Revealing the Nephrogenic Network: The Role of Estrogen Signaling and Estrogen Related Receptor Gamma in Kidney and Multiciliated Cell Development

Kidney disease is a prevalent issue across the United States, with one in seven adults diagnosed with chronic kidney disease. As disease states progress, treatment options are limited to dialysis or transplant, both of which are associated with a host of risks. Growing our understanding of the genetic and molecular cues governing the formation of kidney functional units, known as nephrons, is imperative in the quest to develop innovative and more personalized treatment options. The zebrafish model is an excellent platform to investigate nephron development, as upwards of 70% of genes as well as nephron structure and function are conserved with humans. Using this tractable model, our lab conducted a high-throughput biochemical screen, and identified 17β-estradiol (E2), which is the dominant form of estrogen in vertebrates, as a potential regulator of nephrogenesis. Here, we report that exogenous estrogen treatment results in alterations to distal nephron segmentation. Specifically, E2 treatment causes an increase in the distal early segment and a coordinated decrease in the neighboring distal late domain without changing cell dynamics in this region. Similarly, treatment with xenoestrogens ethinylestradiol and genistein resulted in the same phenotypes. An additional screen of selective estrogen receptor modulators revealed that estrogen receptor 2 (Esr2) was responsible for these segment changes, as pan-antagonism of Esr2 with PHTPP resulted in the opposite phenotypes. Knockdown of Esr2 analog, esr2b, phenocopied PHTPP treatment, further suggesting that Esr2b was the main driver of estrogen signaling in the nephron. Finally, chemical or genetic inhibition of E2 signaling resulted in decreased expression of transcription factor irx3b and its target irx1a, which are both essential for distal segment formation. Together, these data reveal that estrogen is essential for distal nephron cell fate during nephrogenesis. While additional studies are required to determine if estrogen signaling plays a similar role in kidney disease states, this study provides valuable insight to our understanding of hormone function during organogenesis.

A subset of kidney diseases includes ciliopathies, which comprise disorders associated with aberrant function of essential organelles called cilia. Cilia found in the nephron are essential for mechano- and chemo-sensing. While kidney cells have immotile primary cilia, cells with multiple motile cilia (multiciliated cells, MCCs) have also been found in some disease states. The zebrafish pronephros is comprised of both MCCs and transporter cells with a single cilium, making them an ideal model to interrogate factors that affect ciliogenesis. In our study, we found that transcription factor ERRγ ortholog estrogen related receptor gamma a (esrrγa) is essential for renal cell fate choice and ciliogenesis in zebrafish. Deficiency of esrrγa altered nephron segmentation, as the proximal straight tubule increased in length, while the distal late domain and MCC populace decreased. Further, the effects of esrrγa was not sequestered to the kidney, as deficiency disrupted ciliogenesis in the nephron, Kupffer’s vesicle (early patterning organ), and otic vesicle (ear-like structure). These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued by prostaglandin E₂, PGE₂, or the cyclooxygenase enzyme Ptgs1. Further, genetic interaction revealed that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (ppargc1a), has a synergistic relationship with esrrγa upstream of prostaglandin signaling in the ciliogenic pathway. These ciliopathic phenotypes were also observed in mice lacking renal epithelial cell (REC) ERRγ, where significantly shorter cilia formed on proximal and distal tubule cells. Decreased cilia length preceded cyst formation in REC-ERRγ knockout mice, suggesting that altered cilia formation could be the first sign of pathogenesis in this model. These data position esrrγa as a key regulator of development at the nexus of ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with ppargc1a.