RTN1A mediates diabetes-induced AKI-to-CKD transition

Abstract

Diabetic patients have increased susceptibility to acute kidney injury (AKI), and AKI could progress to chronic tubulointerstitial injury and fibrosis, referred to as AKI-to-chronic kidney disease (AKI-to-CKD) transition. However, whether diabetes directly promotes AKI-to-CKD transition is not known. We previously showed that reticulon-1A (RTN1A), a gene highly upregulated in injured renal tubular epithelial cells (RTECs), promotes AKI-to-CKD transition in nondiabetic settings. Therefore, we also examined whether reducing RTN1A expression could attenuate diabetes-induced AKI-to-CKD transition. Diabetes was induced by a high-fat diet and streptozotocin injections, and unilateral ischemic reperfusion injury was created as an AKI model in control, diabetic, and RTEC-specific Rtn1a-knockdown diabetic mice. AKI induced greater renal function decline, tubulointerstitial injury, and fibrosis in diabetic mice than in nondiabetic mice. Reduction of RTN1A markedly reduced the CKD development following AKI in diabetic mice, which was associated with reduced ER stress and mitochondrial dysfunction in RTECs. These findings indicate that diabetes markedly accelerates AKI-to-CKD transition and that RTN1A is a crucial mediator of diabetes-induced AKI-to-CKD transition. The development of RTN1A inhibitors could potentially attenuate AKI-to-CKD transition in diabetic patients.

Keywords: Chronic kidney disease; Diabetes; Mitochondria; Nephrology.

Figure 1. RTN1A expression is increased in RTECs by ischemic and diabetic tubular damage.

(A) Schematics of the experimental outline. Six 8-week-old male C57BL/6J mice were randomized into 4 experimental groups consisting of control mice, mice with unilateral ischemic reperfusion injury (uIRI group), diabetic mice with uIRI (DM+uIRI), and diabetic mice with uIRI with Rtn1a knockdown (DM+uIRI+Rtn1a^KD). Diabetes was induced by high-fat diet (HFD) supplementation and low-dose streptozotocin (STZ) injections. Nondiabetic mice were given a normal diet (ND) with vehicle injections. All mice were euthanized after 20 weeks of each diet, and contralateral kidneys were removed 1 day (uNx, –d1) before endpoint analysis to assess kidney function in mice subjected to uIRI. (B) Body weight and blood glucose levels in the 4 groups are shown. Body weight change was statistically significant in diabetic mice starting at 8 weeks of HFD supplementation, and blood glucose levels were significantly elevated after STZ injection in diabetic mice compared with nondiabetic mice. (C) Representative RTN1A immunofluorescence images of mouse kidney sections. Negative control with IgG control is shown on the bottom. Nuclei are counterstained with DAPI. Scale bar: 20 μm. (D) Quantification of RTN1A⁺ area is shown as fold change relative to the Control group (n = 6 mice per group, 30 fields evaluated per mouse). (E) Real-time PCR analysis of total Rtn1a mRNA expression with primers that detect both mouse and human transcripts (n = 6 mice per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between indicated groups by 2-way ANOVA with Dunnett’s post hoc test (B) or 1-way ANOVA with Tukey’s post hoc test (D and E).

Figure 2. Diabetes predisposes mice to worsened kidney function after uIRI, which is attenuated by RTN1A knockdown.

(A) Kidney function assessment by urinary albumin-to-creatinine ratio (UACR) and blood urea nitrogen (BUN) levels. uIRI significantly accelerated albuminuria development in DM+uIRI mice, but not in DM+uIRI+Rtn1a^KD when examined at endpoint. BUN levels were significantly elevated in all mice with uIRI compared with controls, but only further heightened in DM+uIRI mice (n = 6 mice). (B) Representative images of PAS-stained kidneys. Dotted areas in the top panels are magnified in the bottom panels. Dashed lines in the top panels are magnified in the bottom panels. Scale bars: 50 μm. (C) Quantification of average tubular injury score, glomerular area, and percentage mesangial matrix area are shown (n = 6 mice, 30 fields evaluated for tubular injury score per mouse). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between indicated groups by 2-way ANOVA with Dunnett’s post hoc test (A, left), 1-way ANOVA with Tukey’s post hoc test (A, right), or Kruskall-Wallis test with Dunn’s post hoc test (C, left). Forty glomeruli were evaluated for glomerular area and mesangial fraction. NS by 1-way ANOVA with Tukey’s post hoc test (C, right).

Figure 3. Diabetes predisposes mice to increased renal fibrosis after uIRI, which is attenuated by RTN1A knockdown.

(A) Representative images of kidney sections stained with Masson’s trichrome (MTC) and picrosirius red (PSR) with fast green staining. Scale bar: 50 μm (original magnification, ×200). (B) Quantification of PSR-stained fibrotic area (%). Thirty fields were evaluated per mouse. (C) Real-time PCR analysis of fibrosis markers (Fn1, Acta2, and Mmp2) in mouse kidney cortices. (D) Representative Western blot analysis of fibrosis markers (FN1, α-SMA, and MMP2) and epithelial or mesenchymal markers (E-cadherin or vimentin, respectively) in mouse kidney cortices. (E) Densitometric analysis of proteins in 3D shown as a fold change relative to Control mice (n = 6 mice). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between indicated groups by 1-way ANOVA with Tukey’s post hoc test.

Figure 4. Diabetes exacerbates RTEC senescence in mice with uIRI, which is attenuated by RTN1A knockdown.

(A) Representative images of γ-H2A.X immunofluorescence and quantification. Scale bar: 20 μm. (B) Representative images of p21 immunofluorescence and quantification. Scale bar: 50 μm. n = 6 mice, 30 fields analyzed per mouse. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 between indicated groups by 1-way ANOVA with Tukey’s post hoc test.

Figure 5. Diabetes predisposes mice to increased ER stress in injured tubules, which is attenuated by RTN1A knockdown.

(A) Western blot analysis of ER stress (CHOP, GRP78) and apoptosis (cleaved caspase-3) markers in mouse kidney cortices. (B) Densitometric analysis of CHOP, GRP78, and cleaved caspase-3 expression (normalized to GAPDH) as a fold change relative to Control. (C) Representative immunohistochemistry images of phosphorylated protein kinase R–like ER kinase (p-PERK) in mouse kidneys. Scale bar: 50 μm. (D) Quantification of p-PERK⁺ area. n = 6 mice, 30 fields evaluated per mouse. *P < 0.05; ****P < 0.0001 between indicated groups by 1-way ANOVA with Tukey’s post hoc test.

Figure 6. Diabetes predisposes mice to increased mitochondrial damage in injured tubules, which is attenuated by RTN1A knockdown.

(A) Western blot analysis of mitochondrial markers COX IV and TFAM in the kidney cortices. (B) Densitometric analysis of COX IV and TFAM expression (normalized to GAPDH) as a fold change relative to Control. (C) Representative immunohistochemistry images of mitochondrial proteins COX IV and HK1 in mouse kidneys. Scale bars: 50 μm. (D) Quantification of COX IV⁺ and HK1⁺ areas (%). n = 6 mice per group, 30 fields evaluated per mouse. **P < 0.01; ***P < 0.001; ****P < 0.0001 between indicated groups by 1-way ANOVA with Tukey’s post hoc test.