Purpose: Mutation of the klotho gene in mice elicits a syndrome resembling accelerated human aging. However, there is limited evidence for the role of Klotho in the kidney. We conducted a comparative proteome analysis of wild-type (WT) and klotho-knockout (kl(-/-)) mouse kidneys to identify proteins involved in Klotho deficiency.Experimental design: MALDI imaging MS (MALDI-IMS) of frozen kidney sections from 7-wk-old male WT and kl(-/-) mice was used to determine genotype-specific differences in the MS distribution. Proteins uniquely distributed in kl-/-kidneys were identified by subsequent analysis of adjacent trypsinized sections by MALDI-IMS in combination with LC-MS/MS. Immunohistochemistry and western blotting were adopted in qualitative and quantitation analysis.Results: Ninety-seven and 69 proteins identified by LC-MS/MS were matched to the MALDI-IMS spectra in WT and kl(-/-) mouse kidneys, respectively. Among protein types matched, nucleic acid binding proteins were most abundant, followed by enzymes. We identified secretogranin-1 (SCG1), which was predominately distributed in the glomeruli and renal tubules of kl(-/-) mouse kidneys. Immunohistochemistry for SCG1 mirrored images of MALDI-IMS.Conclusions: SCG1 may be a candidate protein involved in Klotho deficiency. Although further research is needed to investigate the role of SCG1 in the kidney, we show the usefulness of MALDI-IMS combined with LC-MS/MS.