Genetic and pharmacological inhibition of GRPR protects against acute kidney injury via attenuating renal inflammation and necroptosis

Abstract

Gastrin-releasing peptide (GRP) binds to its receptor (GRP receptor [GRPR]) to regulate multiple biological processes, but the function of GRP/GRPR axis in acute kidney injury (AKI) remains unknown. In the present study, GRPR is highly expressed by tubular epithelial cells (TECs) in patients or mice with AKI, while histone deacetylase 8 may lead to the transcriptional activation of GRPR. Functionally, we uncovered that GRPR was pathogenic in AKI, as genetic deletion of GRPR was able to protect mice from cisplatin- and ischemia-induced AKI. This was further confirmed by specifically deleting the GRPR gene from TECs in GRPR^{Flox/Flox//KspCre} mice. Mechanistically, we uncovered that GRPR was able to interact with Toll-like receptor 4 to activate STAT1 that bound the promoter of MLKL and CCL2 to induce TEC necroptosis, necroinflammation, and macrophages recruitment. This was further confirmed by overexpressing STAT1 to restore renal injury in GRPR^{Flox/Flox/KspCre} mice. Concurrently, STAT1 induced GRP synthesis to enforce the GRP/GRPR/STAT1 positive feedback loop. Importantly, targeting GRPR by lentivirus-packaged small hairpin RNA or by treatment with a novel GRPR antagonist RH-1402 was able to inhibit cisplatin-induced AKI. In conclusion, GRPR is pathogenic in AKI and mediates AKI via the STAT1-dependent mechanism. Thus, targeting GRPR may be a novel therapeutic strategy for AKI.

Keywords: GRP; GRPR; STAT1; acute kidney injury; inflammation; necroptosis.

Graphical abstract

Figure 1

GRP/GRPR in patients with AKI, mouse model and in vitro model of AKI (A) Serum ProGRP level in in healthy control and AKI patients were detected. (B) IHC staining of KIM1 and GRPR in kidneys of healthy control and AKI patients. (C) Serum ProGRP level in two different AKI models induced by I/R and cisplatin. (D) GRP mRNA levels in two different AKI models. (E) IF staining of GRPR and lotus tetragonolobus lectin (LTL) in cisplatin-induced AKI mouse kidneys. (F) IHC staining of GRPR in cisplatin-induced AKI mouse kidneys. (G) GRPR mRNA level in kidneys of AKI mice. (H) GRPR protein level in kidneys of AKI mice. (I) GRPR expression was detected in HK2 by real-time PCR. (J) Western blot and quantification of HDAC1/2/3/8. (K) GRPR expression in vitro were determined by real-time PCR. (M) HDAC8 and GRPR protein level in vitro. (M) The binding of HDAC8 on GRPR promoter regions were detected by ChIP assay. Independent experiments were performed throughout the in vitro studies in triplicate. Data represented the mean ± SEM for at least 6 persons or mice. ∗∗p < 0.01, ∗∗∗p < 0.001 vs. sham/saline/NC/EV. ^###p < 0.001 vs. EV + Cis. Cis, cisplatin control; EV, empty vector; NC, normal; OE, overexpression.

Figure 2

Global knockout of GRPR protected against renal dysfunction, renal injury, and inflammation in AKI models induced by cisplatin and I/R (A) Genotyping strategy. (B) GRPR deficiency was confirmed by detecting genomic DNA. (C) Global knockout of GRPR was verified by western blot. (D) Representative PAS staining pictures of kidneys from GRPR WT and KO mice treated with cisplatin and their quantification. Scale bar, 50 μm. (E and F) Serum creatinine and BUN levels. (G) KIM1 protein level detected by western blot and quantification. (H) Relative mRNA levels of KIM1, TNF-α, IL-1β, and CCL2 were determined by real-time PCR. (I) Representative PAS staining of kidneys from GRPR WT and KO mice treated with I/R injury. (J and K) Serum creatinine and BUN levels. Data represented the mean ± SEM for six mice. ∗∗∗p < 0.001 vs. WT + saline/WT + sham. ^##p < 0.01, ^###p < 0.001 vs. WT + Cis/WT + I/R. Cis, cisplatin; WT, wild type.

Figure 3

Conditional knockout of GRPR from kidney TECs protected against renal dysfunction, renal injury, and inflammation in AKI models induced by cisplatin and I/R (A) The construction of GRPR^{Flox/Flox/KspCre} mice. (B) Conditional knockout of GRPR from kidney TECs was confirmed by western blot. (C) GRPR expression was determined by real-time PCR. (D) Representative PAS staining pictures of kidneys from GRPR^Flox/Flox and GRPR^{Flox/Flox/KspCre} mice treated with cisplatin and their quantification. (E, F) Serum creatinine and BUN levels. (G) KIM1 protein level was detected by Western blot. (H) Relative mRNA levels of KIM1, TNF-α, IL-1β, and CCL2 were determined by real-time PCR. (I) Representative electron micrographs of kidneys are shown. (J) Representative PAS staining pictures of kidneys from GRPR^Flox/Flox and GRPR^{Flox/Flox/KspCre} mice treated with I/R injury and their quantification. (K, L) Serum creatinine and BUN levels. (M) Relative mRNA levels of KIM1, TNF-α, IL-1β, and CCL2 were determined by real-time PCR. (N) IF staining of F4/80. Data represented the mean ± SEM for six mice. ∗∗p < 0.01, ∗∗∗p < 0.001 vs. FF/FF + saline/FF + sham. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. FF + Cis/FF + I/R. Cis, cisplatin.

