Aqp2 gene transcription is strongly regulated by vasopressin in the renal collecting duct. However, the transcription factors (TFs) responsible for regulation of expression of aquaporin-2 (Aqp2) remain largely unknown. We used Bayes' Theorem to integrate several -omics datasets to stratify the 1344 TFs present in the mouse genome with regard to probabilities of regulating Aqp2 gene transcription. Also, we carried out new RNA-Seq experiments mapping the time course of vasopressin-induced changes in the transcriptome of mpkCCD cells to identify TFs that change in tandem with Aqp2. The analysis identified 17 TFs out of 1344 that are most likely to be involved in regulation of Aqp2 gene transcription. These TFs included eight that have been proposed in prior studies to play a role in Aqp2 regulation, viz. Cebpb, Elf1, Elf3, Ets1, Jun, Junb, Nfkb1, and Sp1. The remaining nine represent new candidates for future studies (Atf1, Irf3, Klf5, Klf6, Mef2d, Nfyb, Nr2f6, Stat3, Nr4a1). Conspicuously absent is CREB (Creb1), which has been widely proposed to mediate vasopressin-induced regulation of Aqp2 gene transcription. Instead, another CREB-like TF, Atf1, ranked fourth among all transcription factors. RNA-Seq time course experiments showed a rapid increase in Aqp2 mRNA, within 3 hours of vasopressin exposure. This response was matched by an equally rapid increase in the abundance of the mRNA coding for Cebpb, which we have shown by ChIP-seq studies to bind downstream from the Aqp2 gene. The identified TFs provide a roadmap for future studies to understand regulation of Aqp2 gene expression.
Glaucoma is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is one of the major risk factors for glaucoma onset and progression, and available pharmaceutical interventions are exclusively targeted at IOP lowering. However, degeneration of retinal ganglion cells (RGCs) may continue to progress despite extensive lowering of IOP. A complementary strategy to IOP reduction is the use of neuroprotective agents that interrupt the process of cell death by mechanisms independent of IOP. Here, we describe an ion complexation approach for formulating microcrystals containing ~50% loading of a protein kinase inhibitor, sunitinib, to enhance survival of RGCs with subconjunctival injection. A single subconjunctival injection of sunitinib-pamoate complex (SPC) microcrystals provided 20 weeks of sustained retina drug levels, leading to neuroprotection in a rat model of optic nerve injury. Furthermore, subconjunctival injection of SPC microcrystals also led to therapeutic effects in a rat model of corneal neovascularization. Importantly, therapeutically relevant retina drug concentrations were achieved with subconjunctival injection of SPC microcrystals in pigs. For a chronic disease such as glaucoma, a formulation that provides sustained therapeutic effects to complement IOP lowering therapies could provide improved disease management and promote patient quality of life.
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