KLF4 in Macrophages Attenuates TNF α-Mediated Kidney Injury and Fibrosis

Abstract

Background: Polarized macrophage populations can orchestrate both inflammation of the kidney and tissue repair during CKD. Proinflammatory M1 macrophages initiate kidney injury, but mechanisms through which persistent M1-dependent kidney damage culminates in fibrosis require elucidation. Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor that suppresses inflammatory signals, is an essential regulator of macrophage polarization in adipose tissues, but the effect of myeloid KLF4 on CKD progression is unknown.

Methods: We used conditional mutant mice lacking KLF4 or TNFα (KLF4's downstream effector) selectively in myeloid cells to investigate macrophage KLF4's role in modulating CKD progression in two models of CKD that feature robust macrophage accumulation, nephrotoxic serum nephritis, and unilateral ureteral obstruction.

Results: In these murine CKD models, KLF4 deficiency in macrophages infiltrating the kidney augmented their M1 polarization and exacerbated glomerular matrix deposition and tubular epithelial damage. During the induced injury in these models, macrophage-specific KLF4 deletion also exacerbated kidney fibrosis, with increased levels of collagen 1 and α-smooth muscle actin in the injured kidney. CD11b⁺Ly6C^hi myeloid cells isolated from injured kidneys expressed higher levels of TNFα mRNA versus wild-type controls. In turn, mice bearing macrophage-specific deletion of TNFα exhibited decreased glomerular and tubular damage and attenuated kidney fibrosis in the models. Moreover, treatment with the TNF receptor-1 inhibitor R-7050 during nephrotoxic serum nephritis reduced damage, fibrosis, and necroptosis in wild-type mice and mice with KLF4-deficient macrophages, and abrogated the differences between the two groups in these parameters.

Conclusions: These data indicate that macrophage KLF4 ameliorates CKD by mitigating TNF-dependent injury and fibrosis.

Keywords: KLF4; macrophages; necroptosis; renal fibrosis.

Graphical abstract

Figure 1.

LysM-expressing myeloid cells accumulate in kidney during NTN. (A) Representative images of kidney sections from mT/mG and LysM-Cre⁺ mT/mG mice at day 14 of NTN. (B) Representative images of KLF4 flox and Lysz-Cre determination. (C) mRNA expression for KLF4 in isolated T cells, B cells, CD11b⁺ splenic macrophages (Macro), and in whole kidney and heart from WT and KLF4 MKO mice (n≥6 for each group). Scale bars, 100 μm. Data represent the mean±SEM. ***P<0.001.

Figure 2.

Myeloid KLF4 attenuates glomerular and tubular injury. (A and B) Renal pathology in WT and KLF4 MKO mice at day 14 of NTN. (A) Representative images of kidney sections from WT and KLF4 MKO mice with summary data of (B) glomerular matrix deposition (%), (C) BUN, (D) ACR, and (E) tubular injury scores. (F and G) Renal mRNA expression of (F) PAI1 and (G) NGAL (n=16 for WT and n=22 for KLF4 MKO in NTN). Scale bars, 50 μm. Data represent the mean±SEM. *P<0.05.

Figure 3.

KLF4 deficiency in macrophages exacerbates kidney fibrogenesis. (A–G) Renal fibrosis in WT and KLF4 MKO mice at day 14 of NTN. (A) Representative images of COLI immunostaining in kidney sections from WT and KLF4 MKO mice at day 14 of NTN. (B–D) mRNA expression of COLI, TGFβ, and fibronectin (FN) in WT and KLF4 MKO kidneys at day 14 of NTN. (E) Western blot for COLI and αSMA in whole kidney at day 14 of NTN. (F and G) Semiquantification of COLI and αSMA protein expression (n=3 for sham control and n=10 for WT/KLF4 MKO in NTN). (H–M) Renal fibrosis in WT and KLF4 MKO mice at day 7 of UUO. (H) Representative images of COLI immunostaining in kidney sections from WT and KLF4 MKO mice at day 7 of UUO. (I) Renal mRNA expression of COLI in WT and KLF4 MKO mice at day 7 of UUO (n=10 for WT and KLF4 MKO). (J) Western blot for COLI and αSMA in obstructed and contralateral control kidney at day 7 of UUO. (K and L) Semiquantification of (K) COLI and (L) αSMA protein expression (n=3 for contralateral control and n=10 for WT/KLF4 MKO in UUO). (M) Hydroxyproline content in obstructed WT and KLF4 MKO kidneys at day 7 of UUO (n=10 for WT and KLF4 MKO). Scale bars, 100 μm. Data represent the mean±SEM. *P<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4.

