Targeting Phospholipase D4 Attenuates Kidney Fibrosis

Abstract

Phospholipase D4 (PLD4), a single-pass transmembrane glycoprotein, is among the most highly upregulated genes in murine kidneys subjected to chronic progressive fibrosis, but the function of PLD4 in this process is unknown. Here, we found PLD4 to be overexpressed in the proximal and distal tubular epithelial cells of murine and human kidneys after fibrosis. Genetic silencing of PLD4, either globally or conditionally in proximal tubular epithelial cells, protected mice from the development of fibrosis. Mechanistically, global knockout of PLD4 modulated innate and adaptive immune responses and attenuated the upregulation of the TGF-β signaling pathway and α1-antitrypsin protein (a serine protease inhibitor) expression and downregulation of neutrophil elastase (NE) expression induced by obstructive injury. In vitro, treatment with NE attenuated TGF-β-induced accumulation of fibrotic markers. Furthermore, therapeutic targeting of PLD4 using specific siRNA protected mice from folic acid-induced kidney fibrosis and inhibited the increase in TGF-β signaling, decrease in NE expression, and upregulation of mitogen-activated protein kinase signaling. Immunoprecipitation/mass spectrometry and coimmunoprecipitation experiments confirmed that PLD4 binds three proteins that interact with neurotrophic receptor tyrosine kinase 1, a receptor also known as TrkA that upregulates mitogen-activated protein kinase. PLD4 inhibition also prevented the folic acid-induced upregulation of this receptor in mouse kidneys. These results suggest inhibition of PLD4 as a novel therapeutic strategy to activate protease-mediated degradation of extracellular matrix and reverse fibrosis.

Keywords: TGF-beta; chronic kidney disease; fibrosis; immunology.

Graphical abstract

Figure 1.

Expression of fibrotic markers as well as PLD4 increases in FA- and UUO-induced kidney fibrosis. Protein expression of the fibrotic markers α-SMA, fibronectin (FN), and collagen 1α1 (Col 1α1) in the kidneys of mice (A) injected with FA and (D) subjected to UUO (n=5/group). (B and E) mRNA levels of PLD1, PLD2, PLD3, and PLD4 and (C and F) protein level of PLD4 in the kidneys of mice injected with FA and subjected to UUO, respectively. Data were normalized to GAPDH and are presented as mean±SEM (n=5/group) of the fold change over normal. *P<0.05. (G) Immunostaining and the relative quantitation of α-SMA and PLD4 in the kidney sections from patients with biopsy-proven tubulointerstitial fibrosis versus patients with no evidence of fibrosis (n=5/group). Scale bars, 50 µm. Data are presented as mean±SEM of the fold change over normal. *P<0.05. C, contralateral; CoK, contralateral kidney; N, normal; U, UUO.

Figure 2.

PLD4^−/− mice are protected from FA- and UUO-induced kidney fibrosis. Protein expression of the fibrotic markers α-SMA, fibronectin (FN), and collagen 1α1 (Col 1α1) in the kidneys of PLD4^+/+ and PLD4^−/− mice injected with (A) FA and (B) subjected to UUO (n=5–7/group). (C) Representative photomicrographs depicting the expression of fibrotic markers (α-SMA, FN, and Col 1α1) in mouse kidneys (n=5/group), and Picrosirius Red and Masson’s trichrome staining depicting collagen deposition in mouse kidneys at baseline and after fibrosis (n=3/group). Scale bars, 50 µm. Relative quantitation of immunostaining and Picrosirius Red and Masson’s trichrome staining. Data are presented as mean±SEM of the fold change over PLD4^+/+ normal. *P<0.05. (D) Schematic representation of the timeline of tamoxifen and FA administration in mice. Mice were injected with FA (250 mg/kg, ip) followed by treatment with tamoxifen (2 mg, diluted in corn oil and 3% ethanol, ip) at 1, 3, and 5 days after FA injection. Mice were euthanized on day 7 after FA injection (n=5/group). (E) Specificity of PLD4 knockdown in kidney proximal tubular epithelial cells was confirmed by coimmunostaining PLD4 with LTL, a proximal tubular marker (thick arrows, LTL-positive tubules; thin arrows, LTL-negative tubules), using the kidneys obtained from SLC34a1^GCE/+ PLD4^fl/fl (PLD4^fl/fl) and wild-type mice SLC34a1^GCE/+ PLD4^wt/wt (PLD4^wt/wt) (n=5/group). Scale bars, 50 μm. (F) Protein levels of α-SMA, FN, and Col 1α1 in the kidneys of PLD4^wt/wt and PLD4^fl/fl mice injected with FA (n=5/group). C, contralateral; ip, intraperitoneally; N, normal; U, UUO; +/+, PLD4 wild-type; −/−, PLD4 knockout.

