SESN2 inhibits tubular exosome secretion and diabetic kidney disease progression by restoring the autophagy‒lysosome pathway

Abstract

During diabetic kidney disease (DKD), tubulointerstitial fibrosis persists, although several methods have been applied to reduce albuminuria levels. In this research, we found that bovine serum albumin (BSA)-induced renal tubular cell injury could also spread to normal tubular cells through exosomes, which may explain why tubulointerstitial fibrosis persists. Our previous studies revealed that SESN2 overexpression alleviates tubular dysfunction. In this study, we showed that SESN2 overexpression in donor HK2 cells interrupted this "doom loop" and confirmed that SESN2 may mediate this process by reducing exosome secretion. By using RNA-seq and IP-MS, we found that SESN2 could inhibit BSA-induced Rab-7a ubiquitination, thus promoting autophagosome and lysosome fusion and accelerating MVB degradation. We also showed that SESN2 promotes the nuclear translocation of TFEB through the mTOR pathway, thus further alleviating lysosomal function and promoting MVB degradation. We also found that SESN2 not only slowed DKD progression but also promoted renal tubular cell secretion of protective exosomes, which also slowed DKD progression. In conclusion, SESN2 can interrupt the progression of albuminuria-induced tubular injury by inhibiting exosome secretion and promoting MVB degradation. Thus, SESN2 may be a new therapeutic target for DKD treatment.

Keywords: Diabetic kidney disease; SESN2; autophagy; exosomes; lysosome.

Figure 1

Exosomes from BSA-induced tubular cells promote recipient tubular cell injury. HK-2 cells were co-cultured with Control HK2 cells (co-Control-HK-2) and BSA-treated HK2 cells (co-BSA-HK2): (A) mRNA expression levels of TNF-α, IL-6, FN, Col-I and α-SMA (n=3). (B) Protein expression levels of Col-I and FN (n=3). (C) Representative immunostaining images of KIM-1. Scale bars: 50 μm. *p < 0.05 vs. the co-Control-HK-2 group. HK-2 cells were co-cultured with BSA-treated HK2 cells (co-BSA-HK2) and BSA-treated GW4869-treated HK2 cells (co-BSA-HK2+GW4869): (D) mRNA expression levels of TNF-α, IL-6, FN, Col-I and α-SMA (n=3). (E) Protein expression levels of Col-I and FN (n=3). (F) Representative immunostaining images of KIM-1. Scale bars: 50 μm. *p < 0.05 vs. the co-BSA-HK-2 group. (G) Representative TEM images of exosomes isolated from HK-2 cells. Scale bars: 1.0 μm. (H) Detection of exosome markers in both cell lysates and exosomes. (I) NTA analysis diameters of exosomes isolated from HK-2 cells. HK-2 cells were co-cultured with exosomes from Control HK2 cells (Control-exo) and BSA-treated HK2 cells (BSA-exo): (J) mRNA expression levels of TNF-α, IL-6, FN, Col-I and α-SMA (n=3). (K) Protein expression levels of Col-I and FN (n=3). (L) Representative immunostaining images of KIM-1. Scale bars: 50 μm. *p < 0.05 vs. the Control-exo group. Db/db mice were injected with Control-exo (db/db+Control-exo) and BSA-exo (db/db+BSA-exo): (M) IVIS images of different organs harvested 24 h after db/db mice were injected with Did-labelled exosomes. (N) Fasting blood glucose. (O) Urinary ACR levels. (P) Masson staining of kidney cortex. Scale bars: 100 μm. (Q) mRNA expression levels of TNF-α, IL-6, FN, Col-I and α-SMA (n=5). (R) Protein expression levels of Col-I and FN (n=3). (S) Representative immunostaining images of KIM-1(n=5). Scale bars: 50 μm. *p < 0.05 vs. the db/db+Control-exo group.