KLF4 in infiltrating Ly6C^hi myeloid cells suppresses their generation of TNFα. (A) Representative images of F4/80 immunostaining in kidney sections from WT and KLF4 MKO mice at day 14 of NTN. (B) Representative flow cytometry gating strategy for single-cell suspensions from NTN kidneys. (C) Number of CD11b⁺Ly6C^hi macrophages in kidneys from WT and KLF4 MKO mice at day 14 of NTN (n=8 for WT and n=6 for KLF4 MKO). (D) mRNA expression for TNFα, TGFβ, IFNγ, and IL1β in CD11b⁺Ly6C^hi macrophages sorted from NTN kidneys of WT and KLF4 MKO mice (n≥3 for each group). Scale bars, 50 μm. Data represent the mean±SEM. *P<0.05.

Figure 5.

TNFα in infiltrating myeloid cells aggravates nephrotoxic nephritis. (A and B) Renal pathology in WT and Lysz-Cre⁺ TNF^fl/fl (TNF MKO) mice at day 14 of NTN. (A) Representative images of kidney sections from WT and TNF MKO mice with summary of (B) glomerular matrix deposition (%), (C) BUN, (D) ACR, and (E) renal tubular injury scores in WT and TNF MKO mice. (F and G) Renal mRNA expression of (F) PAI1 and (G) NGAL (n=10 for WT and n=13 for TNF MKO in NTN). Scale bars, 50 μm. Data represent the mean±SEM. *P<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PAS, Periodic acid–Schiff.

Figure 6.

TNF derived from macrophages contributes to renal fibrogenesis. (A–E) Renal fibrosis in WT and TNF MKO mice at day 14 of NTN. (A) Representative images of COLI immunostaining in kidney sections from WT and TNF MKO mice at day 14 of NTN. (B) Renal mRNA expression for COLI in WT and TNF MKO mice at day 14 of NTN (n=10 for WT and n=13 for TNF MKO). (C) Western blot for COLI and αSMA in whole kidney at day 14 of NTN. (D and E) Semiquantification of COLI and αSMA protein expression (n=3 for sham control and n=10 for WT/TNF MKO in NTN). (F) Representative images of COLI immunostaining in kidney sections from WT and TNF MKO mice at day 7 of UUO. (G) Renal mRNA expression of COLI in WT and TNF MKO mice at day 7 of UUO (n=7 for WT and n=10 for TNF MKO in UUO). (H) Western blot for COLI and αSMA in obstructed and contralateral control kidney at day 7 of UUO. (I and J) Semiquantification of (I) COLI and (J) αSMA protein expression (n=3 for contralateral control, n=7 for WT, and n=10 for TNF MKO in UUO). (K) Hydroxyproline content in obstructed WT and TNF MKO kidneys at day 7 of UUO (n=7 for WT and n=10 for TNF MKO in UUO). Scale bars, 100 μm. Data represent the mean±SEM. *P<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 7.

Augmented renal fibrosis and necroptosis in KLF4 MKO mice is abrogated by TNFα receptor inhibition during NTN. (A) BUN in WT and KLF4 MKO mice with/without R-7050 treatment during NTN. (B and C) Renal mRNA levels of (B) PAI1 and (C) COLI in WT and KLF4 MKO mice with/without R-7050 treatment. (D) Western blot for COLI and αSMA in whole kidney at day 14 of NTN. (E and F) Semiquantification of (E) COLI and (F) αSMA protein expression. (J) Representative images of TUNEL staining in kidney sections from WT and KLF4 MKO mice at day 14 of NTN. (K) Semiquantification of TUNEL-positive cells per high-powered field (n=10 for WT and KLF4 MKO in NTN alone, n=6 for WT and KLF4 MKO in NTN+R-7050). Scale bars, 50 μm. Data represent the mean±SEM. *P<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pMLKL, phosphorylated MLKL; pRIPK3, phosphorylated RIPK3.