Acute renal venous obstruction is more detrimental to the kidney than arterial occlusion: implication for murine models of acute kidney injury

Abstract

In this study, we compared the traditional murine model with renal pedicle clamp with models that clamped the renal artery or vein alone as well as to a whole body ischemia-reperfusion injury (WBIRI) model. Male C57BL/6J mice underwent either clamping of the renal artery, vein, or both (whole pedicle) for 30 or 45 min followed by reperfusion, or 10 min of cardiac arrest followed by resuscitation up to 24 h. After 30 min of ischemia, the mice with renal vein clamping showed the mostly increased serum creatinine and the most severe renal tubule injury. After 45 min of ischemia, all mice with renal vasculature clamping had a comparable increase in serum creatinine but the renal tubule injury was most severe in renal artery-clamped mice. Renal arterial blood flow was most decreased in mice with a renal vein clamp compared with a renal artery or pedicle clamp. A 30-or 45-min renal ischemia time led to a significant increase in the protein level of interleukin-6, keratinocyte-derived chemokine (KC), and granular colony-stimulating factor in the ischemic kidney, but the KC was the highest in the renal pedicle-clamped kidney and the lowest in the renal vein-clamped kidney. Of note, 10 min of WBIRI led to kidney dysfunction and structural injury, although less than longer time clamping of isolated renal vasculature. Our data demonstrate important differences in ischemic AKI models. Understanding these differences is important in designing future experimental studies in mice as well as clinical trials in humans.

Fig. 1.

Renal function of acute kidney injury (AKI) mice. Male C57BL/6 mice underwent 10 min of whole body ischemia-reperfusion injury (WBIRI) or 30 or 45 min of kidney ischemia (KI) by clamping the bilateral renal artery, vein, or pedicle followed by cardiopulmonary resuscitation or reperfusion. Blood samples were taken at 24 h after the procedure for measurement of serum creatinine levels. All the AKI mice had significant increases in the serum creatinine (SCr) level compared with sham-operated mice. However, WBIRI mice had a significant lower rise in SCr than kidney ischemia-reperfusion injury (IRI) mice except those with 30 min of renal artery clamping. *P < 0.05 vs. 30-min renal artery group. #P < 0.05 vs. all other KI groups except 30-min renal artery; n = 6 in 30-min KI and WBI; n = 5 in 45-min KI.

Fig. 2.

Representative photomicrograph of mouse kidney histology. Male C57BL/6 mice underwent 10 min of whole body ischemia (WBIRI), 30 or 45 min of clamping of the bilateral renal artery, vein, or pedicle followed by 24 h of reperfusion. A: sham. B: renal artery-clamped kidney. C: renal vein-clamped kidney. D: renal pedicle-clamped kidney. E: WBIRI. The kidneys were harvested upon euthanasia and stained with hematoxylin and eosin for histological examination under a light microscope. AKI mice had profound renal tubule necrosis compared with sham-operated mice. However, WBIRI mice had less acute tubular necrosis than kidney IRI mice. Arrows indicate necrotic tubules. Original magnification: ×20. Scale bars = 50 μm.

Fig. 3.

Semiquantification of kidney tubular injury. Male C57BL/6 mice underwent 10 min of whole body ischemia (WBIRI), 30 or 45 min of KI by clamping the bilateral renal artery, vein, or pedicle followed by cardiopulmonary resuscitation or reperfusion. The kidneys were harvested upon euthanasia 24 h after the procedure and stained with hematoxylin and eosin for histological examination. The percentage of necrotic tubules of the total tubules examined in both the cortex (CO; A) and medulla (OM; B) was calculated as a marker of kidney tubular injury in each mouse. In A, *P < 0.05 vs. renal vein-CO group. #P < 0.05 vs. 45 min of renal artery clamping. In B, *P < 0.05 vs. 45 min of renal vein or pedicle clamping. #P < 0.05 vs. 30 min of renal pedicle clamping and all 45-min groups; n = 6 in 30-min KI and WBI; n = 5 in 45-min KI.

Fig. 4.

Representative photographs of renal blood flow pulses. Male C57BL/6 mice underwent 10 min of WBIRI, 30 min of kidney IRI by clamping the bilateral renal artery, vein, or pedicle followed by resuscitation or reperfusion. At 24 h after the procedure, mice were lightly anesthetized to measure the renal blood flow by using a Sequoia C256 ultrasound. A: sham kidney. B: renal artery-clamped kidney. C: renal vein-clamped kidney. D: renal pedicle-clamped kidney. E: WBIRI. Scale = 2.0 m/s.

Fig. 5.

Measurement of mouse renal blood flow (RBF). Male C57BL/6 mice underwent 10 min of whole body ischemia (WBI), 30 min of kidney IRI by clamping the bilateral renal artery, vein, or pedicle followed by resuscitation or reperfusion. At 24 h after the procedure, mice were lightly anesthetized to measure RBF by using a Sequoia C256 ultrasound. The kidney IRI mice had significantly decreased RBF compared with sham-operated mice. However, WBIRI mice had significantly increased RBF compared with isolated kidney IRI mice. Values are means ± SE; n = 6 per group. *P < 0.05 vs. sham group. #P < 0.05 vs. all KI groups.

Fig. 6.

Proinflammatory mediator protein array in the kidney. Male C57BL/6 mice underwent 10 min of WBI, 30 or 45 min of kidney IRI by clamping the bilateral renal artery, vein, or pedicle followed by 24 h of resuscitation or reperfusion. The kidneys were harvested upon euthanasia and measured for IL-1b, IL-6, IL-10, TNF-α, INF-γ, keratinocyte-derived chemokine (KC), granular colony-stimulating factor (G-CSF), and monocyte chemoattractant protein-1 (MCP-1) protein by using a Bio-Plex multiplex protein array. Data are presented as fold-change of each protein in ischemic kidneys over that protein in sham-operated kidney (scale = 1). All kidney IRI mice had increased levels of renal IL-6, KC, G-CSF, and MCP-1 compared with sham-operated mice. However, the WBIRI mice had significant increased IL-10 and IFN-γ compared with 30- or 45-min kidney IRI mice. *P < 0.05 vs. vein-clamped group. #P < 0.05 vs. IL-10 in 30- or 45-min renal pedicle-clamped group vs. IL-6 in 45-min renal artery- or vein-clamped groups; n = 6 in 30-min KI and WBI; n = 5 in 45-min KI. ^, These proteins were not measured in the WBI mice.