Hyalinosis is a vascular lesion affecting the renal vasculature and contributing to ageing-related renal function decline. We assessed whether arteriolar hyalinosis is caused by Klotho deficiency - a state known to induce both renal and vascular phenotypes associated with ageing. Histochemistry was used to assess hyalinosis in Klotho-/- kidneys, compared to Klotho+/- and wild-type littermates. Immunohistochemistry was used to investigate vascular lesion composition and the different layers of the vascular wall. Finally, spironolactone was used to inhibit calcification in kl/kl mice and vascular lesions were characterized in the kidney. Arteriolar hyalinosis was detected in Klotho-/- mice, which was present up to the afferent arterioles. Hyalinosis was accompanied by loss of α-smooth muscle actin expression, while the endothelial lining was mostly intact. Hyalinous lesions were positive for IgM and iC3b/c/d, indicating subendothelial leakage of plasma proteins. The presence of extracellular matrix proteins suggested increased production by smooth muscle cells. Finally, in Klotho-/- mice with marked vascular calcification, treatment with spironolactone allowed for replacement of calcification by hyalinosis. Klotho deficiency potentiates both endothelial hyperpermeability and smooth muscle cell de-differentiation. Absent a calcification-inducing stimulus, smooth muscle cells assume a synthetic phenotype in response to subendothelial leakage of plasma proteins. In the kidney, this results in arteriolar hyalinosis, which contributes to the decline in renal function. Klotho may play a role in preventing ageing-related arteriolar hyalinosis. Copyright © 2019. Published by Elsevier Inc.