basic research www.kidney-international.

org

Dexmedetomidine reduces norepinephrine

requirements and preserves renal oxygenation and
function in ovine septic acute kidney injury
Yugeesh R. Lankadeva1,6, Shuai Ma1,2,6, Naoya Iguchi1, Roger G. Evans3, Sally G. Hood1,
David G.S. Farmer1, Simon R. Bailey4, Rinaldo Bellomo5 and Clive N. May1
1
Preclinical Critical Care Unit, Florey Institute of Neuroscience and Mental Health, University of Melbourne, Victoria, Australia; 2Division of
Nephrology, Shanghai Ninth People’s Hospital, University School of Medicine, Shanghai, China; 3Cardiovascular Disease Program,
Biomedicine Discovery Institute and Department of Physiology, Monash University, Victoria, Australia; 4Faculty of Veterinary Science,
University of Melbourne, Victoria, Australia; and 5Centre of Integrated Critical Care, School of Medicine, University of Melbourne,
Melbourne, Victoria, Australia

Norepinephrine exacerbates renal medullary hypoxia in
experimental septic acute kidney injury. Here we examined
whether dexmedetomidine, an a2-adrenergic agonist, can
restore vasopressor responsiveness, decrease the
requirement for norepinephrine and attenuate medullary
hypoxia in ovine gram-negative sepsis. Sheep were
instrumented with pulmonary and renal artery flow probes,
and laser Doppler and oxygen-sensing probes in the renal
cortex and medulla. Conscious sheep received an infusion of
live Escherichia coli for 30 hours. Eight sheep in each group
were randomized to receive norepinephrine, norepinephrine
with dexmedetomidine, dexmedetomidine alone or saline
vehicle, from 24-30 hours of sepsis. Sepsis significantly
reduced the average mean arterial pressure (84 to 67 mmHg),
average renal medullary perfusion (1250 to 730 perfusion
units), average medullary tissue pO2 (40 to 21 mmHg) and
creatinine clearance (2.50 to 0.78 mL/Kg/min). Norepinephrine
restored baseline mean arterial pressure (to 83 mmHg) but
worsened medullary hypoperfusion (to 330 perfusion units)
and medullary hypoxia (to 9 mmHg). Dexmedetomidine (0.5
mg/kg/h) co-administration significantly reduced the
norepinephrine dose (0.8 to 0.4 mg/kg/min) required to
restore baseline mean arterial pressure, attenuated medullary
hypoperfusion (to 606 perfusion units), decreased medullary
tissue hypoxia (to 29 mmHg), and progressively increased
creatinine clearance (to 1.8 mL/Kg/min). Compared with
vehicle time-control, dexmedetomidine given alone
significantly prevented the temporal reduction in mean
arterial pressure, but had no significant effects on medullary
perfusion and oxygenation or creatinine clearance. Thus, in
experimental septic acute kidney injury, dexmedetomidine
reduced norepinephrine requirements, attenuated its adverse
effects on the renal medulla, and maintained renal function.
Correspondence: Clive N. May, Florey Institute of Neuroscience and Mental
Health, University of Melbourne, 30 Royal Parade, Parkville, Victoria, 3052
Australia. E-mail: clive.may@florey.edu.au
6
These authors contributed equally to this work.
Received 19 March 2019; revised 10 June 2019; accepted 14 June 2019;
published online 10 July 2019
Kidney International (2019) 96, 1150–1161; https://doi.org/10.1016/
j.kint.2019.06.013
KEYWORDS: acute kidney injury; dexmedetomidine; norepinephrine; renal
hypoperfusion; renal hypoxia; sepsis
Copyright ª 2019, International Society of Nephrology. Published by
Elsevier Inc. All rights reserved.
Translational Statement
The renal medulla appears to be particularly susceptible
to hypoxia in multiple forms of acute kidney injury,
including in sepsis. Medullary hypoxia is thought to be
critical for the development of acute kidney injury. In
sepsis, resuscitation with norepinephrine further
worsens the renal medullary hypoperfusion and hypoxia.
Our current findings in ovine sepsis indicate that coad-
ministration of dexmedetomidine reduces the require-
ment for norepinephrine, resulting in the preservation of
renal medullary perfusion, oxygenation, and kidney
function. A randomized clinical trial is now warranted to
test the efficacy of dexmedetomidine as an adjunct
therapy with norepinephrine in patients with septic
acute kidney injury.
cute kidney injury (AKI) develops in up to 50% of
A critically ill patients with sepsis, significantly compli-
cates patient management and is associated with
increased mortality.1–3 Moreover, survivors of septic AKI have
a greater risk of developing chronic and end-stage kidney dis-
ease in later life.4–7 Although the pathophysiology of septic
AKI is unclear, it has been proposed that AKI is a heteroge-
neous group of syndromes that varies depending on the etiol-
ogy.8 Indeed, we recently described increases in renal blood
flow (RBF) and renal oxygen delivery (RDO2) in sepsis, but
decreases during cardiopulmonary bypass.9–14 Interestingly,
despite these opposite effects on whole-organ blood flow
and oxygen delivery, in both settings renal medullary tissue
hypoperfusion and hypoxia occurred. Thus, medullary
ischemia and hypoxia may be common pathophysiological
features critical to the development of AKI.

