Iron deficiency-responsive element 1 (IDE1) and IDE2 are cis-acting elements that are responsible for Fe-deficiency-inducible and root-specific expression of the barley (Hordeum vulgare L.) gene IDS2 (Fe-deficiency-specific clone no. 2). Using these cis-acting elements, we aimed to construct super-promoters that would induce prominent gene expression in the roots of Fe-deficient rice plants (Oryza sativa L.). Modules containing IDE1 and IDE2 of the IDS2 promoter were used as repeats or were linked to the Fe-deficiency-responsive promoter of barley IDS3, and were connected to known enhancer-like sequences. Five artificial promoters, as well as the native promoters of barley IDS2 or IDS3, were connected individually upstream of β-glucuronidase (GUS) and were introduced into rice. Transgenic rice plants were grown under control or Fe-deficient conditions, and GUS expression was analyzed. The artificial promoter that contained one module of IDE1 and IDE2 conferred strong Fe-deficiency-inducible GUS expression to the roots of rice plants. Each of the five artificial promoters induced a similar level of GUS expression in Fe-deficient roots, which did not exceed the GUS expression driven by the native IDS2 or IDS3 promoter. Artificial and native promoters induced GUS expression in response to Fe-deficiency in leaves, although the level of expression was lower than that in roots. Histochemical observations revealed that GUS expression driven by artificial and native promoters was spatially similar, and expression was dominant within vascular bundles and root exodermis. These findings suggest that there is coordinated expression of the genes that are involved in Fe-deficiency-induced Fe uptake in rice.

Chronic kidney disease (CKD) is characterized by progressive glomerulosclerosis and interstitial fibrosis, ultimately leading to irreversible renal dysfunction. Tacrolimus, a calcineurin inhibitor widely used after kidney transplantation, is known to accelerate chronic renal injury, whereas everolimus has been introduced as a calcineurin inhibitor–sparing agent. However, their direct effects on renal fibrogenesis remain incompletely defined. In this study, we established a chronic renal failure model using 5/6 nephrectomized rats to evaluate tacrolimus-accelerated renal interstitial fibrosis and the impact of concomitant everolimus treatment. Low-dose tacrolimus (1 mg/kg every other day) administered for two weeks starting four weeks after nephrectomy markedly accelerated interstitial fibrotic progression, accompanied by histological deterioration and increased albuminuria. Co-administration of low-dose everolimus (1 mg/kg every other day) significantly attenuated tacrolimus-accelerated fibrosis and improved functional and structural renal parameters. Histological analyses demonstrated reduced interstitial fibrosis and glomerular alterations in the everolimus-treated group. Although α-smooth muscle actin expression was not significantly suppressed, everolimus restored autophagic activity, as indicated by normalization of the LC3-II/LC3-I ratio in both in vivo and in vitro experiments. These findings indicate that tacrolimus accelerates renal fibrogenesis in a remnant kidney model and that concomitant low-dose everolimus effectively mitigates this progression, providing a time- and cost-efficient experimental platform for evaluating antifibrotic strategies in CKD.