Figure 2

SESN2 delays DKD progression. (A) SESN2 was detected using western blot (B) and immunostaining (Scale bars: 100 μm.) in the kidney cortices of db/m and db/db mice. *p < 0.05 vs. the db/m group. Db/m and db/db mice were injected with AAV-vector or AAV-SESN2 via the tail vein: (C) Fasting blood glucose. (D) Urinary ACR levels. (E) Masson staining of kidney cortex. Scale bars: 100 μm. (F) mRNA expression levels of SESN2, TNF-α, IL-6, FN, Col-I and α-SMA (n=5). (G) Representative immunostaining images of KIM-1(n=5). Scale bars: 50 μm. (H) Protein expression levels of Col-I and FN (n=3). *p < 0.05 vs. the db/m+AAV-vector group, #p < 0.05 vs. the db/db+AAV-vector group. Db/m and db/db mice were injected with AAV-sh-NC or AAV-sh-SESN2 via the tail vein: (I) Fasting blood glucose. (J) Urinary ACR levels. (K) Masson staining of kidney cortex. Scale bars: 100 μm. (L) mRNA expression levels of SESN2, TNF-α, IL-6, FN, Col-I and α-SMA (n=5). (M) Representative immunostaining images of KIM-1(n=5). Scale bars: 50 μm. (N) Protein expression levels of Col-I and FN (n=3). *p < 0.05 vs. the db/m+AAV-sh-NC group, #p < 0.05 vs. the db/db+AAV-sh-NC group. HK-2 cells were co-cultured with BSA-HK2 cells stably overexpressing Vector (co-BSA+Vector-HK2) or SESN2 (co-BSA+LV-SESN2-HK2): (O) mRNA expression levels of TNF-α, IL-6, FN, Col-I and α-SMA (n=3). (P) Representative immunostaining images of KIM-1. Scale bars: 50 μm; (Q) Protein expression levels of Col-I and FN (n=3). *p < 0.05 vs. the co-BSA+Vector-HK2 group.

Figure 3

SESN2 inhibits exosome secretion from renal tubular epithelial cells. Exosomes were isolated from Vector or SESN2 stably overexpressed HK-2 cells and analyzed using (A) Representative TEM images (Scale bars: 1.0 μm) and (B) NTA analysis. (C) Exosome numbers from Vector or SESN2 stably overexpressing HK-2 cells treated with or without BSA (n=3). *p < 0.05 vs. the Vector group, #p < 0.05 vs. the Vector+BSA group. (D) Exosome numbers from Si-NC- or Si-SESN2-transfected HK-2 cells (n=3). *p < 0.05 vs. the Si-NC group. (E) Representative immunostaining images of CD63 in Vector or SESN2 stably overexpressing HK-2 cells treated with or without BSA. Scale bars: 50 μm. (F) Representative immunostaining images of CD63 in Si-NC- or Si-SESN2-transfected HK-2 cells. Scale bars: 50 μm. (G) Exosome numbers from db/m and db/db mice injected with AAV-vector or AAV-SESN2 via the tail vein (n=5). *p < 0.05 vs. the db/m+AAV-vector group, #p < 0.05 vs. the db/db+AAV-vector group. (H) Exosome numbers from db/m and db/db mice injected with AAV-sh-NC or AAV-sh-SESN2 via the tail vein (n=5). *p < 0.05 vs. the db/m+AAV-sh-NC group, #p < 0.05 vs. the db/db+AAV-sh-NC group. (I) Representative immunostaining images of LTL and CD63 in db/m and db/db mice injected with AAV-vector or AAV-SESN2 via the tail vein. Scale bars: 50 μm. (J) Representative immunostaining images of LTL and CD63 in db/m and db/db mice injected with AAV-sh-NC or AAV-sh-SESN2 via the tail vein. Scale bars: 50 μm.