Figure 4

GRPR promoted renal injury and inflammation in STAT1-dependent mechanisms (A) KEGG pathway analysis of RNA-seq in GRPR overexpression and control mTECs. (B) Heatmap. (C) Correlated signaling pathways in GRPR knockdown and control HK2 cells. (D) Correlated signaling pathways in GRPR WT and KO mice. (E) IF staining of GRPR and TLR4 in TECs. (F) Co-IP of GRPR and TLR4 in cisplatin-treated TECs. (G) Protein levels of P-STAT1/STAT1/TLR4/GRPR and quantification. (H) The binding of P-STAT1 on MLKL or CCL2 promoter regions were detected by ChIP assay. (I) Relative mRNA levels of STAT1, MLKL and CCL2 in STAT1 knockdown mTECs. (J) The mRNA level of GRP in GRPR knockdown or overexpressed mTECs. (K) The binding of P-STAT1 to GRP promoter by ChIP assay. (L) The mRNA level of GRP in STAT1 knockdown mTECs. (M) IF staining of P-MLKL. Independent experiments were performed throughout the in vitro studies in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. EV + NC/WT + saline/TLR4-KD/P-STAT1+IgG. ^#p < 0.05, ^###p < 0.001 vs. EV + Cis/WT + Cis/TLR4-KD/EV + Cis. ^$$p < 0.01, ^$$$p < 0.001 vs. GRPR-OE. Cis, cisplatin; EV, empty vector; KD, knockdown; NC, normal control; OE, overexpression; WT, wild type.

Figure 5

Rescue of STAT1 expression in conditional KO mice or KO cell restored renal damage (A) P-STAT1/STAT1, PP65/P65, KIM1 protein level was determined by western blot. (B) Relative mRNA levels of STAT1, KIM1 and proinflammatory genes. (C) Representative PAS staining of kidney in lentivirus-mediated STAT1 overexpression in mice treated with cisplatin. (D, E) Serum creatinine and BUN levels. (F) IF staining of KIM1, F4/80, and TNF-α. (G) Protein levels of P-STAT1/STAT1, PP65/P65, and quantification. (H) Relative mRNA levels of STAT1, KIM1, and proinflammatory indexes were determined by real-time PCR. (I) IF staining of P-STAT1 in TECs. Data represented the mean ± SEM for six mice. ∗p < 0.05, ∗∗∗p < 0.001 vs. FF + Cis. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. KspCre+Cis. Independent experiments were performed throughout the in vitro studies in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. WT + Cis. ^#p < 0.05, ^###p < 0.001 vs. KO + Cis. Cis, cisplatin; WT, wild type.

Figure 6

Lentivirus-mediated in vivo GRPR knockdown protected against cisplatin-induced AKI (A) Construction of GRPR-KD mice. (B) Lentivirus-mediated GRPR knockdown in mice was confirmed by western blot. (C) Representative PAS staining of kidney in NC, EV, and GRPR KD mice treated with cisplatin. (D and E) Serum creatinine and BUN levels. (F) KIM1 protein level was determined by western blot and quantitative analysis. (G) Relative mRNA levels of KIM1 and proinflammatory genes. (H) IF staining of F4/80 and TNF-α. (I) Protein levels of P-STAT1/STAT1 and PP65/P65. Data represented the mean ± SEM for six mice. ∗p < 0.05, ∗∗∗p < 0.001 vs. EV + saline. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. EV + Cis. Cis, cisplatin; EV, empty vector.

Figure 7

RH-1402 attenuated cisplatin-induced cell injury and inflammation by antagonizing GRPR in HK2 cells (A) The potential binding of GRPR and RH-1402 was conducted by molecular docking. (B) The binding affinity between GRPR and RH-1402 was confirmed by radioligand binding assays. (C) The thermal stability of RH-1402 to GRPR was indicated by CETSA assay. (D, E) The impacts of RH-1402 on cell viability in HK2 cells with or without cisplatin were analyzed by MTT assay. (F) KIM1 protein level was determined by western blot, while cisplatin-induced HK2 cells were treated with RH-1402. (G) Relative mRNA levels of KIM1, TNF-α, IL-1β, and CCL2 were determined by real-time PCR. (H) The effect of RH-1402 on macrophage migration by transwell assay. (I) Protein levels of P-STAT1/STAT1 and PP65/P65 in cisplatin-induced HK2 with or without RH-1402. (J) PI/Hoechst double staining and IF of p-MLKL in HK2 cells. (K) The effect of RH-1402 on programmed cell death induced by cisplatin was detected by flow cytometric analysis. (L) Protein levels of RIPK1/RIPK3/MLKL axis with quantification in vitro. (M) The effect of RH1402 on NF-κB promoter activity. (N) Protein levels of PP65/P65 in cisplatin-induced HK2 with or without RH-1402 while GRPR was silenced. (O) Relative mRNA levels of KIM1 and proinflammatory indexes. Independent experiments were performed throughout the in vitro studies in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. NC/EV + Con. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Cis/EV + Cis. ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001 vs. EV+Cis+RH-1402. Cis, cisplatin; EV, empty vector.

Figure 8

The preventive and therapeutic effects of RH-1402 in three AKI mouse models (A) Experimental timeline of prevention. (B) Representative PAS staining pictures of kidneys from AKI mice in which RH-1402 was applied before cisplatin injection. (C, D) Serum creatinine and BUN levels. (E) IF and IHC staining of TNF-α and F4/80 and their quantification. (F) Experimental timeline of treatment. (G) Representative PAS staining pictures of kidneys from AKI mice in which RH-1402 was applied after model establishment. (H and I) Serum creatinine and BUN levels. (J) IF staining of KIM1, F4/80, and TNF-α and their quantification. (K) PAS staining and scores in the kidneys of I/R-induced AKI mice subjected to I/R treatment. (L and M) BUN levels and serum creatinine levels. Data represented the mean ± SEM for six mice. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. Saline. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Cis/Cis D3. Cis, cisplatin; D, day.