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YR Lankadeva et al.: DEX and norepinephrine therapy in sepsis
Clinically, norepinephrine remains the primary vasopressor
used to restore mean arterial pressure (MAP) in patients with
sepsis.15 However, restoration of blood pressure with norepi-
nephrine can worsen underlying renal medullary hypoperfusion
and hypoxia in experimental gram-negative sepsis associated with
AKI.11 In addition, catecholamines have deleterious effects on
immune function, thrombogenicity, and metabolic efficiency,
and cause myocardial injury.16 This suggests that, during gram-
negative sepsis, the use of catecholamine-sparing strategies that
maintain MAP while decreasing norepinephrine dose may be
desirable.
Dexmedetomidine (DEX) is a potent a2-adrenergic receptor
agonist that has been used to reduce agitation and delirium in
critically ill patients.17,18 Currently, DEX is not recommended as
a standard-of-care sedative due to its central action to inhibit
sympathetic nerve activity,19,20 which would be expected to
reduce blood pressure in sepsis. However, in both clinical21–23
and experimental19,24,25 sepsis, there is evidence that treat-
ment with a2-adrenergic receptor agonists improves respon-
siveness to exogenous vasopressors such as norepinephrine,
phenylephrine, and angiotensin II, which likely accounts for
their ability to preserve blood pressure. A multinational, double-
blinded, randomized clinical trial is currently assessing DEX as a
primary sedative agent,26 making this agent of clinical interest
and relevance. In rodent models of lipopolysaccharide-induced
sepsis, pretreatment with supratherapeutic doses of DEX
(25–40 mg/kg) have been reported to protect against AKI.27–30
However, there is a paucity of data regarding the effects of
clinically relevant doses of DEX in combination with norepi-
nephrine on the renal macro- and microcirculation and on renal
function in the setting of established sepsis-associated AKI.
Accordingly, in a large animal model of gram-negative sepsis
associated with AKI, we compared the efficacy of a hemody-
namic support strategy consisting of norepinephrine alone with
one consisting of DEX and norepinephrine together. We hy-
pothesized that adjunctive DEX therapy would decrease the
dose of norepinephrine required to restore baseline MAP,
attenuate renal medullary hypoperfusion and hypoxia, and
improve renal function.
RESULTS
Two sheep from each group of 10 animals reached 2 or more of
the predefined ethical end-point criteria between 12 and 30
hours of bacteremia (i.e., lactate >2 mmol/l, partial pressure of
arterial blood oxygen <65 mm Hg, MAP <55 mm Hg, or heart
rate >200 beats/min). In accord with the requirements of the
Animal Ethics Committee, these 8 sheep were humanely killed
with pentobarbital (100 mg/kg, i.v.); results from these animals
were excluded from the analysis. Figures showing data expressed
as means
SD are presented in the paper, and in addition the
same data showing the responses in individual sheep for all 4
treatments are presented in the Supplementary Figures S1–S5.
Gram-negative sepsis with AKI
After infusion of E. coli for 24 hours, a hyperdynamic
circulatory state developed with decreased MAP, increased
Kidney International (2019) 96, 1150–1161
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cardiac output (CO), and tachycardia (Table 1). Sheep also
developed hypocapnia (partial pressure of arterial carbon
dioxide <25 mm Hg), fever (>41.5  C), and increased
arterial lactate >1.5 mmol/l (all P < 0.05 compared with
baseline).
At 24 hours after commencement of E. coli infusion, stage
1 AKI developed. According to the Kidney Disease:
Improving Global Outcomes clinical criteria,31 stage 1 AKI is
characterized by a >1.5-fold increase in plasma creatinine,
>60% reduction in urine flow and creatinine clearance, and
a 50% decrease in fractional sodium excretion (all P < 0.01)
(Table 1). These functional changes occurred despite
increased RBF, increased renal vascular conductance, and
increased RDO2 (Table 1). Renal hyperemia was associated
with increased cortical tissue perfusion and oxygen tension
(pO2) and large reductions in renal medullary tissue
perfusion and medullary tissue pO2 (Table 1). There were
increases in plasma interleukin (IL)-6 (to 5.6
1.7 ng/ml)
and IL-10 (to 5.8
2.0 ng/ml) from undetectable levels
during baseline (Table 1). The sepsis-induced changes in
systemic hemodynamics, renal perfusion, oxygenation and
function, and plasma cytokines were not significantly
different between the 4 treatment groups at 24 hours of
sepsis (PTime  Sepsis always > 0.05) prior to administration of
norepinephrine-saline, norepinephrine-DEX, DEX-saline,
or vehicle-saline (Supplementary Table S1).
Effects of treatment on systemic hemodynamics
Infusion of norepinephrine in combination with saline from
24 to 30 hours of sepsis restored MAP to premorbid levels (68

