AKI induces cell senescence in TECs. To test whether cellular senescence is a common event after kidney injury, we looked for 2 established hallmarks of senescence: an increase in activity of the enzyme senescence-associated β-galactosidase (SA-β-Gal), and a reduction in abundance of lamin B1 (LAMNB1) protein in the nuclear envelope (23). We used 3 mouse models of kidney injury: folic acid–induced (FA-induced) nephrotoxicity, ischemia/reperfusion injury (IRI), and cisplatin-induced (CP-induced) nephrotoxicity. We assessed senescence 28 days after the initial insult. In TECs of all 3 injury models, SA-β-Gal activity increased (Figure 1, A–F) and LAMNB1 levels decreased (Figure 1, G–L). These results suggest that TEC senescence is common to several forms of AKI.

Figure 1 AKI induces cell senescence in kidney tubular cells. (A–C) SA-β-Gal staining of kidneys in 3 mouse models of AKI (FA, IRI, and CP) 28 days after injury, compared with controls and relative quantification (D–F). Scale bars: 500 μm. (G–I) Representative immunofluorescence confocal images of LAMNB1 28 days after FA, IRI, and CP, and relative digital image analysis qualification of LAMNB1-positive cells (J–L). Scale bars: 20 μm. Data are presented as mean ± SD. P values were calculated with 2-tailed Student’s t test. Ten images per mouse. The numbers of experimental mice are indicated in each panel.

Tubular cells undergo senescence early after kidney injury. To further characterize the onset of cellular senescence in tubular cells after AKI, we took advantage of the p16-3MR–transgenic mouse line (24). These mice had been generated by cloning the gene encoding the trimodality reporter (3MR) fusion protein, which contains functional domains of a synthetic Renilla luciferase (LUC), monomeric red fluorescent protein (mRFP), and truncated herpes simplex virus 1 (HSV-1) thymidine kinase (HSV-TK) (25), in frame with the promoter of the tumor suppressor gene p16Ink4a, the expression of which is activated in senescent cells. mRFP permits flow cytometry–based sorting of the expressing cells from tissues, and HSV-TK allows their killing by administration of ganciclovir (GCV), a nucleoside analog that has a high affinity for TK from HSV but a low affinity for its mammalian counterpart (cellular TK). HSV-TK converts GCV into a toxic DNA chain terminator that induces fragmentation of mitochondrial DNA in nondividing senescent cells and causes death by apoptosis (26). For the experiments described below, we chose not to use either the CP or IRI injury model because CP induces DNA damage and senescence and IRI-induced damage is not confined to tubular cells. We therefore focused on the FA model, using a single injection of FA that is toxic to rodent TECs because it causes intraluminal precipitation of folate crystals and oxidative stress (27–29). By confocal immunofluorescence microscopy, we consistently observed expression of mRFP, in tubular segments mostly at the cortico-medullary junction (Figure 2A and Supplemental Video 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.125490DS1). This signal frequently colocalized with the proximal-tubule marker Lotus tetranoglobus lectin (LTL, Figure 2B) but not with the collecting-duct marker Dolichos biflorus agglutinin (DBA, not shown). Surprisingly, quantification of mRFP fluorescence in explanted kidneys (Figure 2C) showed that, in the context of FA injury, levels of senescence were significantly higher than in vehicle-injected controls as early as 3 days after FA injury, and that the levels increased slowly at later time points (Figure 2D). The appearance of senescent cells in the tubules between day 2 and day 3 after injury was confirmed by staining for SA-β-Gal activity (Supplemental Figure 1), indicating that TEC senescence is an early event after injury.

