TXNIP deficiency attenuates renal fibrosis by modulating mTORC1/TFEB-mediated autophagy in diabetic kidney disease

Figures & data

Table 1. The sequences of the PCR primers in this study.

Gene	Forward primers (5′–3′)	Reverse primers (5′–3′)
Mouse fibronectin	AGGCTGGATGATGGTGGACT	CGGCTGAAGCACTTTGTAGAG
Collagen-I	CAAGAAGACATCCCT GAAGTC	ACAGTCCAGTTCTTCATTGC
β-actin	CTGAGAGGGAAATCGTGCGT	CCACAGGATTCCATACCCAAGA
Human TXNIP	CCGTTAGGATCCTGGCTTGC	GGCGCCTTGTACTCATATTTGTTTC
α-SMA	CGCCCTCGCCACCAGATCTG	TAGCCTTCATAGATGGGGAC
E-cadherin	GCCGGAGCCCTGCCACCCTG	CTTTCTGTAGGTGGAGTCCC
Fibronectin	ACAAGCATGTCTCTCTGCCA	CCAGGGTGATGCTTGGAGAA
Collagen I	ATGCCTGGTGAACGTGGT	AGGAGAGCCATCAGCACCT
ATP6V1H	CTGCTCACGATGTTGGAGAA	GGCCAGCAGAGCATTATAGC
VPS11	CGGCGCTTCGTTTTCTTCG	CCCGTAGTTTGTAGGCTTGGAA
18s	ACACGGACAGGATTGACAGA	GGACATCTAAGGGCATCACAG

Table 2. Animal characteristics.

Parameters	WT	DM	TKO + DM	TKO
Blood glucose (mmol/L)	5.863 ± 0.514	30.32 ± 1.356*	29.91 ± 1.138	6.203 ± 0.617
Body weight (g)	32.63 ± 1.796	24.57 ± 0.822*	27.56 ± 0.729^#	31.67 ± 0.540
Kidney weight (g)	0.384 ± 0.005	0.388 ± 0.011	0.381 ± 0.007	0.385 ± 0.009
Kidney/body weight (mg/g)	11.8 ± 0.648	15.79 ± 0.691*	13.82 ± 0.458^#	12.15 ± 0.335
Scr (μmol/L)	21.82 ± 1.448	52.35 ± 2.159*	38.70 ± 2.356^#	20.9 ± 1.159
BUN (mmol/L)	11.32 ± 0.922	32.49 ± 1.711*	19.65 ± 0.829^#	10.76 ± 0.929
UAE (mg/24 h)	7.83 ± 0.92	41.2 ± 1.99*	23.34 ± 1.50^#	8.16 ± 0.95
Systolic BP (mmHg)	109.8 ± 3.25	113.3 ± 1.21	111.7 ± 1.86	106.3 ± 2.25

Notes: Data are presented as mean ± SD (n = 6). TKO: TXNIP knockout; Scr: serum creatinine; BUN: blood urea nitrogen; UAE: urine albumin excretion; BP: blood pressure.

*p < 0.05, versus WT group.

^#p < 0.05, versus DM group.

Figure 1. Effects of TXNIP deficiency on renal fibrosis in diabetic kidneys. (A) Renal pathological changes were examined by PAS staining, Sirius red staining and Masson trichrome staining (Bar = 50 μm). Glomerular injury index for PAS staining (B) and tubulointerstitial injury index for Masson trichrome staining (C) were measured. (D) The expression of collagen I was detected by immunofluorescence (Bar = 50 μm). (E) The mRNA levels of collagen I and fibronectin were analyzed by RT-qPCR. (F, G) The expression of TXNIP, collagen I and fibronectin was analyzed by western blotting. ****p < 0.0001; 0.0001 <***p < 0.001; 0.01<*p < 0.05.

