Dicer deficiency in proximal tubules exacerbates renal injury and tubulointerstitial fibrosis and upregulates Smad2/3

Abstract

Renal fibrosis is a common pathological feature in chronic kidney disease (CKD), including diabetic kidney disease (DKD) and obstructive nephropathy. Multiple microRNAs have been implicated in the pathogenesis of both DKD and obstructive nephropathy, although the overall role of microRNAs in tubular injury and renal fibrosis in CKD is unclear. Dicer (a key RNase III enzyme for microRNA biogenesis) was specifically ablated from kidney proximal tubules in mice via the Cre-lox system to deplete micoRNAs. Proximal tubular Dicer knockout (PT- Dicer KO) mice and wild-type (WT) littermates were subjected to streptozotocin (STZ) treatment to induce DKD or unilateral ureteral obstruction (UUO) to induce obstructive nephropathy. Renal hypertrophy, renal tubular apoptosis, kidney inflammation, and tubulointerstitial fibrosis were examined. Compared with WT mice, PT- Dicer KO mice showed more severe tubular injury and renal inflammation following STZ treatment. These mice also developed higher levels of tubolointerstitial fibrosis. Meanwhile, PT- Dicer KO mice had a significantly higher Smad2/3 expression in kidneys than WT mice (at 6 mo of age) in both control and STZ-treated mice. Similarly, UUO induced more severe renal injury, inflammation, and interstitial fibrosis in PT- Dicer KO mice than WT. Although we did not detect obvious Smad2/3 expression in sham-operated mice (2-3 mo old), significantly more Smad2/3 was induced in obstructed PT- Dicer KO kidneys. These results supported a protective role of Dicer-dependent microRNA synthesis in renal injury and fibrosis development in CKD, specifically in DKD and obstructive nephropathy. Depletion of Dicer and microRNAs may upregulate Smad2/3-related signaling pathway to enhance the progression of CKD.

Keywords: Dicer; Smad2/3; diabetic kidney disease; fibrosis; microRNA; unilateral urethral obstruction.

Fig. 1.

Dicer deficiency in proximal tubules aggravates renal tubular injury and interstitial inflammation in diabetic mice. Eight weeks old proximal tubule (PT)-Dicer wild-type (WT) and knockout (KO) mice were treated with streptozotocin (STZ) or without STZ (Control, CT) and examined after 16 wk. A: kidney weight/body weight. Data are expressed as means ± SD. B: representative images of periodic acid-Schiff (PAS) staining showing the vacuolization (arrowheads) in tubules in PT-Dicer KO diabetic. Scale bar: 50 µm. C: representative images of TUNEL assay and TUNEL-positive signal counting showing more apoptotic tubular cells in renal cortex and outer medulla in PT-Dicer KO diabetic compared with PT-Dicer WT diabetic mice. D: representative images of immunohistochemistry staining of macrophage showing more obvious macrophage infiltration in kidney cortex from PT-Dicer KO diabetic mice. E: urine albumin-to-creatinine ratio (ACR). n ≥ 7.*P < 0.05, vs. WT-Control (WT-CT); #P < 0.05, vs. STZ-treated PT-Dicer WT mice.

Fig. 2.

Dicer deficiency in proximal tubules aggravates renal interstitial fibrosis in diabetic mice. Eight-week-old proximal tubule (PT)-Dicer wild-type (WT) and PT-Dicer knockout (KO) mice were treated with streptozotocin (STZ) or without STZ (Control, CT) and examined after 16 wk. A: representative images of Masson trichrome staining and quantification of collagen deposition area ratio. Scale bar: 50 µm. B: representative images of immunohistochemistry staining of fibronectin and quantification of fibronectin staining positive area percentage. Scale bar: 50 µm. C: representative images of immunohistochemistry staining of collagen I- and quantification of collagen I-positive area percentage. Scale bar: 50 µm. D: representative immunoblots and densitometry quantification showing a significant increase of α-SMA in kidney cortex samples from PT-Dicer KO mice compared with WT diabetic mice. Cyclophilin B was used as loading control. n ≥ 7. *P < 0.05, vs. WT-CT; #P < 0.05, vs. PT-Dicer WT diabetic mice.

Fig. 3.

