Pyridoxamine reduces postinjury fibrosis and improves functional recovery after acute kidney injury

Abstract

Acute kidney injury (AKI) is a common and independent risk factor for death and chronic kidney disease (CKD). Despite promising preclinical data, there is no evidence that antioxidants reduce the severity of injury, increase recovery, or prevent CKD in patients with AKI. Pyridoxamine (PM) is a structural analog of vitamin B6 that interferes with oxidative macromolecular damage via a number of different mechanisms and is in a phase 3 clinical efficacy trial to delay CKD progression in patients with diabetic kidney disease. Because oxidative stress is implicated as one of the main drivers of renal injury after AKI, the ability of PM to interfere with multiple aspects of oxidative damage may be favorable for AKI treatment. In these studies we therefore evaluated PM treatment in a mouse model of AKI. Pretreatment with PM caused a dose-dependent reduction in acute tubular injury, long-term postinjury fibrosis, as well as improved functional recovery after ischemia-reperfusion AKI (IR-AKI). This was associated with a dose-dependent reduction in the oxidative stress marker isofuran-to-F2-isoprostane ratio, indicating that PM reduces renal oxidative damage post-AKI. PM also reduced postinjury fibrosis when administered 24 h after the initiating injury, but this was not associated with improvement in functional recovery after IR-AKI. This is the first report showing that treatment with PM reduces short- and long-term injury, fibrosis, and renal functional recovery after IR-AKI. These preclinical findings suggest that PM, which has a favorable clinical safety profile, holds therapeutic promise for AKI and, most importantly, for prevention of adverse long-term outcomes after AKI.

Keywords: oxidative stress; renal fibrosis; renal function.

Fig. 1.

CONSORT flow diagram of preclinical studies. This diagram shows how the animals in this study were used. We performed three sets of experiments with 99 mice. Of these, a total of 3 mice died: 2 mice at the time of contralateral nephrectomy, both in the pyridoxamine (PM) treatment groups, and 1 mouse died after nephrectomy in one of the PM treatment groups. All of the assays, including histology, quantitative RT-PCR, and serum creatinine assays, were performed in all of the surviving mice, with the exception of serum creatinine analyses in 5 mice for which serum sample volumes were insufficient for the analyses. Vehicle and pretreatment with 1,000 mg·kg⁻¹·day⁻¹ PM from experiment 3 were included with data from experiment 2 for analysis of long-term dose dependence follow-up.

Fig. 2.

Dose-dependent effects of PM pretreatment on renal oxidative stress and early injury after ischemia-reperfusion acute kidney injury (IR-AKI). A: experimental design. Mice were either left uninjured or underwent unilateral renal pedicle clamping to induce AKI. The AKI mice were pretreated for 3 days with either vehicle or 500 or 1,000 mg·kg⁻¹·day⁻¹ PM in drinking water. Treatment was continued until the end of the experiment. Mice were killed, and kidneys were harvested for analysis 3 days after the initial injury. B: effect of PM treatment on renal oxidative stress. Isofuran-to-F2-isoprostane ratios were determined in renal tissues. C: plasma PM levels 3 days after injury. D and E: effect of PM pretreatment on renal injury markers. Renal Kim1 and N-Gal mRNA expression levels normalized to Gapdh mRNA. Results are expressed as fold change relative to uninjured controls. F and G: effect of PM pretreatment on acute renal tubular injury. F: representative images of the outer medulla (OM) from periodic acid-Schiff (PAS)-stained kidneys (scale bars, 50 μm). G: tubular injury scores in the OM (0–4, arbitrary units). Results are expressed as means ± SE, n = 10 mice/group; 3 uninjured controls. Data were analyzed using 1-way ANOVA with post hoc Tukey's correction for multiple pairwise comparisons. Results were only considered significant by ANOVA where P < 0.05. Post hoc analysis in these experiments is indicated above the error bars: *P < 0.05, **P < 0.01, ***P < 0.001, and #P < 0.0001 vs. uninjured controls. The differences indicated with brackets are PM-treated AKI vs. vehicle-treated AKI.

Fig. 3.