Figure 2. Effects of TXNIP deficiency on activation of mTORC1 and TFEB, and autophagy in diabetic kidneys. (A, B) The expression of p-p70S6K and p70S6K was analyzed by western blotting. (C) Immunohistochemical staining with TFEB antibody (Bar = 50 μm), and semi-quantitative analysis for TFEB (D). (E) Immunofluorescence staining with TFEB antibody, and yellow arrows displayed nuclear TFEB or tubular nuclear(Bar = 20 μm). Western blotting was implemented to examined the levels of total TFEB (F, G), nuclear TFEB and cytoplasmic TFEB (H, I), LC3B and p62 (J, K). (L) Autophagic vacuoles in renal tubular cells were observed by the transmission electron microscope, and yellow arrows represented autophagic vacuoles (bar = 2 μm, or 1 μm). ****p < 0.0001; 0.0001 <***p < 0.001; 0.001 <**p < 0.01; 0.01 <*p < 0.05.

Figure 3. Effects of TXNIP deficiency on fibrotic responses in HK-2 cells exposed to HG. The mRNA and protein levels of TXNIP were analyzed by RT-qPCR and western blotting respectively (A–C). (D) The mRNA levels of fibronectin, collagen I, α-SMA and E-cadherin were assessed by RT-qPCR. (E–G) The expression of fibronectin, collagen I, E-cadherin and α-SMA was analyzed by western blotting. (H) E-cadherin and α-SMA was detected by immunoinfluorence (bar = 25 μm). (I) Morphological alterations of HK-2 cells were observed by the inverted microscope (bar = 50 μm). ****p < 0.0001; 0.0001 < ***p < 0.001; 0.001 <**p < 0.01; 0.01 <*p < 0.05.

Figure 4. Effects of TXNIP deficiency on activation of mTORC1 and TFEB, as well as autophagy in HK-2 cells exposed to HG. (A, B) The expression of p-p70S6K and p70S6K was analyzed by western blotting. (C) The expression of TFEB, LC3B and p62 was examined in HK-2 cells by immunofluorescence (Bar = 25 μm). The expression of cytoplasmic TFEB, nuclear TFEB and total TFEB (D–G), LC3B and p62 (H, I) was analyzed by western blotting. ****p < 0.0001; 0.0001 <***p < 0.001; 0.001 <**p < 0.01; 0.01 <*p < 0.05.

Figure 5. Effects of inhibition of mTORC1 on activation of TFEB and autophagy in HK-2 cells under high glucose conditions. (A, B) The expression of p-p70S6K and p70S6K was analyzed by western blotting. (C, D) The expression of cytoplasmic TFEB and nuclear TFEB was analyzed by western blotting. (E) Immunofluorescence staining for TFEB was conducted in HK-2 cells (Bar = 25 μm). Western blotting (F, G) and immunofluorescence staining (H) were performed to examine the expression of LC3B and p62 (Bar = 25 μm). ****p < 0.0001; 0.0001 <***p < 0.001; 0.001 <**p < 0.01; 0.01 <*p < 0.05.

Figure 6. Effect of activated TFEB on autophagy flux in HK-2 cells exposed to HG. (A, B) The expression of cytoplasmic TFEB and nuclear TFEB was analyzed by western blotting. (C, D) Western blotting analysis for the expression of LC3B and p62. (E) Autophagic vacuoles in HK-2 cells were observed by the transmission electron microscope, and yellow arrows represented autophagic vacuoles (×4,000, or ×30,000). ****p < 0.0001; 0.0001 <***p < 0.001; 0.001 <**p < 0.01.

Figure 7. Effect of activated TFEB on EMT of HK-2 cells under HG conditions. (A) The mRNA levels of fibronectin, collagen I, α-SMA and E-cadherin were assessed by RT-qPCR. (B–D) The expression of fibronectin, collagen I, E-cadherin and α-SMA was analyzed by western blotting. (E) E-cadherin and α-SMA was detected by immunoinfluorence staining (Bar = 25 μm). (F) Morphological alterations of HK-2 cells were observed by the inverted microscope (Bar = 50 μm). (G) The mRNA levels of VPS11and ATP6V1H were assessed by RT-qPCR. ****p < 0.0001; 0.0001 <***p < 0.001; 0.001 <**p < 0.01; 0.01 <*p < 0.05.

Data availability statement

The authors confirm that all the data supporting the results of this study are available within the article and from the corresponding authors upon reasonable request.