Sex as a biological variable. Our study examined male mice, because male animals exhibited less variability in phenotype.

Expression constructs. The expression constructs for V5-tagged hHIPK2 were previously described (3, 21). Flag-HIPK2-CT, V5-HIPK2-CT, GFP-HIPK2, GFP-HIPK2-CT, mCherry-p65, caspase-6, and cleaved caspase-6 (amino acids 193–294) were generated using a PCR-based standard procedure, and detailed information and primers used to generate cDNA for each expression constructs are described in the Supplemental Methods.

Cell culture and transfection. HEK293T cells were obtained from ATCC and cultured according to the manufacturer’s specifications in DMEM medium (Gibco) containing 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco). For transient transfection, HEK293T cells were transfected using PolyJet reagent (SignaGen, SL100688).

Luciferase reporter gene assays. HEK293T cells were cotransfected with pcDNA4B or HIPK2-CT, as well as Renilla expression vector to normalize transfection efficiency, and p65 luciferase reporter construct or Smad3-binding element luciferase reporter construct or p53 luciferase reporter construct (p65-luc construct was a gift from the laboratory of Huabao Xiong [Department of Medicine, Precision Immunology Institute, Icahn School of Medicine at Mount Sinai], ref. 28, and SBE-luc and p53-luc were obtained from Addgene). Twenty-four to 36 hours after transfection, cells were stimulated with 10 ng/mL TNF-α (p65), 10 ng/μL TGF-β1 (Smad3), or 0.5 μg/mL adriamycin (p53) for 16 hours. The luciferase assays were performed with the Dual-Luciferase Reporter Assay Kit (Promega, E1910).

Immunocytochemistry and confocal imaging. HEK293T cells cotransfected with GFP-HIPK2-WT or GFP-HIPK2-CT and mCherry-p65 plasmids were treated with or without 10 ng/mL TNF-α for 30 minutes. Cells were then fixed with freshly made 4% formaldehyde (pH 7.5) for 15 minutes at room temperature. After a brief wash, DNA was counterstained with DAPI, and slides were mounted with the anti-fade medium (Vectashield, H1200). Images were acquired using Zeiss LSM880 Airyscan confocal microscope (Carl Zeiss).

Western blot analysis. Protein lysate preparation and Western blot analysis were performed according to the standard protocol using 30–60 μg total protein. For densitometric analysis, the density for each target protein was normalized to GAPDH or β-actin. The following antibodies were used in this study: anti-V5 (GenScript, A01724), anti-FLAG (MilliporeSigma, F3165), p-p65 (Cell Signal Technology, 3033), p65 (Cell Signal Technology, 8242), p-IκB (Cell Signal Technology, 2859), IκB (Abcam, ab32518), β-actin (Millipore Sigma, A4700), and GAPDH (Cell Signal Technology, A2118).

Immunoprecipitation. Cells were washed in ice-cold PBS and lysed in lysis buffer (Thermo Fisher Scientific, 87788) supplemented with a protease inhibitor cocktail (MilliporeSigma, P8340). After incubating on ice for 15 minutes with periodic mixing, cell lysates were centrifuged, collected, and incubated with the following antibodies/reagents for immunoprecipitation: anti-p65 antibody (1:50; Santa Cruz, sc8008), anti-IκB antibody (1:50; Santa Cruz, sc1643), protein G–agarose, or anti-FLAG mAb magnetic beads (MilliporeSigma, M8823).

Mouse model. Human HIPK2-CT cDNA corresponding to NM_022740.5 was subcloned with a C-terminal HA-tag into the pTRE-Tight vector (Clontech, 631059) expressing the TRE. The sequence of the HIPK2-CT insert was confirmed by restriction endonuclease and DNA sequencing. The linearized DNA fragment of pTRE-Tight-hHIPK2-CT-HA was used for microinjection to generate the TRE-HIPK2-CT–transgenic mice in the FVB/NJ background. To generate tubular epithelial cell–specific overexpression of HIPK2-CT mice, TRE-HIPK2-CT–transgenic mice were crossed with Pax8-rtTA–transgenic mice, which express the reverse tetracycline-controlled trans-activator (rtTA) protein under the control of murine Pax8 promotor, which directs the expression in all renal tubular cells (19). Tubular cell–specific HIPK2-CT expression was induced by the administration of dox-supplemented chow (625 g/kg chow, Envigo).

The UUO model was created according to a previous protocol (20). Briefly, the left ureter of each mouse was exposed through a midline abdominal incision and ligated using 4.0 silk. Contralateral sham-operated kidneys (exposure of ureters, without ligation) were used as controls. All surgeries were performed under general anesthesia with isoflurane. To identify renal inflammation in acute injury and fibrosis in chronic kidney injury, mice were sacrificed 3 days and 14 days after UUO surgery. Kidneys were perfused in situ with PBS, and tissue samples from both kidneys were collected for histology and Western blot analysis.

For LPS-induced AKI, mice were administrated as described previously (29, 30). Age- and sex-matched WT and Pax8-HIPK2-CT mice received a single low dose of LPS (5 mg/kg) injection intraperitoneally. Twenty-four hours after injection, serum and samples were harvested and processed for BUN and immunostaining.

Mouse kidney histology. Kidney tissues were fixed in 10% formalin, embedded in paraffin, and sectioned to 4 μm thickness. Periodic acid–Schiff–stained and Sirius Red/Fast Green-stained kidney sections were used for kidney histology. Histological scoring was performed by the renal pathologists, with experimental groups and mouse genotypes masked. To evaluate tubular injury score, 10 random tissue sections (×20 magnification) per mouse were assessed for PAS staining and scored semiquantitatively as follows: 1, none; 1, <25%; 2, 25%–50%; 3, 50%–75%; 4, >75%. Results are presented as average for each mouse. To evaluate interstitial fibrosis, 10 tissue sections per mouse were obtained randomly (×20 magnification) and assessed after Sirius Red/Fast green staining (Chondrex, 9046). Tissue fibrosis, as defined by red staining, was scored, and the average values of the fibrosis scores are reported.

Immunohistochemical and immunofluorescence staining. Immunofluorescence staining was conducted on frozen sections using standard procedures using the anti-p65 antibody (Cell Signal Technology, 8242) or anti-F4/80 antibody (Abcam, ab6640), nuclei were counterstained with DAPI, and slides were mounted with anti-fade medium (Vectashield, H1200).

For immunohistochemical staining, deparaffinized kidney sections were incubated with anti-F4/80 antibody (1:100) and processed using the Vectastain Elite ABC kit (Vector Laboratories, PK-6100). Images were acquired using an AxioVision II microscope with a digital camera (Carl Zeiss).

Quantitative real-time PCR assay. The total RNA from cells or kidney cortex was extracted using TRIzol (Life Technologies). The cDNA was then synthesized using a PrimeScript RT reagent kit (Takara, RR037A). qPCR was performed with the TB Green Premix Ex Taq kit (Takara, RR420A) using a 7500 real-time PCR System (Applied Biosystems). All the primer sequences are listed in Supplemental Methods.

Statistics. Data are presented as the mean ± SD. Comparison between 2 groups was performed with a 1-tailed, unpaired t test. Three or more groups were analyzed with 1-way or 2-way ANOVA, as appropriate. All statistical analyses were performed using GraphPad Prism software (version 9). P values of less than 0.05 were considered statistically significant.

Study approval. All mouse protocols were approved by the Institutional Animal Care and Use Committee at Icahn School of Medicine at Mount Sinai (no. LA09-00377).

Data availability. All the data are available from the corresponding author upon request. Values for all data points in graphs are reported in the Supporting Data Values file.