Integrin-Linked Kinase Deficiency in Collecting Duct Principal Cell Promotes Necroptosis of Principal Cell and Contributes to Kidney Inflammation and Fibrosis

Abstract

Background: Necroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied.

Methods: We performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown.

Results: Ilk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF-β signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals' injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice.

Conclusions: The study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney.

Keywords: collecting duct principal cells; integrin-linked kinase (ILK); necroptosis.

Graphical abstract

Figure 1.

Renal failure and kidney tubular injury are present in PC Ilk KO mice. (A) Representative images of wild-type (WT) and PC Ilk KO mice at 4 weeks old. (B) Body weight. *P<0.05 versus 4-week-old wild type; ^#P<0.05 versus 8-week-old wild-type mice. (C) Survival rate was shown by a Kaplan–Meier curve. n=8–10 mice per group. (D and E) Elevated serum creatinine (Scr) and BUN in PC Ilk KO mice. n=6–10. **P<0.01, ***P<0.001 versus 4-week-old wild-type mice; ^##P<0.01, ^###P<0.001 versus 8-week-old wild-type mice; ^&P<0.05, ^&&P<0.01 versus 4-week-old KO mice. (F) Increased urinary excretion of NGAL in PC Ilk KO mice as shown by immunoblotting. (G) Representative images of H&E staining of kidney cortex and outer medulla (OM) of wild-type and KO mice. Tubular dilation and microcysts were present in Ilk KO kidney (arrows). Detached and dead tubular cells were observed within tubular lumen (arrowheads). Scale bar, 50 μm. (H) Tubular injury score was calculated based on the percentage of damaged tubules. The degree of injury was graded blindly in ten randomly chosen fields as follows: 0, normal; 1, <10%; 2, 11%–25%; 3, 26%–75%; 4, >75%. ***P<0.001 versus 4-week-old wild-type mice; ^###P<0.001 versus 8-week-old wild-type mice; ^&P<0.05 versus 4-week-old KO mice. n=4–7. Original magnification, 200×. (I) Immunofluorescence staining using an Airyscan confocal microscope confirmed deletion of ILK in PCs in KO kidney. ILK (red) was expressed on the basal membrane that was costained with wheat germ agglutinin (WGA)–FITC (green) in both PCs (stained purple with anti-AQP2 antibody) and ICs (negative for AQP2 staining) in wild-type kidney, whereas membrane expression of ILK (red) was significantly reduced or even absent in Ilk KO PCs. Arrows indicate ILK expression in WGA-positive basal membranes in wild-type and KO CDs, respectively. Scale bar, 5 μm. (J) Immunofluorescence staining revealed reduced ILK expression (red) in PCs in the KO kidney. PC-specific marker AQP2 stained green and IC-specific marker V-ATPase stained blue. ICs had low level of expression of ILK in general and there was no detectable change in ILK expression in KO kidney compared with the wild type. Arrows indicate ILK expression in basal membranes in wild-type and KO PCs, respectively. Scale bar, 10 μm. Data are presented as mean±SEM. Statistics were performed using the t test.

Figure 2.