Figure 3.

PLD4^−/− mouse kidneys have differentially expressed innate and adaptive immune genes and decreased level of a protease inhibitor, AAT, compared with the PLD4^+/+ mice. (A) Heat map depicting differentially expressed genes between PLD4^+/+ and PLD4^−/− mouse kidneys at baseline and after UUO (days 5 and 10) (n=4/group). (B) Pathway analysis of differentially expressed genes between PLD4^+/+ and PLD4^−/− mouse kidneys at baseline. (C) Flow cytometric analysis of innate and adaptive immune system components in the kidneys of mice at baseline and after UUO (day 5) (n=3/group). (D) mRNA levels of cytokines in the kidneys of mice at baseline and after UUO (days 5 and 10). Protein expression of (E) TGF-β signaling molecules, pSmad2, Smad2, pSmad3, and Smad3; and (F) AAT and NE in mouse kidneys. Data were normalized to GAPDH and are presented as mean±SEM (n=5–7/group) of the fold change over PLD4^+/+ normal. *P<0.05. C, contralateral; N, normal; U, UUO; +/+, PLD4 wild-type; −/−, PLD4 knockout.

Figure 4.

Therapeutic targeting of PLD4 by specific siRNA protects mice from kidney fibrosis. (A) Schematic representation of the treatment protocol with FA and siRNA (scrambled or PLD4). Mice were injected with FA (250 mg/kg, ip) followed by treatment with scrambled or PLD4 siRNA (30 µg/200 µl, iv) at 2, 20, 38, 62, and 110 hours after FA injection. Mice were euthanized on day 7 after FA injection (n=5/group). (B) Protein levels of PLD4 and the fibrotic markers α-SMA, fibronectin (FN), and collagen 1α1 (Col 1α1) in mouse kidneys (n=5/group). (C) Picrosirius Red and Masson’s trichrome staining depicting collagen deposition in mouse kidneys (n=3/group). Scale bars, 50 µm. Data are presented as mean±SEM (n=5/group) of the fold change over vehicle+scrambled siRNA. *P<0.05. Protein expression of (D) TGF-β signaling molecules, pSmad2, Smad2, pSmad3, and Smad3; and (E) AAT and NE in mouse kidneys (n=5/group). (F) Level of plasma creatinine and BUN in mice and (G) protein level of KIM1 in mouse kidneys (n=4/group). Data are presented as mean±SEM (n=4–5/group) of the fold change over vehicle+scrambled siRNA. *P<0.05. (H) Schematic representation depicting how PLD4 drives fibrosis. PLD4 leads to a decrease in anti-fibrotic cytokines, upregulation of TGFβ signaling and downregulation of neutrophil elastase expression by preventing a decrease in α1-antitrypsin expression. All these result in increased ECM accumulation that progresses to fibrosis. ip, intraperitoneally; iv, intravenously; Scr, scrambled siRNA; V+scr, vehicle+scrambled siRNA.

Figure 5.

PLD4 upregulates MAPK signaling via TrkA pathway to mediate fibrogenesis. (A) Overview of the PLD4 interaction network on the basis of the immunoprecipitation/mass spectrometry (IP/MS) analysis (respective WDN scores indicated in parentheses). (B) Diagrammatic representation of the localization of interacting partners of PLD4. Orange circles represent PLD4 and gray circles represent the binding partners of PLD4. Immunofluorescence analysis of the subcellular localization of PLD4 by costaining PLD4 with ER (Calnexin)-, Golgi (Golga1)-, and mitochondria (Tom20)-specific proteins in (C) HeLa cells and (D) HEK293 cells. Scale bars, 20 µm. (E) Coimmunoprecipitation of the top three interacting partners of PLD4 (CLGN, LMAN2, and SEL1L, selected on the basis of the WDN score) using PLD4-overexpressed HEK293T cells. Protein expression of CLGN, LMAN2, SEL1L, TrkA, FRS2α, pERK, and ERK in the kidneys of (F) PLD4^+/+ and PLD4^−/− mice and (G) siRNA (scr and PLD4)-treated mice injected with FA (n=5/group). ER, endoplasmic reticulum; IP, immunoprecipitation; Mito, mitochondria; scr, scrambled; +/+, PLD4 wild-type; −/−, PLD4 knockout.