which in turn promotes alternatively activated M2 spectrum macrophages. Because RA signaling has an essential role in kidney development but is repressed in the adult, these findings provide evidence of an embryonic signaling pathway that is reactivated after AKI and involved in reducing injury and enhancing repair.

Tachykinins (TKs) are the most prevalent vertebrate brain/gut peptides. In this study, we originally identified authentic TKs and their receptor from a protochordate, Ciona intestinalis. The Ciona TK (Ci-TK) precursor, like mammalian ␥-preprotachykinin A (␥-PPTA), encodes two TKs, Ci-TK-I and -II, including the -FXGLM-NH 2 vertebrate TK consensus. Mass spectrometry of the neural extract revealed the production of both Ci-TKs. Ci-TK-I contains several Substance P (SP)-typical amino acids, whereas a Thr is exceptionally located at position 4 from the C terminus of Ci-TK-II. The Ci-TK gene encodes both Ci-TKs in the same exon, indicating no alternative generation of Ci-TKs, unlike the PPTA gene. These results suggested that the alternative splicing of the PPTA gene was established during evolution of vertebrates. The only Ci-TK receptor, Ci-TK-R, was equivalently activated by Ci-TK-I, SP, and neurokinin A at physiological concentrations, whereas Ci-TK-II showed 100-fold less potent activity, indicating that the ligand selectivity of Ci-TK-R is distinct from those of vertebrate TK receptors. Ci-TK-I, like SP, also elicited the typical contraction on the guinea pig ileum. The Ci-TK gene was expressed in neurons of the brain ganglion, small cells in the intestine, and the zone 7 in the endostyle, which corresponds to the vertebrate thyroid gland. Furthermore, the Ci-TK-R mRNA was distributed in these three tissues plus the gonad. These results showed that Ci-TKs play major roles in sexual behavior and feeding in protochordates as brain/gut peptides and endocrine/paracrine molecules. Taken together, our data revealed the biochemical and structural origins of vertebrate TKs and their receptors.

Phenylthiobutanoic acids (PTBAs) are a new class of histone deacetylase (HDAC) inhibitors that accelerate recovery and reduce postinjury fibrosis after ischemia-reperfusion-induced acute kidney injury. However, unlike the more common scenario in which patients present with protracted and less clearly defined onset of renal injury, this model of acute kidney injury gives rise to a clearly defined injury that begins to resolve over a short period of time. In these studies, we show for the first time that treatment with the PTBA analog methyl-4-(phenylthio)butanoate (M4PTB) accelerates recovery and reduces postinjury fibrosis in a progressive model of acute kidney injury and renal fibrosis that occurs after aristolochic acid injection in mice. These effects are apparent when M4PTB treatment is delayed 4 days after the initiating injury and are associated with increased proliferation and decreased G2/M arrest of regenerating renal tubular epithelial cells. In addition, there is reduced peritubular macrophage infiltration and decreased expression of the macrophage chemokines CX3Cl1 and CCL2. Since macrophage infiltration plays a role in promoting kidney injury, and since renal tubular epithelial cells show defective repair and a marked increase in maladaptive G2/M arrest after aristolochic acid injury, these findings suggest M4PTB may be particularly beneficial in reducing injury and enhancing intrinsic cellular repair even when administered days after aristolochic acid ingestion.

The aged population is currently at its highest level in human history and is expected to increase further in the coming years. In humans, aging is accompanied by impaired angiogenesis, diminished blood flow and altered metabolism, among others. A cellular mechanism that impinges upon these manifestations of aging can be a suitable target for therapeutic intervention. Here we identify cell surface receptor CD47 as a novel age-sensitive driver of vascular and metabolic dysfunction. With the natural aging process, CD47 and its ligand thrombospondin-1 were increased, concurrent with a reduction of self-renewal transcription factors OCT4, SOX2, KLF4 and cMYC (OSKM) in arteries from aged wild-type mice and older human subjects compared to younger controls. These perturbations were prevented in arteries from aged CD47-null mice. Arterial endothelial cells isolated from aged wild-type mice displayed cellular exhaustion with decreased proliferation, migration and tube formation compared to cells from aged CD47-null mice. CD47 suppressed ex vivo sprouting, in vivo angiogenesis and skeletal muscle blood flow in aged wild-type mice. Treatment of arteries from older humans with a CD47 blocking antibody mitigated the age-related deterioration in angiogenesis. Finally, aged CD47-null mice were resistant to age- and diet-associated weight gain, glucose intolerance and insulin desensitization. These results indicate that the CD47-mediated signaling maladapts during aging to broadly impair endothelial self-renewal, angiogenesis, perfusion and glucose homeostasis. Our findings provide a strong rationale for therapeutically targeting CD47 to minimize these dysfunctions during aging.

The recent decoding of a number of animal genomes has provided unprecedented information regarding evolution and gene structures, but this information must be supplemented with precise gene annotations and the temporal and spatial expression patterns of individual genes. In the present study, we systematically identified and characterized 566 zinc finger genes in the genome of Ciona intestinalis, an emerging model system for genome-wide studies of development and evolution. Of these genes, 356 genes encoded a potential transcription factor based on putative nucleic acid binding activity or domains of unknown function. We further examined the expression patterns of 225 genes during embryogenesis, and, when considered with a previous study [Imai, K.S., Hino, K., Yagi, K., Satoh, N., Satou, Y., 2004. Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks. Development 131, 4047-4058], we have characterized the developmental expression patterns of nearly 85% of the potential zinc finger-containing transcription factors. Overall, zinc finger genes are preferentially maternally expressed with little larval expression during development. The present study provides a valuable reference for genome-wide studies in this species and for future studies wishing to examine zinc finger gene expression patterns in other animals.