Klotho is involved in epithelial to mesenchymal transition, cancer metastasis and regulation of endothelial function. Klotho hypomorphic mice (kl/kl) show increased levels of plasminogen activator-1 (Pai-1) and downregulation of endothelial nitric oxide synthase. NO is in turn known to interfere with Tgf-beta signaling thus inhibiting Alk5-Smad2 activity. The kl/kl mice further suffer from volume depletion leading to hyperaldosteronism. The present study explored the effects of the aldosterone antagonist spironolactone on NO/Tgf beta/ Alk5-Smad2 and Pai-1 in kl/kl mice. The spironolactone treatment mitigated the reduction of eNos expression and decreased nitrate tissue levels in kidneys of kl/kl mice. Tgf-beta plasma levels were significantly increased in kl/kl mice but were not significantly decreased by spironolacton treatment. Despite increased Tgf-beta levels, Alk5 mRNA expression and Smad2 phosphorylation were significantly decreased by spironolactone treatment. Furthermore, the increased expression of Runx2 and Pai1 was significantly downregulated by spironolactone treatment of kl/kl mice. In conclusion, increased tgf-beta activity in kl/kl mice is partly spironolactone sensitive. Tgf-beta contributes to epithelial to mesenchymal transition and could contribute to the strong phenotype of premature aging and soft tissue calcification in kl/kl mice. The increased levels of aldosterone in these mice amplify the deranged Tgf-beta1 - Pai-1 signalling, which can be mitigated by spironolactone treatment.