Ilk KO in PCs causes kidney fibrosis and activates the TGF-β/Smad signaling pathway. (A) Immunofluorescence staining revealed the increased expression of α-SMA (red and arrows, top panels), ECM collagen type 1 (Col I; red and arrows, middle panels), and fibronectin (FN; red and arrows, bottom panels) in the cortex of 4-week-old KO mice. CD PCs were highlighted by immunostaining with anti-AQP2 antibody (green). Nuclei were stained blue with DAPI. Scale bar, 10 μm. (B) Deposition of collagen fibrils in 4-week-old wild-type (WT) and 4- and 8-week-old KO kidney was examined by picrosirius red staining. Collagen type 1 (red) and type 3 fibrils (green) were observed under polarized light (bottom three panels). Top three panels are images obtained under the bright light. Scale bar, 50 μm. (C) Masson trichrome staining revealed the excessive deposition of collagen fibers in 4- and 8-week-old KO kidney compared with the control. Scale bar, 50 μm. (D) Representative immunoblotting revealed increased expression of NGAL, FN, α-SMA, phosphorylated Smad3 (Ser423/425, p-Smad3, arrows), and total Smad2/3 (T-Smad2/3) in 4-week-old Ilk KO kidney (left). Individual protein signal intensity was quantified by densitometry and plotted over the intensity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The ratio of p-Smad3 over T-Smad2/3 is also shown in the graph (right). **P<0.01, ***P<0.001 versus wild-type mice. (E–G) Real-time PCR confirmed the augmented mRNA levels of (E) FN, (F) Col I, and (G) TGF-βi in Ilk KO kidney. Expression of individual genes was normalized to β-actin. ***P<0.001 versus 4-week-old wild-type mice; ^#P<0.05, ^##P<0.01 versus 8-week-old wild-type mice. Data presented as mean±SEM, n=6–7 for (D–G). Statistics were performed using the t test. MW, molecular weight.

Figure 3.

PC-specific ablation of Ilk induces renal inflammation. (A, C, and E) Representative immunofluorescence staining revealed increased F4/80-positive macrophages (red and arrows, A), Ly6G-positive neutrophils (red and arrows, C) and CD68-positive macrophages (red and arrows, E) in 4-week-old KO kidney. AQP2 stained green and nuclei stained blue. Scale bar, 20 μm. (B) F4/80-positive cells and (D) Ly6G-positive cells in 4-week-old kidney was quantified in ten randomly chosen fields for each specimen. Data are presented as the number of positive cells per high power field (mean±SEM/field). n=4–6. Original magnification, 400×. (F–J) Increased gene expression of (F) TNF-α, (G) IL-6, (H) IL-1β, (I) IL-33, and (J) CXCL1 in 4-week-old Ilk KO kidney was revealed by real-time PCR. Expression of individual genes was normalized to the expression of β-actin. (K) Representative immunoblotting images showed increased expression of phosphorylated (Ser536) and total NF-κB p65 subunit in 4-week-old KO kidney (left). The ratio of phosphorylated p65 and total NF-κB p65 over glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is plotted in the graph (right). *P<0.05, **P<0.01, ***P<0.001 versus wild-type (WT) mice. Data presented as mean±SEM, n=5–8 for (F–K). Statistics were performed using the t test. MW, molecular weight.

Figure 4.

CD epithelial cell injury and interstitial fibrosis are revealed by TEM in 4- and 8-week-old Ilk KO kidney. (A, a) CD appeared intact in 4-week-old wild-type (WT) kidney. (b and c, arrows) Increased intercellular junction and gaps between PCs and basement membrane were observed in CDs of 4-week-old Ilk KO kidney. (b) Detached tubular cells and cell debris were seen within the tubular lumen. Scale bar, 5 μm. (B) Increased interstitial fibrosis and inflammatory cell infiltration in Ilk KO kidney. (a) Extracellular matrix and fibrils were accumulated around CDs in KO kidney. The CD basement membrane was thickened (arrowhead). (a) Activated fibroblasts (F), (b) monocytes (M), and (c) macrophages (Mφ) were detected in the interstitium next to CDs in KO kidney. Scale bar, 2 μm. (C) Ilk KO PCs were undergoing necroptosis as indicated by cell swelling with (b, double arrows) lucent cytoplasm, (c, hollow arrow) apical membrane rupture, and (c) release of abundant amorphous cellular contents that were filled in the lumen of CDs. (a) Wild-type PCs remained intact. Scale bar, 2 μm. (D) Deformed mitochondria (arrows) and swollen endoplasmic reticulum (arrowheads) were present in (b) Ilk KO PCs, compared with (a) wild-type PCs. Scale bar, 500 nm.

Figure 5.

