Diabetes predisposes mice to aggravated tubular damage after uIRI, which is abrogated by RTN1A inhibition. We previously developed a doxycycline-inducible RTEC-specific Rtn1a-knockdown model, resulting in approximately 80% inhibition of Rtn1a expression (Rtn1aKD) (10, 11, 14). To examine whether diabetes would predispose mice toward accelerated CKD transition after ischemic AKI, diabetes was first induced in Rtn1aKD and control littermate mice (lacking one or more transgenes), and mice were subjected to unilateral ischemic reperfusion injury (uIRI). As Rtn1aKD and control littermates are in the mixed C57BL/6J background that is more resistant to DKD development, diabetes was induced by the administration of a high-fat diet (HFD) for 12 weeks, followed by low-dose streptozotocin (STZ) injections, as shown in Figure 1A. This experimental model is useful in capturing the aspects of both dyslipidemia and insulin deficiency seen in both type 1 and type 2 diabetic individuals, as approximately 60% of type 1 diabetic patients develop obesity, and type 2 diabetic patients often have both obesity and insulin deficiency (22, 23). Nondiabetic groups of mice were administered normal diet and vehicle injections. All mice on HFD gradually gained significant body weight by 8 weeks of HFD, but showed prominent hyperglycemia only after receiving STZ injections (Figure 1B). RTEC-specific knockdown of Rtn1a in diabetic mice did not affect body weight gain or blood glucose levels (Figure 1B). uIRI was induced in control and diabetic mice by clamping the right renal pedicle for 30 minutes at 37°C 4 weeks after STZ or vehicle injection. All mice were euthanized at 20 weeks after initiation of HFD feeding (after 4 weeks of uIRI) (Figure 1A). Unilateral nephrectomy (uNx) of the contralateral (left) kidneys was performed in uIRI mice 1 day before euthanasia to determine the renal function of the ischemic kidney. Thus, the experimental groups consisted of (a) nondiabetic control mice without AKI (Control), (b) nondiabetic mice with uIRI (uIRI), (c) diabetic mice with uIRI (DM+uIRI), and (d) diabetic Rtn1aKD mice with uIRI (DM+uIRI+Rtn1aKD).

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+Rtn1aKD). 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).

Since RTN1A expression increases in RTECs in human DKD and after AKI and is associated with the severity of tubular injury (10, 11), we first examined the expression of RTN1A in mouse kidneys. As expected, immunofluorescent staining indicated that RTN1A expression was hardly detectable in control mouse kidneys, but significantly increased in RTECs by uIRI and even more so by diabetes and uIRI (Figure 1, C and D). RTN1A knockdown effectively reduced its expression in diabetic mice with uIRI, but without a complete abrogation. The change in RTN1A expression was further validated by quantitative PCR (qPCR) of mouse kidney cortices (Figure 1E).

We next examined the kidney function in the 4 groups of mice. At 20 weeks, urine albumin was modestly elevated in both groups of diabetic mice when compared with nondiabetic mice (Figure 2A), but significantly less urine albumin was detected in DM+uIRI+Rtn1aKD mice compared with DM+uIRI mice. Significant elevation in blood urea nitrogen (BUN) levels was observed in all mice subjected to ischemic injury, but this was substantially augmented in DM+uIRI mice compared with uIRI mice (Figure 2A). Notably, DM+uIRI+Rtn1aKD mice, despite having diabetes, displayed BUN levels comparable to nondiabetic uIRI mice, suggesting that the aggravation of tubular damage by diabetes after AKI is abrogated by the reduction in RTN1A. Indeed, the histopathologic analysis of mouse kidneys was consistent with these observations. Periodic acid–Schiff–stained (PAS-stained) kidneys showed sustained tubular injuries at 4 weeks after ischemic injury in uIRI mice, consistent with incomplete recovery after AKI (Figure 2, B and C). Notably, increased severity of tubulointerstitial damage was observed in DM+uIRI mice, with atrophic tubules and thickened tubular basement membranes, which was substantially reduced in DM+uIRI+Rtn1aKD mice (Figure 2, B and C). However, at this early DKD stage (8 weeks after STZ injection), the diabetic mice did not yet show any overt glomerular lesions (Figure 2, B and C). Thus, it is likely that the aggravated tubular damage in DM+uIRI mice contributes to the further increase in albuminuria observed at 20 weeks in comparison with DM+uIRI+Rtn1aKD mice (Figure 2A).

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+Rtn1aKD 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).

