Transcription factor Six1 regulates expression of nephrogenic molecules to promote repair of kidney injury

To investigate the role and mechanism of sine oculis homeobox 1 (Six1), a nephrogenic transcription factor, in regulating injury repair after acute kidney injury (AKI).

C57BL/6J mice were inflicted with renal ischemia reperfusion (IR) injury to establish AKI model, and then the serum samples and kidney tissues were collected at days 1, 2, and 3 after modeling. Serum creatinine (Cr) and blood urea nitrogen (BUN) levels were measured and renal morphology was observed with HE staining. The expression and distribution of nephrogenic molecules (Six1 and Pax2, etc.) and cell proliferation/migration genes (Cyclin A1, MMP9, etc.) were detected with RT-PCR, Western blotting, and immunofluorescence assay and immunohistochemical assay. The expression of apoptosis pathway (Bcl2, Caspase3, etc.) and cell proliferation related molecules were evaluated in Six1 overexpression/knockout human epithelial cells (HK2) with CoCl₂-induced injury. Additionally, after the adeno-associated viruses carrying Six1 overexpression vector were used to overexpress the molecule in the mice, the expression of Six1 and other related molecules were detected after IR injury modeling.

After renal IR injury, Six1 was significantly activated in epithelial cells, the expression of nephrogenic and cell proliferation/migration molecules (Pax2, Cyclin A1, MMP9, etc.) was obviously up-regulated (P < 0.05), which was positively correlated with Six1 expression, while the proliferation/migration molecules (Ki67, MMP9) were also localized within the tubular epithelial cells. In cellular models of Six1 overexpression, the expression levels of nephrogenic and cell proliferation/migration molecules (Pax2, Cyclin A1, MMP9, etc.) were also notably up-regulated (P < 0.05). In the mice with renal overexpression of Six1, the nephrogenic molecules as well as anti-apoptotic ones (Six2, Pax2, Bcl2, etc.) were up-regulated, while the expression of kidney injury-related molecule (Kim1) in kidneys was reduced in the renal tissues. While, in 2 d after IR injury, the anti-apoptotic gene (Bcl2, Stat3) was significantly up-regulated (P < 0.05), and the apoptotic and injury molecules (Kim1, Caspase3) showed remarkable down-regulation (P < 0.05) in the mice with renal Six1 overexpression. Furthermore, CoCl₂-inducion significantly decreased the cell proliferation rate in the Six1 knockout group (TCMK1-Six1^-/-) (P < 0.05) but increased the rate in the Six1 overexpression group (TCMK1-Six1) when compared to the control cells (P < 0.05). And, the expression of nephrogenic, anti-apoptotic pathways and cell proliferation/migration molecules (Pax2, Bcl2, Cyclin A1, MMP9, etc.) were reduced in TCMK1-Six1^-/- group, and apoptosis and kidney injury molecules (Caspase3, Kim1) were significantly down-regulated in TCMK1-Six1 group (P < 0.05).

Activation of Six1 after AKI can promote the proliferation/migration of renal tubular epithelial cells by up-regulating nephrogenic molecules and inducing anti-apoptotic pathway molecules, and then, participate in IR-induced renal injury repair.