Figure 2 Cellular senescence in kidney tubular cells is an early event after AKI. (A) Confocal microscopy images of p16-3MR–transgenic kidney 14 days after FA injury (left) and vehicle-injected control (right). Scale bars: 2 mm. (B) Confocal microscopy image of a kidney section of p16-3MR–transgenic kidney 14 days after FA injury showing colocalization of mRFP-expressing cells with the proximal tubular marker LTL. Scale bar: 50 μm. (C) Epifluorescence images of explanted p16-3MR–transgenic kidneys 28 days after FA injury (upper) and control (lower) and (D) quantification at different time points after FA injury. n = 7 mice per group per time point. All data are presented as mean ± SD. P values were calculated with 2-tailed Student’s t test.

Epithelial cell–specific deletion of Myd88 reduces expression of proinflammatory cytokines after FA injury. Senescent cells express many proinflammatory genes as part of the SASP and whose appearance and persistence are largely controlled by NF-κB (14–16, 23). IL-1R is an important mediator of the paracrine effects of the SASP and activates NF-κB by recruiting the adapter protein Myd88 (30). Myd88-dependent activation of NF-κB is also key to signaling downstream of the TLRs (31, 32). The roles of TLR signaling and Myd88 as mediators of inflammation have been extensively studied in several pathological contexts, including AKI, where this signaling axis controls the migration of pericytes, their conversion to myofibroblasts, and the secretion of proinflammatory cytokines from the latter (33–35). However, TLRs 1–4 are also constitutively expressed in both the proximal and distal tubules, in the thick ascending limb of the loop of Henle, and in the collecting ducts, suggesting that epithelial TLR signaling could also contribute to the development of inflammation and fibrosis after AKI (36). Because epithelial TLR2, TLR4, and Myd88 are upregulated in animal models of AKI (36, 37), we hypothesized that activation of TLR/IL-1R signaling in TECs can sustain the tubule-derived SASP after injury.

To test this hypothesis, we used the FA model of injury in mice with kidney tubule–specific deletion of Myd88 (Ksp-CreMyd88fl/fl), in which expression of the Cre transgene is driven by the promoter of the tubule-specific gene cadherin 16 (Ksp) (38–41), and evaluated the inflammatory components of the SASP. Expression levels of the proinflammatory cytokines IL-1α, IL-1β, IL-6, TNF-α, and MCP-1 were lower in tubules isolated from Ksp-CreMyd88fl/fl versus Ksp-CreMyd88+/+ (control) mice (Figure 3, A–E), indicating that epithelial TLR/IL-1R signaling promotes the expression of proinflammatory cytokines after injury. Interestingly, the expression of Shh, a ligand that induces proliferation and fibrogenic commitment of mesenchymal progenitor cells (6), was also significantly reduced in the tubules of Ksp-CreMyd88fl/fl versus control Ksp-CreMyd88+/+ mice (Figure 3F).

Figure 3 Epithelial cell–specific deletion of Myd88 reduces expression of proinflammatory cytokines after FA injury. (A–E) Expression levels of IL-1α, IL-1β, IL-6, TNF-α, MCP-1, and (F) of the pericyte-activating ligand Shh in isolated tubular cells, measured by real-time quantitative PCR 28 days after FA injury in Ksp-CreMyd88fl/fl mice compared with Ksp-CreMyd88+/+ controls. The numbers of experimental mice are indicated in the corresponding panels. Data are presented as mean ± SD. P values were calculated with 2-tailed Student’s t test.

Senescent tubular cells are a source of profibrotic Hh ligands. Shh and its paralog Ihh are ligands in the Hh signaling pathway. The activity of this pathway is required for cell-fate specification during embryonic development, as well as for the homeostasis of some adult organs, regulating the survival and proliferation of tissue progenitors and stem-cell populations (42). During development, the Hh pathway plays critical roles in kidney morphogenesis, with Shh secreted by the ureteric bud epithelium being essential for the survival, proliferation, and differentiation of the metanephric mesenchyme (43, 44). After kidney development is complete, no Hh ligands are expressed in the epithelial compartment (45). Because tubular cells in the adult kidney do not express Hh-pathway ligands under normal conditions, we tested the possibility that senescent TECs are the source of these proteins following kidney injury. To this end, we isolated mRFP+ and mRFP– cells from the kidneys of p16-3MR mice by flow cytometry and used quantitative real-time PCR to measure the expression of Shh and Ihh in each population. Interestingly, the expression of both of these ligands was significantly higher in the mRFP+ cells (Figure 4), indicating that senescent TECs are a source of profibrotic Hh-pathway ligands after kidney injury.

