Persistent oxidative stress following renal ischemia-reperfusion injury increases ANG II hemodynamic and fibrotic activity

Abstract

ANG II is a potent renal vasoconstrictor and profibrotic factor and its activity is enhanced by oxidative stress. We sought to determine whether renal oxidative stress was persistent following recovery from acute kidney injury (AKI) induced by ischemia-reperfusion (I/R) injury in rats and whether this resulted in increased ANG II sensitivity. Rats were allowed to recover from bilateral renal I/R injury for 5 wk and renal blood flow responses were measured. Post-AKI rats showed significantly enhanced renal vasoconstrictor responses to ANG II relative to sham-operated controls and treatment of AKI rats with apocynin (15 mM, in the drinking water) normalized these responses. Recovery from AKI for 5 wk resulted in sustained oxidant stress as indicated by increased dihydroethidium incorporation in renal tissue slices and was normalized in apocynin-treated rats. Surprisingly, the renal mRNA expression for common NADPH oxidase subunits was not altered in kidneys following recovery from AKI; however, mRNA screening using PCR arrays suggested that post-AKI rats had decreased renal Gpx3 mRNA and an increased expression other prooxidant genes such as lactoperoxidase, myeloperoxidase, and dual oxidase-1. When rats were infused for 7 days with ANG II (100 ng·kg(-1)·min(-1)), renal fibrosis was not apparent in sham-operated control rats, but it was enhanced in post-AKI rats. The profibrotic response was significantly attenuated in rats treated with apocynin. These data suggest that there is sustained renal oxidant stress following recovery from AKI that alters both renal hemodynamic and fibrotic responses to ANG II, and may contribute to the transition to chronic kidney disease following AKI.

Fig. 1.

Experimental schema to determine the potential role of persistent reactive oxygen species (ROS) activity on alterations of renal function following acute kidney injury (AKI). A: summarizes the design of study I to determine the role of ischemia-reperfusion (I/R) injury and recovery on ANG II-induced alterations in renal hemodynamic responses. Rats are maintained on standard 0.4% NaCl diet during 5 wk of recovery from bilateral I/R or sham surgery. Some rats were treated with apocynin-supplemented drinking water (15 mM) during the final week of recovery. At 5 wk, rats were evaluated for renal hemodynamic responses to ANG II. B: summarizes the time line for study II to determine the role of ROS on blood pressure (BP) and renal fibrotic response to chronic ANG II infusion. At 2 wk following recovery from sham or I/R surgery, animals were chronically instrumented with telemetric BP devices and chronic venous infusions lines. After 2 days of baseline measurements under saline infusion, the increase in blood pressure and fibrosis was determined in response to a 7-day infusion of ANG II. RBF, renal blood flow; APO, apocynin.

Fig. 2.

Changes in total RBF (A) and renal vascular resistance (RVR; B) are shown for anesthetized rats following 5 wk of recovery from either sham operation or bilateral renal I/R. Some animals received APO in the drinking water for 1 wk before evaluation of renal hemodynamic response. N for each group is shown in parentheses. A and B: a, b, and c indicate a change in value relative to baseline within each group, P < 0.05; *P < 0.05 in post-AKI group vs. sham-operated and APO-treated groups, using ANOVA and Student-Newman-Keuls post hoc test.

Fig. 3.

Hydroethidium incorporation into rat kidney tissue slices following recovery from I/R-induced AKI. Shown are representative confocal images through renal outer medulla of a sham-operated rat (A) and a rat at 5 wk of recovery from AKI (B). Hydroethidum fluorescence is shown in red while DAPI counterstaining is shown in blue. Hydroethidium signal was rarely observed in sham-operated samples, while multiple structures evidenced incorporation following recovery from AKI, primarily in unidentified interstitial cells (thin white arrow). Fluorescent signal was also apparent in some nuclear structures (thin red arrow) and diffusely (thick red arrow) in many tubular structures. Magnification is shown in A. C: quantitative analysis of hydroethidium incorporation in rat kidney slices comparing dihydroethidium (DHE) signal derived from tissues of rats following sham surgery, or post-AKI rats treated with or without APO in the drinking water. N refers to the number of different animals in each comparison and values for each comparison derived from analysis of 8 images per animal/treatment. *P < 0.05 post-AKI vs. sham. #P < 0.05 treated AKI groups vs. control AKI group, by Student's t-test.

Fig. 4.

Quantitative (Q)-RT-PCR analysis of NADPH oxidase-associated genes in kidney following recovery from AKI. Data are derived from total RNA from whole kidney of sham-operated, AKI-, and AKI-APO-treated rats. Values for 2^ΔCt were generated by normalizing to RPLP1 gene expression. The data are expressed as Log(2) ratios relative to the sham-operated control, such that lower expression in the AKI relative to sham is indicated by a negative value, while a higher expression is indicated by a positive value. No values were significantly different by Student's t-test. Note the Nox4 gene expression is shown in Fig. 5.

Fig. 5.

Q-RT-PCR analysis of oxidant-associated genes in kidney following recovery from AKI. Total RNA derived from whole kidney of sham-operated rats and rats following recovery from I/R using “oxidant stress and anti-oxidant” pathway arrays in kidneys of sham and post-AKI rats (see methods). A: data are expressed as Log(2)-normalized ratios relative to the expression in sham; therefore, greater expression in AKI vs. sham corresponds to a positive value and a lower expression in AKI is represented by a negative value. Shown are data from 14 of the 84 genes evaluated (the 7 most upregulated and 7 downregulated). A 95% confidence interval of Log(2) ratios was determined to be ±1.6 and was used to define significance (dotted line). B, C, and D: data from individual Q-PCR verification of array results for the GPX3, dual-oxidase 1 (Duox1), and lactoperoxidase (LPO) genes, respectively, in a wider number of samples (n = 6 per group) and in APO-treated rats. Data are expressed as means ± SE of 2^ΔCt values normalized to the RPLP1 genes. *P < 0.05 relative to sham control by Student's t-test.

Fig. 6.

Effect of chronic ANG II infusion on renal morphology and interstitial fibrosis in sham-operated and postischemic rats. Representative Masson's trichrome-stained sections through rat renal cortex are shown from a sham-operated rat (A), post-AKI rat supplemented with saline alone (B), a post-AKI rat supplemented with ANG II (C), and a post-AKI rat with ANG II and APO (D). Trichrome-positive interstitial areas were apparent following AKI but more prominent following ANG II infusion (black arrows), and less apparent in APO-treated rats. Magnification is shown in D. E: quantitative analysis of interstitial volume scores based on trichrome-stained sections derived from counting the number of points in arbitrary grid that overlay the interstitial space. Data are means ± SE. * And #P < 0.05 in ANG II-AKI group relative to sham and AKI-ANG II and APO-treated groups, respectively.

Fig. 7.

Effect of chronic ANG II infusion on S100A4-containing cells in kidney following AKI. Representative immunohistochemical images through rat renal outer medulla are shown from a sham-operated rat infused with ANG II (A), post-AKI rat supplemented with saline alone (B), a post-AKI rat supplemented with ANG II (C), and a post-AKI rat with ANG II and APO (D). Black arrows indicate positively stained interstitial cells most prevalent in ANG II infusion into AKI rats. Magnification shown in D.