As in osmoregulation, mineralocorticoid signaling is implicated in the control of brain-behavior actions. Nevertheless, the understanding of this role is limited, partly due to the mortality of mineralocorticoid receptor (MR)-knockout (KO) mice due to impaired Na+ reabsorption. In teleost fish, a distinct mineralocorticoid system has only been identified recently. Here, we generated a constitutive MR-KO medaka as the first adult-viable MR-KO animal, since MR expression is modest in osmoregulatory organs but high in the brain of adult medaka as for most teleosts. Hyper- and hypo-osmoregulation were normal in MR-KO medaka. When we studied the behavioral phenotypes based on the central MR localization, however, MR-KO medaka failed to track moving dots despite having an increase in acceleration of swimming. These findings reinforce previous results showing a minor role for mineralocorticoid signaling in fish osmoregulation, and provide the first convincing evidence that MR is required for normal locomotor activity in response to visual motion stimuli, but not for the recognition of these stimuli per se. We suggest that MR potentially integrates brain-behavioral and visual responses, which might be a conserved function of mineralocorticoid signaling through vertebrates. Importantly, this fish model allows for the possible identification of novel aspects of mineralocorticoid signaling.

To study the critical role of mineralocorticoid signalling, we generated a constitutive mineralocorticoid receptor (MR)-knockout (KO) medaka as the first adult-viable MR-KO animal. This KO medaka displayed abnormal behaviours affected by visual stimuli. In contrast, the loss of MR did not result in overt phenotypic changes in osmoregulation, despite the well-known osmoregulatory functions of MR in mammals. Since glucocorticoid receptor (GR) has been suggested to compensate for loss of MR, we examined expression of duplicated GRs with markedly different ligand sensitivities, in various tissues. qRT-PCR results revealed that the absence of MR induced GR1 in the brain and eyes, but not in osmoregulatory organs. This reinforces the important functions of glucocorticoid signalling, but the minor role of mineralocorticoid signalling, in fish osmoregulation. Because both 11-deoxycorticosterone (DOC) and cortisol are ligands for MR, whereas GRs are specific to cortisol, GR1 signalling may compensate for the absence of cortisol-MR, rather than that of DOC-MR. Thus, this GR expression suggests that our MR-KO model can be used specifically to characterize DOC-MR signalling.

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In vitro, the conditionally immortalized mouse podocytes (MPC5) were used to investigate the effects of Yi Qi Qing Re Gao-containing serum (YQ-S) on actin cytoskeleton reorganization. Podocytes injury were evaluated by released lactic dehydrogenase (LDH) activity assay. Podocytes apoptosis were determined by Annexin V assay. Wound healing assay and transwell migration assay were used to assess podocyte motility. Immunofluorescence was used to demonstrate the formation and distribution of stress fiber. Western Blot was performed to detect the expression of key proteins of RhoA/ROCK signaling pathway. Results: YQQRG significantly decreased urinary protein level, lowered serum lipid level, elevated serum albumin, and ameliorated renal function in Wistar rats, similar to the effects of CsA. Renal pathological lesions, especially podocytes effacement were obviously attenuated. The experiments in vivo and in vitro both displayed that YQQRG could downregulate the mRNA and protein expressions of a-actinin-4, synaptopodin, vimentin, desmin, and nestin in renal tissues and podocytes. Besides, YQ-S alleviated PAN-induced podocytes injury and apoptosis, and reduced podocyte motility and migration. Disruption of F-actin and disorganization of cytoskeleton were largely recovered by YQ-S. Furthermore, YQ-S significantly antagonized PAN-induced downregulation of the protein expression of RhoA and ROCK, decreased the phosphorylation of MLC, LIMK and cofilin, and increased F-actin/Gactin levels. Addition of Y-27632 (a Rho-kinase inhibitor) could weaken the effects of YQ-S on F-actin reorganization and expressions of ROCK, p-MLC, and p-LIMK. Conclusions: YQQRG alleviates actin cytoskeleton reorganization and attenuates podocytes injury in PAN model in vivo and in vitro, and its mechanism may involve the upregulation of RhoA/ROCK signaling pathway. These findings suggest that YQQRG might be a promising natural drug for the treatment of kidney diseases.