Figure 4 Senescent tubular cells are a source of profibrotic Hedgehog ligands. (A and B) Relative expression enrichment of Shh and Ihh in mRFP+ cells compared with mRFP– cells from the same kidneys 14 days after FA injury. The numbers of experimental mice are indicated in the corresponding panels. Data are presented as mean ± SD. P values were calculated with 2-tailed Student’s t test.

Epithelial cell–specific deletion of Myd88 reduces fibrosis and tubular damage after injury. Concordant with the lower expression of cytokines in Myd88-deficient TECs, we also found that kidney fibrosis (Figure 5, A–C) and interstitial infiltration were lower in Ksp-CreMyd88fl/fl versus control mice, as estimated by digital analysis of confocal immunofluorescence images obtained using antibodies that recognize markers of fibroblasts (fibroblast-specific protein 1, i.e., FSP1+; Figure 5, D and E), myofibroblasts (α smooth actin, i.e., α-SMA+; Figure 5, F and G), or macrophages (EGF-like module-containing mucin-like hormone receptor–like 1, i.e., F4/80+; Figure 5, H and I). Kidney damage, quantified based on tubular injury scores of PAS-stained kidney sections assigned by a pathologist, was also significantly lower in Ksp-CreMyd88fl/fl versus control mice 28 days after injury (Figure 5, J and K), indicating that genetic deletion of Myd88 before FA-induced injury is sufficient to protect kidneys from damage. This conclusion was also supported by the reduced mortality of Ksp-CreMyd88fl/fl but not control mice during the first 10 days after injury (Figure 5L). This effect on mortality is likely a consequence of injury being milder in the context of the Myd88 deficiency, as suggested by the lower levels of serum creatinine 2 days after FA administration (Figure 5M).

Figure 5 Epithelial cell–specific deletion of Myd88 reduces kidney damage and fibrosis. (A) Representative images of trichrome-stained kidneys at low (upper panels) and high (lower panels) magnification of Ksp-CreMyd88fl/fl mice compared with Ksp-CreMyd88+/+ controls 28 days after FA injury. Scale bars: 500 μm (upper) and 200 μm (lower). (B) Collagen/total protein content ratios and (C) real-time quantitative PCR of the collagen 1 mRNA levels in kidney cortexes of Ksp-CreMyd88fl/fl mice compared with controls 28 days after FA injury. (D) Representative immunofluorescence confocal images of Ksp-CreMyd88fl/fl mouse kidneys immunostained for the fibroblast marker FSP1 compared with controls 28 days after FA injury, and (E) corresponding quantification by digital image analysis. Scale bars: 20 μm. (F) Representative immunofluorescence confocal images of Ksp-CreMyd88fl/fl mouse kidney immunostained for the myofibroblast marker α-SMA compared with controls 28 days after FA injury, and (G) corresponding quantification by digital image analysis. Scale bars: 20 μm. (H) Representative immunofluorescence confocal images of Ksp-CreMyd88fl/fl mouse kidneys immunostained for the macrophage marker F4/80 compared with controls 28 days after FA injury, and (I) corresponding quantification by digital image analysis. Scale bars: 20 μm. (J) Representative images of PAS-stained kidneys at low (upper panels) and high (lower panels) magnification of Ksp-CreMyd88fl/fl mice compared with controls 28 days after FA injury. Scale bars: 500 μm (upper) and 200 μm (lower). (K) Tubular injury scores in Ksp-CreMyd88fl/fl mice compared with controls 28 days after FA injury. Experimental numbers of mice are reported in each panel. (L) Kaplan-Meier curves of Ksp-CreMyd88fl/fl mice compared with controls. Numbers of experimental mice indicated in the panel. (M) Serum creatinine measurements at different time points after FA injury of Ksp-CreMyd88fl/fl mice compared with controls. n = 7 mice per time point. Data are presented as mean ± SD. P values were calculated using the 2-tailed Student’s t test.