Figure 4

SESN2 inhibits HK2 cell exosome release by promoting autophagic degradation. (A-B) KEGG pathway analysis according to RNA-sequencing results. (C) Protein expression levels of LC3II:I and p62 in the kidney cortices of db/m and db/db mice (n=3). *p < 0.05 vs. the db/m group. (D) Protein expression levels of LC3II:I and p62 in the kidney cortices of db/m and db/db mice injected with AAV-vector or AAV-SESN2 via the tail vein. (n=3) . *p < 0.05 vs. the db/m+AAV-vector group, #p < 0.05 vs. the db/db+AAV-vector group. (E) Protein expression levels of LC3II:I and p62 in the kidney cortices of db/m and db/db mice injected with AAV-sh-NC or AAV-sh-SESN2 via the tail vein. (n=3). *p < 0.05 vs. the db/m+AAV-sh-NC group, #p < 0.05 vs. the db/db+AAV-sh-NC group. (F) Kidney cortex electron microscopic images of db/db mice injected with AAV-vector or AAV-SESN2 via the tail vein. The autophagosome (red arrows) numbers were quantified (n= 3). Scale bars: 5.0 μm. (G) Protein expression levels of LC3II:I and p62 in Vector or SESN2 stably overexpressing HK-2 cells treated with or without BSA (n=3). *p < 0.05 vs. the Vector group, #p < 0.05 vs. the Vector+BSA group. (H) Protein expression levels of LC3II:I and p62 in Si-NC- or Si-SESN2-transfected HK-2 cells treated with or without BSA (n=3). *p < 0.05 vs. the Si-NC group, #p < 0.05 vs. the Si-NC+BSA group. (I) Si-NC- or Si-SESN2-transfected HK-2 cells were infected with RFP-GFP-LC3-expressing adenovirus, and the fluorescence was detected by confocal microscopy. Scale bars: 2.0 μm. (J) Vector or SESN2 stably overexpressing HK-2 cells treated with or without BSA were infected with RFP-GFP-LC3-expressing adenovirus, and the fluorescence was detected by confocal microscopy. Scale bars: 2.0 μm. (K) Representative immunostaining images of CD63 and LAMP1 in Si-NC- or Si-SESN2-transfected HK-2 cells. Scale bars: 50 μm. (L) Representative immunostaining images of CD63 and LAMP1 in Vector or SESN2 stably overexpressing HK-2 cells treated with or without BSA. Scale bars: 50 μm.

Figure 5

SESN2 binds with Rab-7a to enhance its stability and prevent Rab-7a ubiquitination. (A) Cell lysates were pulled down from Vector or SESN2 stably overexpressing HK-2 cells using a SESN2 antibody, and Rab-7a levels were found to be increased in the LV-SESN2 group by mass spectrometry. (B) Diagram of a protein-protein docking model and interfacing residues between SESN2 and Rab-7a proteins (green: Rab-7a; yellow: SESN2). (C) Confocal microscopy images of the co-localization of SESN2 and Rab-7a in HK-2 cells. Scale bars: 20 μm. (D) Co-immunoprecipitation showing the interaction between SESN2 and Rab-7a in HK-2 cells. (E) mRNA expression levels of Rab-7a were measured in Vector or SESN2 stably overexpressing HK-2 cells (n=3). (F) mRNA expression levels of Rab-7a were measured in Si-NC- or Si-SESN2-transfected HK-2 cells (n=3). (G) Protein expression levels of Rab-7a and SESN2 were measured in Vector or SESN2 stably overexpressing HK-2 cells (n=3). *p < 0.05 vs. the Vector group. (H) Protein expression levels of Rab-7a and SESN2 were measured in Si-NC- or Si-SESN2-transfected HK-2 cells (n=3). *p < 0.05 vs. the Vector group. (I) Vector or SESN2 stably overexpressing HK-2 cells were treated with 50 μg/mL cycloheximide, and cell lysates were subjected to Western blotting analysis of Rab-7a. (J) Si-NC- or Si-SESN2-transfected HK-2 cells were treated with 10 μM of MG132, and cell lysates were subjected to Western blotting analysis of Rab-7a. (K) HK-2 cells were transfected with His-Ub, LV-SESN2 or Si-SESN2 and then treated with MG132. The ubiquitylation level of Rab-7a was detected using an anti-His antibody. Vector or SESN2 stably overexpressing HK-2 cells treated with or without BSA were infected with Si-NC or Si-Rab-7a: (L) Protein expression levels of LC3II:I and p62 (n=3). (M) Representative immunostaining images of CD63. Scale bars: 50 μm. (N) Total exosome proteins. *p < 0.05 vs. the Vector+Si-NC group, #p < 0.05 vs. the Vector+Si-NC+BSA group, ^$p < 0.05 vs. the LV-SESN2+Si-NC+BSA group.