5 to 82
6 mm Hg) (Figure 1b). Norepinephrine given in
combination with DEX caused a similar restoration in MAP
(69
4 to 83
7 mm Hg); however, the dose of norepi-
nephrine required to restore baseline MAP was significantly
lower than the dose with norepinephrine-saline treatment
(0.1–0.4 vs. 0.4–0.8 mg/kg per min; PTreatment < 0.001 and
PTreatment  Time < 0.001) (Figure 1a). DEX given in combi-
nation with saline prevented the progressive decline in MAP
(68
8 to 72
7 mm Hg) seen in the vehicle-saline group
(68
2 to 62
8 mm Hg; PTreatment x Time ¼ 0.004)
(Figure 1c). Treatment with norepinephrine-saline and
vehicle-saline caused sustained tachycardia (Figure 1d and e),
while the addition of DEX in combination with norepi-
nephrine or saline resulted in progressive decreases in heart
rate (both PTreatment  Time < 0.05) (Figure 1d and e). There
was an increase in CO from 24 to 30 hours of sepsis during
treatment with norepinephrine-saline and vehicle-saline, but
no increase during norepinephrine-DEX or DEX-saline, with
CO remaining approximately 10% lower than in the
norepinephrine-saline and vehicle-saline groups (Both
PTreatment  Time < 0.001) (Figure 1f and g). Total peripheral
conductance was lower in septic sheep treated with
norepinephrine-DEX (79
17 to 57
13 ml/min per
mm Hg) and DEX-saline (79
14 to 66
17 ml/min per
mm Hg), than with norepinephrine-saline or vehicle-saline
(both PTreatment  Time<0.01) Figure 1h and i).
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Table 1 | Changes in systemic hemodynamics, global and regional kidney perfusion, oxygenation, renal function, and plasma
cytokines from baseline (premorbid) to 24 hours of gram-negative sepsis in conscious sheep
Sheep prior to treatment (N [ 32)
Systemic and renal variables Baseline 24 h sepsis
Mean arterial pressure (mm Hg) 86
5 68
5b
Heart rate (beats/min) 74
11 140
22b
Cardiac output (l/min) 3.5
0.6 5.6
0.8b
Total peripheral conductance (ml/min per mm Hg) 40
8 74
15b
Urine flow (ml/kg per h) 1.5
0.4 0.6
0.2b
Creatinine clearance (ml/kg per h) 2.7
1.0 0.8
0.4b
Plasma creatinine (mmol/l) 61
12 118
25b
Fractional sodium excretion (%) 1.1
0.3 0.5
0.2b
Renal blood flow (ml/min per kg) 6.5
1.5 9.1
1.7b
Renal vascular conductance (ml/min per kg per mm Hg) 0.08
0.02 0.11
0.03b
Renal oxygen delivery (mlO2/min per kg) 0.9
0.2 1.3
0.3b
Renal oxygen consumption (mlO2/min per kg) 0.13
0.04 0.09
0.03a
Cortical tissue perfusion (BPU) 1301
345 1642
470b
Medullary tissue perfusion (BPU) 1212
305 604
228a
Cortical tissue oxygen tension (mm Hg) 40
9 46
12a
Medullary tissue oxygen tension (mm Hg) 42
9 20
8b
Interleukin-6 (ng/ml) 0.0
0.0 5.6
1.7b
Interleukin-10 (ng/ml) 0.0
0.0 5.8
2.0b
BPU, blood perfusion unit.
a
P values <0.01 and b<0.001 indicate significant differences between variables at baseline (premorbid) compared with variables at 24 hours of gram-negative sepsis in all 32
sheep prior to treatment with norepinephrine-saline, norepinephrine-dexmedetomidine, dexmedetomidine-saline, or vehicle-saline (time control). P values were derived from
Student’s 2-tailed paired t test.
Values are between-animal mean
SD. Renal flow variables are corrected for kilograms of body weight.

