Sodium retention is a hallmark of nephrotic syndrome (NS). Puromycin (PAN)-induced NS is associated with high aldosterone levels, increased ENaC expression and apical targeting. However, the mechanisms associated with increased apical targeting of ENaC in NS remain undefined and it is unclear whether this is secondary to high aldosterone levels and whether aldosterone and/or apical ENaC targeting are important for the development of sodium retention. This study aimed at uncovering 1) whether aldosterone is essential for sodium retention in PAN-induced NS, 2) whether ENaC expression or apical targeting is secondary to high aldosterone levels, and 3) to determine the role of aldosterone for dysregulation of sodium transporters in NS. Puromycin treatment of adrenalectomized rats supplemented with dexamethasone induced sodium retention in spite of the absence of aldosterone. Immunocytochemical analyses revealed absence of enhanced apical targeting of ENaC subunits in PAN treated adrenalectomized (ADX-PAN) rats with distribution of labelling similar to adrenalectomized dexamethasone-treated control rats (ADX). Moreover ENaC subunit abundance was increased in ADX-PAN rats. Abundance of Aquaporin 2 was unchanged whereas apical targeting was enhanced. Key sodium transporters were downregulated as previously observed in non-adrenalectomized puromycin-treated rats, whereas the expression of the alpha-1 subunit of the Na,K-ATPase was unchanged. In conclusion, PAN treatment in the absence of aldosterone induced sodium retention, increased ENaC expression but did not change the subcellular distribution of ENaC. This indicates that the previously observed enhanced apical targeting of ENaC in PAN induced NS is caused by aldosterone and that development of sodium retention can occur in the absence of aldosterone in nephrotic syndrome.