Epithelial cell–specific deletion of Myd88 reduces tubular cell senescence after injury. Given that TLR/IL-1–dependent activation of NF-κB is responsible for the paracrine effect of senescent cells (14) and senescent cells are able to induce secondary senescence in neighboring cells, we asked whether epithelial inactivation of Myd88 suppresses the spread of kidney senescence. Indeed, this was the case: tubular deletion of Myd88 led to a reduction in SA-β-Gal activity (Figure 6, A and B), as well as to increases in the abundance of nuclear LAMNB1 (Figure 6, C and D) and proliferating tubular cells (Figure 6, E and F) 28 days after injury. These results indicate that epithelial TLR/IL-1R signaling controls the onset of tubular senescence, and that the inhibition of this signaling is sufficient to limit the accumulation of senescent tubular cells after kidney injury.

Figure 6 Epithelial cell–specific deletion of Myd88 reduces senescence in tubular cells. (A) Representative images of kidneys stained for SA-β-Gal activity at low (upper panels) and high (lower panels) magnification of Ksp-CreMyd88fl/fl mice compared with Ksp-CreMyd88+/+ controls 28 days after FA injury, and (B) corresponding quantification by digital image analysis. Scale bars: 500 μm (upper) and 200 μm (lower). (C) Representative immunofluorescence confocal images of Ksp-CreMyd88fl/fl mouse kidneys probed with an antibody against LAMNB1 compared with controls 28 days after FA injury, and (D) corresponding quantification. Scale bars: 20 μm. (E) Representative immunofluorescence confocal images of Ksp-CreMyd88fl/fl mouse kidneys probed with an antibody against the proliferative marker Ki67 compared with controls 28 days after FA injury, and (F) corresponding quantification. Scale bars: 20 μm. Numbers of experimental mice are reported in each panel. Ten images per mouse. Data are presented as mean ± SD. P values were calculated with 2-tailed Student’s t test.

Inhibition of epithelial innate immunity after injury reduces tubular senescence and fibrosis but does not protect from tubular damage. Because reduced fibrosis, damage, and senescence in constitutive Myd88-knockout mice could be due to less severe injury ab initio, as suggested by the lower serum creatinine levels in Ksp-CreMyd88fl/fl mice 2 days after injury, we tested whether inhibiting Myd88 after senescence is initiated also limits the accumulation of tubular senescent cells. To this end, we used the FA model of injury in KspCre-ERT2Myd88fl/fl mice, in which Cre expression driven by the cadherin 16 promoter is induced upon tamoxifen injection (46). We activated Cre expression 5 days after FA injection, that is, after senescence was detectable in injured kidneys (Figure 2D and Supplemental Figure 1), and looked for the presence of senescent cells 28 days after injury. Deletion of Myd88 in tubular cells beginning 5 days after injury (Figure 7A) reduced the activity of SA-β-Gal (Figure 7, B and C) and increased the abundance of both nuclear LAMNB1 (Figure 7, D and E) and proliferating tubular cells (Figure 7, F and G). Consistent with these findings, interstitial infiltration and fibrosis were also decreased in KspCre-ERT2Myd88fl/fl versus control mice (Figure 8, A–I). However, Myd88 deletion did not confer a significant protective effect with respect to tubular damage (Figure 8, J and K), suggesting that inhibition of TLR/IL-1R signaling is effective at limiting senescence and fibrosis, but not tubular damage, after injury has occurred.