Necroptosis signal is induced in CDs of Ilk KO mice. (A) Representative immunoblotting showed the upregulation of expression of MLKL and RIPK3 in 4-week-old KO mouse kidney (top panel). Expression of MLKL and RIPK3 was quantified by densitometry and normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (bottom panel). *P<0.05, ***P<0.001 versus wild-type (WT) mice. Data presented as mean±SEM, n=6–8. Statistics were performed using the t test. (B) Immunofluorescence staining revealed increased apical membrane expression and accumulation of MLKL (red, arrowheads) in AQP2-positive PCs (green) in Ilk KO mice, whereas the staining of MLKL (red, arrows) was more diffuse inside cells and in the basal region in wild-type PCs (green). Scale bar, 10 μm. (C) Increased apical membrane accumulation of phosphorylated MLKL (Ser345, p-MLKL, red) signal was clearly detected in Ilk KO PCs (AQP2 stained green) by Airyscan fluorescence confocal microscopy. Scale bar, 10 μm. (D and E) Increased expression of phosphorylated RIPK1 (Ser166, p-RIPK1, red) and phosphorylated RIPK3 (Thr231/Ser232, p-RIPK3, red) was also detected in Ilk KO PCs (green) by immunofluorescence staining. Scale bar, 10 μm. MW, molecular weight.

Figure 6.

ILK inhibition and knockdown upregulates necroptotic signal in cultured renal tubular epithelial cells. (A, a) LLC-PK1 relative cell viability was reduced with the treatment of cpd22 in a dose-dependent manner as measured by MTT assay. Cpd22 (0.25–4 μM) was incubated with cells for 24 hours. *P<0.05, ***P<0.001 versus control (con). (b) ILK siRNA knockdown (ILK KD) or treatment with TNF-α or cpd22 (2 μM) caused cell loss as measured by DNA quantification assay in mCCDC11. ***P<0.001 versus control. (B) Increased plasma membrane accumulation of phospho-MLKL (p-MLKL, red) was clearly detected in mCCDC11 cells transfected with ILK siRNA. Similar membrane accumulation of phospho-MLKL was also present in cells treated with cpd22 or TNF-α as detected by immunofluorescence staining. Scale bar, 10 μm. (C) Immunofluorescence staining revealed that cpd22 treatment caused translocation of MLKL signals (red) from cytosol to the plasma membrane in LLC-PK1, compared with the control. Nuclei were stained blue with DAPI. Scale bar, 20 μm. (D) Representative immunoblotting showed the reduction of phosphorylated Akt (Ser 473, p-Akt) and upregulation of MLKL and RIPK3 in LLC-PK1 with cpd22 treatment for 24 hours (left panel). Bar graph represents the ratio of p-Akt over total Akt, and the ratio of MLKL and RIPK3 over β-actin expression as quantified by densitometry (right panel). *P<0.05, **P<0.01 versus control. (E) Nec-1 treatment of LLC-PK1 cells reversed the reduction of cell viability induced by cpd22. LLC-PK1 cells were treated with cpd22 (2 μM) in the presence or absence of Nec-1 (50 μM) for 24 hours. Relative cell viability was assessed by measuring the cellular DNA content. *P<0.05 versus control; ^###P<0.001 versus 2 μM cpd22 treatment group. Data presented as mean±SEM. Experiments were repeated at least three times. Statistics were performed using one-way ANOVA.

Figure 7.

