No abstract

Podocyte injury is the inciting event in primary glomerulopathies, such as minimal change disease and primary FSGS, and glucocorticoids remain the initial and often, the primary treatment of choice for these glomerulopathies. Because inflammation is not readily apparent in these diseases, understanding the direct effects of glucocorticoids on the podocyte, independent of the immunomodulatory effects, may lead to the identification of targets downstream of glucocorticoids that minimize toxicity without compromising efficacy. Several studies showed that treatment with glucocorticoids restores podocyte differentiation markers and normal ultrastructure and improves cell survival in murine podocytes. We previously determined that Krüppel-like factor 15 (KLF15), a kidney-enriched zinc finger transcription factor, is required for restoring podocyte differentiation markers in mice and human podocytes under cell stress. Here, we show that in vitro treatment with dexamethasone induced a rapid increase of KLF15 expression in human and murine podocytes and enhanced the affinity of glucocorticoid receptor binding to the promoter region of KLF15 In three independent proteinuric murine models, podocyte-specific loss of Klf15 abrogated dexamethasone-induced podocyte recovery. Furthermore, knockdown of KLF15 reduced cell survival and destabilized the actin cytoskeleton in differentiated human podocytes. Conversely, overexpression of KLF15 stabilized the actin cytoskeleton under cell stress in human podocytes. Finally, the level of KLF15 expression in the podocytes and glomeruli from human biopsy specimens correlated with glucocorticoid responsiveness in 35 patients with minimal change disease or primary FSGS. Thus, these studies identify the critical role of KLF15 in mediating the salutary effects of glucocorticoids in the podocyte.

Elucidating the molecular mechanisms that created ancient complex traits like insect wings is difficult. Fortunately, some complex traits have arisen recently. For example, hermaphroditic reproduction evolved independently many times during recent nematode evolution. Although C. elegans hermaphrodites require fog-2, which encodes an F box protein that regulates the translation of tra-2 mRNAs, the related species C. briggsae lacks fog-2. We identified a critical regulator of hermaphrodite development in C. briggsae, named she-1. Analysis of double mutants indicates that she-1 acts upstream of tra-2 in C. briggsae, just as fog-2 does in C. elegans. Molecular cloning shows that she-1 encodes a novel F box protein that was created by a recent gene duplication. Whereas FOG-2 acts through GLD-1 in C. elegans, SHE-1 does not bind GLD-1 in C. briggsae. Thus, both species recruited F box genes produced by recent duplication events into the sex-determination pathway to control hermaphrodite development, but these genes have distinct activities. This result implies that some gene families are more likely to give rise to novel regulatory genes than other families. Finally, we note that null mutations of she-1 are temperature sensitive, so C. briggsae might once have been a facultative hermaphrodite.

The Sox9 gene encodes a transcription factor that is critical for testis determination and chondrogenesis in vertebrates. Mutations in human SOX9 cause campomelic dysplasia, a dominant skeletal dysmorphology syndrome often associated with male to female sex reversal. Here we show that the Sox9a gene was duplicated during evolution of the rice field eel, Monopterus albus, a freshwater fish which undergoes natural sex reversal from female to male during its life, and has a haploid genome size (0.6-0.8 pg) that is among the smallest of the vertebrates. The duplicated copies of the gene (named Sox9al and Sox9a2) fit within the Sox9 clade of vertebrates, especially in the Sox9a subfamily, not in the Sox9b subfamily. They have similar structures as revealed by both genomic and cDNA analysis. Furthermore, both Sox9al and Sox9a2 are expressed in testis, ovary, and ovotestis; and specifically in the outer layer (mainly gonocytes) of gonadal epithelium with bipotential capacity to form testis or ovary, suggesting that they have similar roles in gonadal differentiation during sex reversal in this species. The closely related gene structure and expression patterns of the two sox9a genes in the rice field eel also suggest that they arose in recent gene duplication events during evolution of this fish lineage.

Morphologically distinct males and females are observed throughout the animal kingdom. Why and how sex evolved and is maintained in most living organisms remains a key question in cellular and evolutionary biology. Here we report that four isoforms of dmrt1 (dsx- and mab3-related transcription factor 1) are generated in testis, ovotestis, and ovary by alternative splicing in the rice field eel, a fresh water fish that undergoes natural sex reversal from female to male during its life cycle. These transcripts encode four different size proteins with 301, 196, 300, and 205 amino acids. Like fly doublesex splicing, the dmrt1 of the rice field eel is also alternatively spliced at the 3' region, which generates diverse isoforms in gonads by alternative use of 3' sequences. Not only is dmrt1 expressed specifically in gonads, but its multiple isoforms are differentially coexpressed in gonadal epithelium during gonad transformation. Expression levels of a and b isoforms of dmrt1 ranged from low to high (ovary < ovotestis I < ovotestis II < ovotestis III < testis), based on comparisons of mean values from real-time fluorescent quantitative reverse transcription-polymerase chain reaction analysis. The overall expression level of dmrt1 b was much lower than that of dmrt1 a. Expression of dmrt1 d was not only low, but it also did not change significantly during sex transformation. The differential expression of dmrt1 isoforms may also be regulated by their 3' untranslated regions (UTRs), although these 3' UTRs do not contribute to intracellular localization of the Dmrt1 protein. These results provide new insight into roles of regulation at the level of splicing of dmrt1 in governing the sex differentiation cascade.