Persistent subclinical renal injury in female rats following renal ischemia-reperfusion injury

Abstract

The incidence of acute kidney injury (AKI) continues to rise in both men and women. Although creatinine levels return to normal quicker in females following AKI than in males, it remains unclear whether subclinical renal injury persists in young females post-AKI. This study tested the hypothesis that AKI results in subclinical renal injury in females despite plasma creatinine returning to sham levels. For the present study, 12-13-week-old female Sprague-Dawley (SD) rats were randomized to sham or 45-minute warm bilateral ischemia-reperfusion surgery as an experimental model of ischemic AKI. Rats were euthanized 1, 3, 7, 14, or 30 days post-AKI/sham. Plasma creatinine, cystatin C, kidney injury molecule 1 (KIM-1), and NGAL were quantified via assay kits or immunoblotting. Kidneys were processed for histological analysis to assess tubular injury and fibrosis, and for electron microscopy to examine mitochondrial morphology. Immunoblots on kidney homogenates were performed to determine oxidative stress and apoptosis. Plasma creatinine levels were increased 24 hours post-AKI but returned to sham control levels three days post-AKI. However, cystatin C, KIM-1, and NGAL were increased 30 days post-AKI compared with sham. Tubular injury, tubulointerstitial fibrosis, and mitochondrial dysfunction were all increased in 30-day post-AKI rats compared with sham. Additionally, 30-day post-AKI rats had higher p-JNK expression and lower antioxidant enzyme glutathione peroxidase and catalase levels compared with sham. AKI resulted in higher expression of cleaved caspase 3, TUNEL+ cells, and caspase 9 than sham. Despite the normalization of creatinine levels, our data support the hypothesis that subclinical renal injury persists following ischemia-reperfusion injury in young female rats.

Keywords: apoptosis; fibrosis; mitochondrial damage; oxidative stress; tubular injury.

Figure 1. Female rats exhibit persistent subclinical renal injury 30 days post-AKI, despite resolution of plasma creatinine levels by three days post-AKI.

For this study, 12–13-week-old female SD rats were randomized to sham or 45-minute bilateral ischemia-reperfusion surgery (AKI) and allowed to recover 1, 3, 7, 14, or 30 days post-AKI. Plasma creatinine was measured at each timepoint (A), and plasma cystatin C was measured in samples collected 1, 7, and 30 days post AKI (B); n = 5–7. Data are expressed as mean ± SEM and compared via one-way ANOVA with Tukey post-hoc test for multiple comparisons, *P < 0.05, **P < 0.01, ****P < 0.0001. KIM-1 (C,E) and NGAL (D,F) expression were visualized via immunohistochemical analysis: 20× magnification, 50 µm, and measured by Western blot analysis in rats 30 days post-sham or AKI surgery; n = 6–7. Data are expressed as mean ± SEM and compared via Student t-test, *P < 0.05 vs. sham. AKI, acute kidney injury; SD, Sprague-Dawley.

Figure 2. AKI induces sustained tubular injury and fibrosis in young female rats.

Renal histology was evaluated using H&E (A) and renal fibrosis was assessed via Masson’s Trichrome (B) in 12–13-week old female SD rats randomized to sham or 45-minute bilateral ischemia-reperfusion surgery (AKI) and allowed to recover 1, 3, 7, 14, or 30 days post-AKI; n = 5. Shown are representative images with data expressed as mean ± SEM and compared via one-way ANOVA with Tukey post-hoc test for multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. sham. AKI, acute kidney injury; H&E, hematoxylin and eosin; SD, Sprague-Dawley.

Figure 3. AKI female rats exhibit mitochondrial ultrastructural anomalies compared with sham controls 30 days post-AKI.

For this study, 12–13-week-old female SD rats were randomized to sham or 45-minute bilateral ischemia-reperfusion surgery (AKI) and allowed to recover 30 days post-AKI; n = 3 rats, n = 75–85 images analyzed/rat with each data point representing an average for all mitochondria analyzed. Shown are representative EM microphotographs (A), mitochondrial area (B), mitochondrial circularity (C), mitochondrial numbers (D), mitochondrial density (E), and mitochondrial health scores (F). Data are expressed as mean ± SEM and compared via a Student’s unpaired t-test, *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. sham. AKI, acute kidney injury; SD, Sprague-Dawley.

Figure 4. Female rats with a history of AKI have greater renal protein levels of MAPKs, markers of oxidative stress than sham controls.

Protein levels of p-JNK/SAPK, p-ERK1/2, and p-P38 were measured via Western blot analysis in whole kidney homogenates from 12- to 13-week-old female SD rats randomized to sham or 45-minute bilateral ischemia-reperfusion surgery (AKI) and allowed to recover 30 days post-AKI; n = 6–7. Representative images and densitometric analysis of protein expression are shown for p-JNK/SAPK (A), p-ERK1/2 (B), and p-P38 (C). Data are expressed as mean ± SEM and compared via Student’s unpaired t-test, *P < 0.05 vs. sham. AKI, acute kidney injury; MAPKs, mitogen-activated protein kinases; SD, Sprague-Dawley.

Figure 5. Female rats with a history of AKI have less GPx1 and catalase protein levels vs. sham controls.

Antioxidant enzyme protein levels were measured via Western blot analysis in whole kidney protein homogenates from 12- to 13-week-old female SD rats randomized to sham or 45-minute bilateral ischemia-reperfusion surgery (AKI) and allowed to recover 30 days post-AKI; n = 6–7. Representative images and densitometric analysis of protein expression are shown show for GPx1 (A), catalase (B), and SOD2 (C). Total SOD activity was measured via SOD Assay kit according to the manufacturer’s instructions (D). Data are expressed as mean ± SEM and compared via a Student’s unpaired t-test, *P < 0.05, **P < 0.01 vs. sham. AKI, acute kidney injury; GPx1, glutathione peroxidase; SD, Sprague-Dawley; SOD, super oxide dismutase.

Figure 6. AKI induces renal apoptosis in female rats.

Markers of renal cell death were measured via Western blot analysis in whole kidney protein homogenates from 12- to 13-week-old female SD rats randomized to sham or 45-minute bilateral ischemia-reperfusion surgery (AKI) and allowed to recover 30 days post-AKI; n = 6–7. Representative images and densitometric analysis of protein expression are shown for cleaved caspase-3 (A) and caspase-9 (B). TUNEL-positive nuclei were assessed using the ApopTag plus Peroxidation in situ Apoptosis Detection kit (C). Shown are representative images and mean data. Data are expressed as mean ± SEM and compared via a Student’s unpaired t-test, *P < 0.05, ***P < 0.001 vs. sham. AKI, acute kidney injury; SD, Sprague-Dawley.