According to RNA-Seq results, further analyses were conducted to compare immune cell infiltrate and cytokine expression in WT and CalpTG mice. Immunohistochemistry evidenced that macrophage infiltration of the renal tissue increased with aging in WT mice but not in TG mice (Fig. 10A–C, p = 0.028). Since immune cell infiltrate was heterogeneous, tissue flow cytometry experiments have been performed in another set of aged and young CalpTG and WT mice to count immune cells and to identify whether macrophages polarization would be influenced by calpains. The proportion of CD45+ immune cells/prominin 1+ proximal tubular cells increased significantly with aging in WT mice but not in CalpTG mice (Fig. 10D–F, p < 0.05). The number of macrophages and lymphocytes infiltrating kidney tissue was significantly increased with aging in WT mice only (Fig. 10G,H, p < 0.05). The proportion of CD11c positive (M1) macrophages was similar in both groups, suggesting that calpain inhibition did not modify macrophages polarization (Fig. 10I,J). The proportion of M2 macrophages (CD206+) was too low to be quantified appropriately (not shown). At last, we confirmed that the expression of cytokines related to inflammasome activation and the main alarmins IL1-α or IL-1β increased with aging in kidneys of WT but not CalpTG mice (Fig. 10K,L, p < 0.05).Figure 10Calpastatin overexpression protects against kidney inflammaging. Kidney infiltration by F4/80+ macrophages was assessed first by immunohistochemistry, evidencing a significant increase with aging in WT mice only (A–C, *p < 0.05, n = 8/group, magnification ×400). To improve immune cell quantification, tissue flow cytometry has been performed in another set of experiments to assess the CD45+ (immune cells)/prominin1+ (proximal tubular cells) ratio, evidencing that immune cell infiltrate increased significantly in WT mice only (D–F, *p < 0.05, n = 4–6/group). The proportion of macrophages, including M1 CD11+ macrophages, and lymphocytes was significantly increased in old WT mice kidneys only (G,H,I *p < 0.05, n = 4–6/group). Nevertheless, the proportion of M1 macrophages was similar in both WT and CalpTG kidneys (J, n = 4–6/group). Il-1α expression was very faint in kidney tissue but significantly higher at 2 years in WT mice kidneys when compared to young mice kidneys (K, *p < 0.05, n = 6/group). Il-1β was quantitatively more expressed but its expression differed significantly only between aged WT and young CalpTG (TG) (L, *p < 0.05, n = 6/group).