Effects of treatment on renal function
There were significant interactions between treatment
(norepinephrine-saline vs. norepinephrine-DEX) and time
for urine flow (PTreatment  Time < 0.001), creatinine clearance
(PTreatment  Time < 0.001), and fractional sodium excretion
(PTreatment  Time ¼ 0.003), such that, after 2 hours, mean
urine flow (0.6
0.2 to 2.4
1.3 ml/kg per h) and creatinine
clearance (0.9
0.2 to 2.8
1.0 ml/kg per min) increased
with norepinephrine-saline, but then gradually declined to-
ward preintervention levels (Figure 2). In contrast, treatment
with norepinephrine-DEX progressively increased urine flow
(0.6
0.2 to 1.1
0.3 ml/kg per h), creatinine clearance (0.8

0.4 to 1.8
0.4 ml/kg per min), and fractional sodium
excretion (0.3
0.1% to 0.7
0.3%; Figure 2). Nevertheless,
the reduction in plasma creatinine from 24 to 30 hours of
sepsis was similar between the 2 treatment groups
(PTreatment  Time ¼ 0.2; Figure 2e). Treatment with DEX-
saline resulted in progressive increases in urine flow (0.5

0.2 to 1.3
0.6 ml/kg per h; PTreatment  Time ¼ 0.005) and
fractional sodium excretion from 24 to 30 hours of sepsis (0.5

0.3% to 0.9
0.4%; PTreatment  Time ¼ 0.02), although
there were no significant changes in creatinine clearance or
plasma creatinine compared with those of the vehicle-saline
group (Figure 2).
Effects of treatment on global and regional-kidney
hemodynamics and oxygen handling
Neither norepinephrine nor norepinephrine-DEX signifi-
cantly changed renal vascular conductance, RBF, RDO2, or
renal oxygen consumption (Figure 3). DEX given in combi-
nation with saline prevented the further increases in RBF
(PTreatment  Time ¼ 0.009) and renal vascular conductance
(PTreatment  Time < 0.001) seen in the vehicle-saline group
from 24 to 30 hours of sepsis, but had no significant effects on
RDO2 or renal oxygen consumption (Figure 3). Renal cortical
perfusion and oxygenation remained increased in the
norepinephrine-saline and norepinephrine-DEX treatment
groups (Figure 4a and c). However, norepinephrine-saline
(0.4–0.8 mg/kg per min) worsened the degree of renal med-
ullary hypoperfusion (561
178 to 330
150 blood
perfusion units; PTreatment  Time ¼ 0.001) and medullary
hypoxia (25
6 to 9
5 mm Hg; PTreatment ¼ 0.001 and
PTreatment  Time < 0.001) (Figure 4e and g). In contrast,
norepinephrine combined with DEX allowed the dose of
norepinephrine to be reduced (0.1–0.4 mg/kg per min)
(Figure 1b), resulting in preserved renal medullary tissue
perfusion (734
176 to 606
300 blood perfusion units)
and oxygenation (21
6 to 29
7 mm Hg) (Figure 4e and
g). In contrast, DEX given alone had no significant effects on
renal cortical and medullary perfusion and oxygenation
compared with the vehicle-saline group from 24 to 30 hours
of sepsis (Figure 4).
Effects of treatment on inflammatory and anti-inflammatory
cytokines
Norepinephrine-saline had no significant effect on the high
level of IL-6 from 24 to 30 hours, but treatment with
norepinephrine-DEX significantly reduced plasma IL-6 (5.7

0.9 to 2.6
0.7 ng/ml; PTreatment ¼ 0.03 and PTreatment 
Time ¼ 0.003; Figure 5a). Norepinephrine-saline reduced
plasma IL-10 (5.8
2.1 to 2.7
0.9 ng/ml), but this effect
was prevented by the addition of DEX (6.1
1.9 to 6.5
1.3