Figure 7 Epithelial cell–specific deletion of Myd88 after injury reduces senescence in tubular cells. (A) Schematic representation of the protocol for tamoxifen-induced expression of the Cre transgene 5 days after injury in KspCre-ERT2Myd88fl/fl mice. TMX, tamoxifen; PBS, phosphate-buffered saline. (B) Representative images of kidneys stained for SA-β-Gal activity at low (upper panels) and high (lower panels) magnification of KspCre-ERT2Myd88fl/fl mice compared with controls (KspCre-ERT2Myd88+/+) 28 days after FA injury, and (C) corresponding digital image analysis. Scale bars: 500 μm (upper) and 200 μm (lower). (D) Representative immunofluorescence confocal images of KspCre-ERT2Myd88fl/fl mouse kidneys probed with an antibody against LAMNB1 compared with controls 28 days after FA injury, and (E) corresponding quantification. Scale bars: 20 μm. (F) Representative immunofluorescence confocal images of KspCre-ERT2Myd88fl/fl mouse kidneys probed with an antibody against the proliferation marker Ki67 compared with controls 28 days after FA injury, and (G) corresponding quantification. Scale bars: 20 μm. Numbers of experimental mice are reported in each panel. Ten images per mouse. Data are presented as mean ± SD. P values were calculated with 2-tailed Student’s t test.

Figure 8 Epithelial cell–specific deletion of Myd88 after injury reduces fibrosis but does not protect from tubular damage. (A) Representative images of trichrome-stained kidneys at low (upper panels) and high (lower panels) magnification of KspCre-ERT2Myd88fl/fl mice compared with KspCre-ERT2Myd88+/+ controls 28 days after FA injury. Scale bars: 500 μm (upper) and 200 μm (lower). (B) Collagen/total protein content ratios and (C) real-time quantitative PCR of the collagen 1 mRNA levels in kidney cortexes of KspCre-ERT2Myd88fl/fl mice compared with controls 28 days after FA injury. (D) Representative immunofluorescence confocal images of KspCre-ERT2Myd88fl/fl mouse kidneys immunostained for the fibroblast marker FSP1 compared with controls 28 days after FA injury, and (E) corresponding quantification by digital image analysis. Scale bars: 20 μm. (F) Representative immunofluorescence confocal images of KspCre-ERT2Myd88fl/fl mouse kidneys immunostained for the myofibroblast marker α-SMA compared with controls 28 days after FA injury, and (G) corresponding quantification by digital image analysis. Scale bars: 20 μm. (H) Representative immunofluorescence confocal images of KspCre-ERT2Myd88fl/fl mouse kidneys immunostained for the macrophage marker F4/80 compared with controls 28 days after FA injury and (I) corresponding quantification by digital image analysis. Scale bars are 20 μm. (J) Representative images of PAS-stained kidneys at low (upper panels) and high (lower panels) magnification of KspCre-ERT2Myd88fl/fl mice compared with controls 28 days after FA injury. Scale bars: 500 μm (upper) and 200 μm (lower). (K) Tubular injury scores for KspCre-ERT2Myd88fl/fl mice compared with controls 28 days after FA injury. Experimental numbers of mice are reported in each panel. Data are presented as mean ± SD. P values were calculated using the 2-tailed Student’s t test.