Inhibition of necroptosis attenuates tubular injury and renal function decline in PC Ilk KO mice. (A) Diagram of Nec-1 administration schedule for wild-type (WT) and Ilk KO mice. After genotyping, 2-week-old wild-type and KO mice were treated with 1.65 mg/kg Nec-1 or vehicle (1% DMSO in PBS) by intraperitoneal injection every 2 days for 2 weeks. (B) Nec-1 reduced the elevation of serum creatinine (Scr) and (C) BUN in Ilk KO mice. **P<0.01 versus wild-type with DMSO mice; ^#P<0.05, ^##P<0.01 versus KO with DMSO mice. (D) Representative immunoblotting showed that Nec-1 reduced expression of NGAL, MLKL, and RIPK3 in Ilk KO kidney (left). The expression of NGAL, MLKL, and RIPK3 was quantified by densitometry, and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (right). ***P<0.001 versus wild-type with DMSO mice; ^#P<0.05, ^##P<0.01, ^###P<0.001 versus KO with DMSO mice. (E) Representative images of H&E staining in kidney cortex. Nec-1–treated Ilk KO mice showed less tubular dilation and microcyst formation, compared with wild-type kidney treated with or without Nec-1. Scale bar, 50 μm. (F) Nec-1 treatment improved the tubular injury score in Ilk KO kidney. ***P<0.001 versus wild type with DMSO mice; ^###P<0.001 versus KO with DMSO mice. (G) Immunofluorescence staining showed that Nec-1 blocked translocation of MLKL signal (red) from cytosol to plasma membrane in Ilk KO PCs (AQP2 stained green). Scale bar, 10 μm. (H) Gene expression of MLKL and RIPK3 was evaluated by real-time PCR. Expression of individual genes was normalized to β-actin. **P<0.01, ***P<0.001 versus wild-type with DMSO mice; ^###P<0.001 versus KO with DMSO mice. Data presented as mean±SEM, n=5–7. Statistics were performed using one-way ANOVA. KO+D, KO mice treated with 1% DMSO in PBS; KO+N, KO mice treated with Nec-1; P, postnatal day; WT+D, wild-type mice treated with 1% DMSO in PBS; WT+N, wild-type mice treated with Nec-1.

Figure 8.

Nec-1 blocks inflammatory response and fibrosis in PC Ilk KO kidney. (A) Nec-1 suppressed gene expression of TNF-α, IL-6, and IL-1β in Ilk KO kidney as revealed by real-time PCR. Expression of individual genes was normalized to β-actin. ***P<0.001 versus wild-type (WT) with DMSO mice; ^###P<0.001 versus KO with DMSO mice. (B) Representative immunoblotting showed that Nec-1 inhibited the upregulation of phosphorylated NF-κB p65 (p-p65), total p65, fibronectin (FN), and α-SMA in Ilk KO kidney (left). Protein expression of p-p65, total p65, FN, and α-SMA was quantified by densitometry and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (right). ***P<0.001 versus wild-type with DMSO mice; ^##P<0.01, ^###P<0.001 versus KO with DMSO mice. (C) Representative immunofluorescence staining showed reduced infiltration of F4/80-positive macrophages (red) in Nec-1–treated KO kidney. CD PCs were indicated by staining of AQP2 (green). Scale bar, 20 μm. (D) The number of F4/80-positive cells was quantified per field (mean±SEM/field). ***P<0.001 versus wild-type with DMSO mice; ^###P<0.001 versus KO with DMSO mice. Original magnification, 400×. (E) Representative immunofluorescence images revealed decreased expression of α-SMA (red), FN (red), and collagen type 1 (Col I, red) in Nec-1–treated Ilk KO kidney. Green fluorescence signal indicates the AQP2-positive CDs. Nuclei were stained blue with DAPI. Scale bar, 20 μm. (F) Nec-1 decreased gene expression of FN, Col I, and TGF-βi in Ilk KO kidney by real-time PCR. Expression of individual genes was normalized to β-actin. ***P<0.001 versus wild-type with DMSO mice; ^###P<0.001 versus KO with DMSO mice. Data presented as mean±SEM, n=5–7. Statistics were performed using one-way ANOVA. KO+D, KO mice treated with 1% DMSO in PBS; KO+N, KO mice treated with Nec-1; WT+D, wild-type mice treated with 1% DMSO in PBS; WT+N, wild-type mice treated with Nec-1.