Figure 6

SESN2 promotes MVB degradation through the regulation of TFEB subcellular localization. (A) Representative fluorescence microscopy images of Vector or SESN2 stably overexpressing HK-2 cells treated with or without BSA stained with LysoTracker probe (n=3). Scale bars: 50 μm. *p < 0.05 vs. the Vector group, #p < 0.05 vs. the Vector+BSA group. (B) Representative fluorescence microscopy images of Si-NC- or Si-SESN2-transfected HK-2 cells stained with LysoTracker probe (n=3). Scale bars: 50 μm. *p < 0.05 vs. the Si-NC group. (C) Representative immunostaining images of Lamp1 (n=5) in the kidney cortices of db/m and db/db mice injected with AAV-vector or AAV-SESN2 via the tail vein. Scale bars: 50 μm. *p < 0.05 vs. the db/m+AAV-vector group, #p < 0.05 vs. the db/db+AAV-vector group. (D) Representative immunostaining images of Lamp1 (n=5) in the kidney cortices of db/m and db/db mice injected with AAV-sh-NC or AAV-sh-SESN2 via the tail vein. Scale bars: 50 μm.*p < 0.05 vs. the db/m+AAV-sh-NC group, #p < 0.05 vs. the db/db+AAV-sh-NC group. (E) Representative immunostaining images of TFEB in Vector or SESN2 stably overexpressing HK-2 cells treated with or without BSA. Scale bars: 50 μm. TFEB and DAPI colocalization was analyzed (n=3). *p < 0.05 vs. the Vector group, #p < 0.05 vs. the Vector+BSA group. (F) Representative immunostaining images of TFEB in Si-NC- or Si-SESN2-transfected HK-2 cells. Scale bars: 50 μm. TFEB and DAPI colocalization was analyzed (n=3). *p < 0.05 vs. the Si-NC group. (G) Representative immunostaining images of TFEB in Si-SESN2-transfected HK-2 cells treated with or without rapamycin. Scale bars: 50 μm. TFEB and DAPI colocalization was analyzed (n=3). *p < 0.05 vs. the Si-SESN2 group. (H) Exosome numbers from SESN2 stably overexpressing HK-2 cells transfected with Si-NC or Si-TFEB (n=3). *p < 0.05 vs. the Vector group, #p < 0.05 vs. the Vector+BSA group.

Figure 7

SESN2-overexpressing HK2 cell-derived exosomes alleviate tubular injury. (A) Vector or SESN2 stably overexpressing HK-2 cells were treated with or without BSA, and miR-199a-5p expression in HK-2 cell-derived exosomes was measured by RT-PCR. Db/db mice were injected with Vector or SESN2 stably overexpressing HK-2 cell-derived exosomes (db/db+Vector-exos and db/db+SESN2-exos). (B) Fasting blood glucose. (C) Urinary ACR levels. (D) Protein expression levels of LC3II:I and p62. (E) Masson staining of kidney cortex. Scale bars: 100 μm. (F) mRNA expression levels of TNF-α, IL-6, FN, Col-I and α-SMA (n=5). (G) Representative immunostaining images of KIM-1 (n=5). Scale bars: 50 μm. *p < 0.05 vs. the db/db+Vector-exos group.