Genetic and pharmacologic elimination of senescent cells after FA injury partially protects against fibrosis but does not affect tubular damage. The above-described experiments in FA-injured KspCre-ERT2Myd88fl/fl mice indicate that limiting the tubule-derived SASP is sufficient to prevent kidney inflammation and fibrosis after FA injury. To test whether the elimination of senescent cells from kidneys after injury prevents the progression of kidney damage and fibrosis, we selectively induced apoptosis in HSV-TK-p16Ink4A–expressing senescent cells of p16-3MR mice 5 days after FA injury, injecting GCV daily for 10 days (Figure 9A) (17). An increase in the number of apoptotic cells (positive for activated caspase-3 and TUNEL staining) 10 days after the first GCV injection confirmed that cell death was efficiently induced (Supplemental Figure 2, A–D). Consistent with these findings, TEC senescence (assessed based on SA-β-Gal activity and nuclear LAMNB1 abundance) declined significantly (Supplemental Figure 2, E–H) and cell proliferation increased (Supplemental Figure 2, I and J) 28 days after injury. In addition, fibrosis (Figure 9, B–D), as well as interstitial infiltration of fibroblasts (FSP-1+; Figure 9, E and F), macrophages (F4/80+; Figure 9, G and H), and myofibroblasts (α-SMA+; Figure 9, I and J), were reduced in GCV-treated versus vehicle-treated (control) mice. However, no significant difference in tubular damage was observed (Figure 9, K and L).

Figure 9 Genetic clearance of senescent cells ameliorates interstitial fibrosis but not tubular damage after AKI. (A) Schematic representation of the protocol for inducing clearance of senescent cells by ganciclovir administration to FA-injured p16-3MR mice. GCV, ganciclovir; PBS, phosphate-buffered saline. (B) Representative images of trichrome-stained kidneys at low (upper panels) and high (lower panels) magnification of GCV-treated mice compared with PBS-treated controls 28 days after FA injury. Scale bars: 500 μm (upper) and 200 μm (lower). (C) Collagen/total protein content ratios and (D) real-time quantitative PCR of the collagen 1 mRNA levels in kidney cortexes of GCV-treated and vehicle-treated mice 28 days after FA injury. (E) Representative immunofluorescence confocal images of GCV-treated mouse kidneys immunostained for the fibroblast marker FSP1 compared with controls 28 days after FA injury, and (F) corresponding quantification by digital image analysis. Scale bars: 20 μm. (G) Representative immunofluorescence confocal images of GCV-treated mouse kidneys immunostained for the macrophage marker F4/80 compared with controls 28 days after FA injury, and (H) corresponding quantification by digital image analysis. Scale bars: 20 μm. (I) Representative immunofluorescence confocal images of GCV-treated mouse kidneys immunostained for the myofibroblast marker α-SMA compared with controls 28 days after FA injury, and (J) corresponding quantification by digital image analysis. Scale bars: 20 μm. (K) Representative images of PAS-stained kidneys at low (upper panels) and high (lower panels) magnification of GCV-treated mice compared with controls 28 days after FA injury. Scale bars: 500 μm (upper) and 200 μm (lower). (L) Tubular injury scores of GCV-treated mice compared with controls 28 days after FA injury. Experimental numbers of mice are reported in each panel. Data are presented as mean ± SD. P values were calculated with 2-tailed Student’s t test.

Given the apparent discrepancy between the effects on fibrosis and tubular damage in the above-described experiments, we eliminated senescent cells using a second approach, applying the synthetic peptide FOXO4-DRI, which induces apoptosis of senescent cells by disrupting the interaction between FOXO4 and p53 (Supplemental Figure 3A) (24, 47). FOXO4-DRI was as effective as GCV in both inducing apoptosis (Supplemental Figure 3, B–E) and reducing the number of senescent cells (Supplemental Figure 3, F–K) within 28 days of injury. However, treatment with FOXO4-DRI did not lead to reduced interstitial fibrosis (Supplemental Figure 4, A–C), cellular infiltration generally (reduction of only the number of FSP1+ fibroblasts) (Supplemental Figure 4, D–I), or tubular damage (Supplemental Figure 4, J and K). Overall, these results suggest that eliminating senescent cells at early stages after injury may have beneficial effects with regard to inflammation and fibrosis, but that this does not protect against tubular damage regardless of whether the actions are early or late. They also suggest that GCV and FOXO4-DRI might target distinct subpopulations of senescent cells.