Diabetes predisposes mice to increased fibrosis, inflammation, and tubular senescence after uIRI, which is abrogated by RTN1A inhibition. Given the marked tubulointerstitial damage sustained after uIRI in diabetic mice, we next examined the effects of diabetes on the ensuing renal fibrosis development. Masson’s trichome and picrosirius red with fast green staining showed mild renal fibrosis in the nondiabetic uIRI mice, but much more pronounced fibrosis development in DM+uIRI mice (Figure 3, A and B). RTN1A knockdown markedly attenuated renal fibrosis in diabetic mice with uIRI, consistent with reduced tubular injury. We further validated the histologic findings with the expression of fibrosis markers, fibronectin (FN1), α-smooth muscle actin (α-SMA), and matrix metallopeptidase 2 (MMP2) by qPCR and Western blot analyses. Both analyses confirmed a significant increase in the expression of profibrotic markers in DM+uIRI mouse kidneys in comparison with uIRI, and that RTN1A knockdown counteracted these changes (Figure 3, C–E). We also observed an increased expression of vimentin and reduced expression of E-cadherin in kidney cortices of DM+uIRI mice, reflecting a partial de-differentiation of RTECs (Figure 3, D and E), which were also normalized by RTN1A knockdown. Another important aspect of the AKI-to-CKD transition is the increased senescence of tubular epithelial cells due to maladaptive repair response (7). Immunofluorescent staining of senescence markers, γ-H2A.X and p21, showed a marked increase in these markers in DM+uIRI mice (Figure 4, A and B), which was mitigated by RTN1A knockdown. Consistent with these findings, RTN1A knockdown also reduced the severity of renal inflammation in diabetic mice with uIRI, as shown by F4/80 immunostaining and qPCR of inflammatory cytokines (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.185826DS1). These results are consistent with the above findings that underlying diabetes promotes and accelerates AKI-to-CKD transition by aggravating tubular damage, fibrosis, and inflammation and that RTN1A is a major culprit in this process.

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.

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.

ER stress and mitochondrial dysfunction in RTECs underlie the accelerated AKI-to-CKD transition in diabetic mice, which are reversed by RTN1A inhibition. We previously demonstrated that RTN1A promotes CKD transition by promoting sustained ER stress and mitochondrial dysfunction by regulating the ER-mitochondria contact (EMC) sites (10, 11, 15). Therefore, we next examined the expression of ER stress and mitochondrial markers in the kidney cortices of mice with uIRI. For ER stress, we examined the expression of ER chaperone protein, glucose-regulated protein 78 (GRP78), also known as BiP, and downstream molecule in the unfolded protein response (UPR), C/EBP homologous protein (CHOP), which triggers apoptosis under sustained UPR and ER stress. We previously showed that GRP78 and CHOP are increased in RTECs in AKI patient kidneys (11). Western blot analysis showed a modest increase in GRP78 expression in kidney cortices of all mice with uIRI compared with healthy controls, but significantly higher levels of GRP78 in DM+uIRI kidneys compared with the other uIRI groups (Figure 5, A and B). Importantly, the expression of CHOP, a marker of the apoptotic response in UPR, and apoptosis marker cleaved caspase-3 was significantly elevated in DM+uIRI kidneys in comparison with uIRI kidneys, indicative of increased RTEC apoptosis in DM+uIRI mice (Figure 5, A and B), while RTN1A knockdown effectively reduced RTEC apoptosis. By immunostaining, we confirmed that these marked changes were occurring in RTECs. As shown in Figure 5, C and D, levels of phosphorylated PKR-like ER kinase (PERK), an ER transmembrane protein kinase that oligomerizes and is autophosphorylated in response to UPR, was not detectable in healthy control kidneys, but increased in the tubules in uIRI kidneys. p-PERK expression was markedly upregulated in the tubules of DM+uIRI mice, but attenuated by RTN1A knockdown, consistent with the above Western blot analysis. These results are also consistent with our previous observation that RTN1A directly interacts with PERK and that its increased expression results in increased PERK and CHOP expression, leading to RTEC apoptosis (10, 11).

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.

In addition to inducing ER stress, RTN1A was recently identified to mediate ER and mitochondrial crosstalk as a component of EMC sites (15, 24). Increasing evidence supports EMCs as important regulators of mitochondrial homeostasis, apoptosis, and autophagy and that EMC integrity is disrupted in tubular cells in diabetic kidneys (25, 26). We further demonstrated that increased RTN1A expression in RTECs concomitantly worsened ER stress and mitochondrial function in diabetic kidneys (15). Therefore, we next examined the expression of mitochondrial proteins, COX IV and TFAM, by Western blot analysis. As anticipated, a marked reduction in COX IV and TFAM was observed in DM+uIRI kidneys in comparison with the other uIRI groups (Figure 6, A and B). Immunostaining for mitochondrial proteins COX IV and hexokinase-1 (HK1) further corroborated these observations and indicated a marked decrease in mitochondrial proteins by uIRI, which was compounded by underlying diabetes but attenuated by RTN1A knockdown (Figure 6, C and D). Transmission electronic microscopy also revealed markedly altered mitochondrial morphology in kidney cortices of DM+uIRI mice compared with nondiabetic uIRI mice (Supplemental Figure 2), which was markedly reduced by RTN1A knockdown. These results are also consistent with our previous observation that ER-bound RTN1A interacted with mitochondrial HK1, causing its degradation and resulting in mitochondrial dysfunction and the induction of inflammasome and apoptosis pathways in RTECs (15). Thus, RTN1A knockdown restrained the diabetes-mediated aggravation of tubular injury and loss following ischemic injury by reducing ER stress and mitochondrial dysfunction in RTECs.

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.