Dose-dependent effects of PM pretreatment on long-term renal fibrosis after IR-AKI. A: experimental design. Mice were either left uninjured or underwent unilateral renal pedicle clamping, followed by contralateral nephrectomy 8 days after the initial surgery. The AKI mice were pretreated for 3 days with vehicle or 500 or 1,000 mg·kg⁻¹·day⁻¹ PM in drinking water. The treatment was continued until the end of the experiment. Mice were killed, and kidneys were harvested for analysis 28 days after the initial injury. B: plasma PM levels 28 days after the initial injury. C–E: effect of PM pretreatment on renal expression of fibrosis markers: collagen1-α1 (Col1-α1, C), collagen3-α1 (Col3-α1, D), and α-smooth muscle actin (α-SMA, E) mRNA levels were normalized to Gapdh mRNA. Results are expressed as fold change relative to uninjured controls. F: representative Western blot for collagen IV α₂ chain (Col IV), α-SMA, and β-actin loading control in whole kidney lysates 28 days after injury from uninjured mice, mice after IR-AKI treated with vehicle, and mice with IR-AKI treated with 1,000 mg·kg⁻¹·day⁻¹ PM in drinking water. G and H: quantification of α-SMA (G) and Col IV (H) Western blots normalized to β-actin. Results are expressed as mean ± SE fold changes compared with uninjured controls: 4 uninjured controls, 10 IR-AKI treated with vehicle; 7 IR-AKI treated with PM. I: representative images showing collagen-specific polarized light birefringence of Sirius red-stained tissues [outer medulla (OM); scale bars 50 μm]. J: quantification of Sirius red staining expressed as fold change relative to uninjured controls. K: representative images of PAS-stained kidneys (OM, scale bars 100 μM). L: chronic tubular injury scores in the OM (0–4, arbitrary units). Results are expressed as means ± SE, 8 uninjured controls, 20 vehicle treated; 10 PM treated at 500 mg·kg⁻¹·day⁻¹ and 10 PM-treated at 1,000 mg·kg⁻¹·day⁻¹. Data were analyzed using 1-way ANOVA with post hoc Tukey's correction for multiple pairwise comparisons. Results were only considered significant by ANOVA where P < 0.05. Post hoc analysis in these experiments is indicated above the error bars: *P < 0.05, **P < 0.01, ***P < 0.001, and #P < 0.0001 vs. uninjured controls. The differences indicated with the brackets are PM-treated IR-AKI vs. vehicle-treated IR-AKI.

Fig. 4.

Dose-dependent effects of PM pretreatment on functional recovery after IR-AKI. The experimental design was the same as in Fig. 3. Mice were pretreated for 3 days with either vehicle or PM at 500 or 1,000 mg·kg⁻¹·day⁻¹ in drinking water, which was continued until the end of the experiment. A: serum creatinine 9 days after the initial injury. Serum creatinine was measured using an enzymatic cascade assay that only detects changes in serum creatinine ≥0.3 mg/dl. B: serum creatinine 28 days after injury. Serum creatinine was measured using a mass spectrometry-based approach, which is a more sensitive assay able to detect changes in serum creatinine of ≥0.1 mg/dl. Individual data points and means for each group are shown. Data were analyzed using 1-way ANOVA with post hoc Tukey's correction for multiple pairwise comparisons. Results were only considered significant by ANOVA where P < 0.05. Post hoc analysis in these experiments is indicated above the data points: *P < 0.05, **P < 0.01, and #P < 0.0001 vs. uninjured controls. The differences indicated with the brackets are PM-treated AKI vs. vehicle-treated AKI.

Fig. 5.

Effects of delayed treatment with PM on long-term renal fibrosis and functional recovery after IR-AKI. The experimental design was the same as in Fig. 3, except mice were treated with PM at 1,000 mg·kg⁻¹·day⁻¹ starting 24 h after the initial injury. A–C: effect of PM treatment on renal expression of fibrosis markers: Col1-α1 (A), Col3-α1 (B), and α-SMA (C) mRNA levels normalized to Gapdh mRNA. Results are expressed as fold change relative to uninjured controls. D: representative images showing collagen-specific polarized light birefringence of Sirius red-stained tissues (OM; scale bars 50 μm). E: quantification of Sirius red staining expressed as fold change relative to uninjured controls. F: representative images of PAS-stained kidneys (OM, scale bars 100 μM). G: chronic tubular injury scores in the OM (0–4, arbitrary units). Effect of PM treatment on serum creatinine at day 9 (H) and day 28 (I). Results are expressed as means ± SE, 8 uninjured controls, 10 vehicle- and 10 PM-treated mice. Individual data points and means for each group are shown in H and I. Data were analyzed using 1-way ANOVA with post hoc Tukey's correction for multiple pairwise comparisons. Results only indicated if ANOVA P < 0.05. Post hoc analysis in these experiments is indicated above the error bars or data points: *P < 0.05, **P < 0.01, and #P < 0.0001 vs. uninjured controls. The differences indicated with the brackets are PM-treated AKI vs. vehicle-treated AKI.