Dicer deficiency in proximal tubules aggravates renal tubular injury and interstitial inflammation in unilateral ureteral obstruction (UUO). Proximal tubule (PT)-Dicer wild-type (WT) and PT-Dicer knockout (KO) mice were subjected to either sham operation (Sham) or UUO surgery. Injured mice were euthanized 1 wk (UUO1w) or 2 wk (UUO2w) after UUO, and left kidneys were collected for histological and immunoblotting analyses. A: representative images of Periodic acid-Schiff (PAS) staining showing more severe renal injury in PT-Dicer KO mice after UUO. Scale bar: 50 µm. Arrowheads and asterisks stand for atrophic and atresic tubules, respectively. B: representative images of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and quantification of TUNEL-positive signals showing more apoptotic tubular cells in renal cortex and outer medulla in PT-Dicer KO mice compared with PT-Dicer WT after UUO injury. Scale bar: 50 µm. *Statistically significant difference compared with WT group. C: representative images of immunohistochemistry staining of macrophage showing more macrophage infiltration in kidney cortex in PT-Dicer KO mice compared with WT mice after UUO injury. Scale bar: 50 µm. n ≥ 10. *P < 0.05, vs. WT-sham; #P < 0.05, vs. PT-Dicer WT UUO.

Fig. 4.

Dicer deficiency in proximal tubules aggravates renal interstitial fibrosis in unilateral ureteral obstruction (UUO). Proximal tubule (PT)-Dicer wild-type (WT) and PT-Dicer knockout (KO) mice were subjected to either sham operation (sham) or UUO surgery. Mice were euthanized at 1 wk (UUO1w) or 2 wk (UUO2w) after UUO injury, and left kidneys were collected for histological and immunoblotting analyses. A: representative Immunoblots showing more fibronectin induced in kidney cortex from PT-Dicer KO mice at 1 wk after UUO. Cyclophilin B was used as a loading control. B: representative immunoblots showing more induction of fibronectin and collagen I in PT-Dicer KO mice compared with PT-Dicer WT at 2 wk after UUO. Cyclophilin B was used as a loading control. D: representative images of Masson Trichrome staining and quantification of collagen deposition area. Scale bar = 50 µm. n ≥ 10.*P < 0.05, vs. WT-sham; #P < 0.05, vs. PT-Dicer WT UUO.

Fig. 5.

Increased Smad2/3 expression in proximal tubule (PT)-Dicer knockout (KO) kidney may contribute to enhanced renal interstitial fibrosis in diabetic and unilateral ureteral obstruction (UUO) nephropathy. A: representative immunoblots of Smad2/3 in mouse kidney. C57BL/6 mice were subjected to either sham operation or UUO surgery. Mice were euthanized at 4 days (UUO4d), 1 wk (UUO1w), or 2 wk (UUO2w) after surgery, and left kidneys were collected for immunoblotting analyses of Smad2/3. Cyclophilin B was used as loading control. Representative immunoblots are presented. The results showed a time-dependent increase of Smad2/3 in kidney cortex following UUO. B: representative immunoblots and densitometry quantification of Smad2/3 levels. PT-Dicer wild-type (WT) and PT-Dicer KO mice were subjected to either sham operation or UUO surgery. Mice were euthanized at 1 wk after surgery, and left kidneys were collected for immunoblot analysis of Smad2/3 expression and densitometry quantification. The results showed the significantly increased Smad2/3 in kidney cortex in both PT-Dicer WT and PT-Dicer KO mice subjected to UUO compared with sham controls. The levels of Smad2/3 were significantly higher in PT-Dicer KO mice than in PT-Dicer WT mice after UUO. n ≥ 10. *P < 0.05, vs WT-sham; #P < 0.05, vs. PT-Dicer WT UUO. Cyclophilin B was used as loading control. C: representative immunoblots and densitometry quantification of Smad2/3 levels. Eight-week-old PT-Dicer WT and PT-Dicer KO mice were treated with streptozotocin (STZ) or without STZ (Control, CT) and examined after 16 wk. The results showed that Smad2/3 levels were significantly elevated in PT-Dicer KO mice compared with PT-Dicer WT mice in both control and diabetic mice. n ≥ 7. *P < 0.05, vs. WT-CT; #P < 0.05, vs. PT-Dicer WT diabetic mice. Cyclophilin B was used as loading control. D: representative immunoblot of Smad2/3. Left kidneys from 6-mo-old PT-Dicer WT and PT-Dicer KO mice were collected for immunoblot analyses. The results showed the remarkably increased Smad2/3 levels in PT-Dicer KO kidney cortex than in WT cortex. Cyclophilin B was used as loading control. E: Smad2 and Smad3 mRNA levels in the renal cortex in 6-mo-old PT-Dicer KO mice by real-time PCR; n = 5. No significant difference in Smad2/3 mRNA levels was found between WT and PT-Dicer KO mice under the control condition. F: representative images of Masson Trichrome staining of kidney samples from 7-mo-old PT-Dicer WT and PT-Dicer KO mice showing obvious deposition and accumulation of collagen in PT-Dicer KO mice compared with PT-Dicer WT mice; n = 6. Scale bar: 50 µm.