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Figure 1 | Systemic hemodynamic responses to dexmedetomidine and norepinephrine treatment in ovine gram-negative
bacteremia. Norepinephrine dose (a), mean arterial pressure (b,c), heart rate (d,e), cardiac output (f,g), and total peripheral conductance (h,i)
during infusion of Escherichia coli from 0 to 30 hours and subsequent treatment with norepinephrine þ dexmedetomidine (continued)

Kidney International (2019) 96, 1150–1161 1153

basic research
ng/ml; PTreatment ¼ 0.02 and PTreatment  Time ¼ 0.005;
Figure 5c). Similarly, DEX given in combination with saline
significantly reduced plasma IL-6 (5.1
1.2 to 2.6
0.7 ng/
ml; PTreatment  Time ¼ 0.01) and prevented the decline in
plasma IL-10 (5.6
2.7 to 6.5
1.3 ng/ml; PTreatment 
Time ¼ 0.001) compared with vehicle-saline treatment
(Figure 5b and d).
DISCUSSION
In a large animal model of experimental gram-negative
sepsis with hypotension and AKI, we studied the effects of
a clinically relevant dose of DEX as adjunct therapy to
norepinephrine infusion on systemic and global renal
hemodynamics, renal function, cortical and medullary
perfusion and oxygenation, and plasma levels of 2 key
cytokines. We found that addition of DEX halved the dose
of norepinephrine required to restore MAP to premorbid
levels, prevented the exacerbation of renal medullary
hypoperfusion and hypoxia induced by norepinephrine
alone, and was associated with progressive improvements in
creatinine clearance. Moreover, in contrast to norepineph-
rine alone and vehicle-saline, adjunctive DEX therapy
attenuated sepsis-induced increases in heart rate and CO,
reduced the high level of IL-6, and preserved the increased
level of IL-10.
In agreement with previous findings,10–12 infusion of live
E. coli for 24 hours induced a hyperdynamic circulatory
state associated with stage 1 AKI, as defined by the Kidney
Disease: Improving Global Outcomes criteria.31 In this
model of septic AKI, renal medullary hypoperfusion and
hypoxia occur within the first hour of gram-negative
infection, which is 12 to 24 hours prior to the detection
of AKI using currently available biomarkers.9,11,12 We have
recently provided compelling evidence that renal medullary
hypoperfusion and hypoxia also occur during experimental
cardiopulmonary bypass in sheep,14 although this occurred
in the presence of reduced RBF and RDO2, the opposite of
the changes in sepsis. In sepsis, renal macrocirculatory
measures of blood flow and oxygen delivery appear to be
poor predictors of microcirculatory abnormalities because,
despite increased RBF and RDO2 and preserved renal
cortical perfusion and oxygenation, localized hypoperfusion
and hypoxia occur within the renal medulla.9,11,12 Such
early onset of progressive renal medullary hypoxia, leading
to oxidative stress and inflammation, can initiate a vicious
cycle of cellular injury contributing to AKI under different
pathophysiological settings.8,32,33 In ovine sepsis, restoring
MAP with norepinephrine worsens renal medullary hypo-
perfusion and hypoxia and fails to induce a sustained
=
Figure 1 | (continued) (n ¼ 8, red circles), norepinephrine þ saline (n ¼ 8, open circles), dexmedetomidine þ saline (n ¼ 8, blue squares), or
vehicle-saline (n ¼ 8, open squares) in conscious sheep. Norepinephrine (0.1–1.0 mg/kg per min) and dexmedetomidine (0.5 mg/kg per h) were
infused from 24 to 30 hours of sepsis. Time 0 is the mean of the 24th hour of the baseline period and times 24 to 30 hours are means of 1-hour
periods. Data are within-animal mean
SD. P values represent treatment-time interactions from a 2-way repeated-measures analysis of
variance from 24 to 30 hours of sepsis. bpm, beats per minute.
1154
YR Lankadeva et al.: DEX and norepinephrine therapy in sepsis
improvement in renal function.11 These previous findings
indicate the need to develop alternative resuscitation ap-
proaches that preserve renal medullary oxygenation in
pathophysiological situations where medullary hypoxia is
suspected.
In our study, a clinical dose of DEX (0.5 mg/kg per h),
administered in established septic AKI, substantially reduced
the dose of norepinephrine required (from 0.8
0.4 to 0.4

0.2 mg/kg per min) to restore target MAP, indicating that
coadministration of an a2-adrenergic agonist may allow the
maintenance of MAP with lower doses of vasopressor drugs.
This is likely due to an effect of a2-adrenergic receptor ago-
nists to improve vascular reactivity to catecholamines and
angiotensin II.19,24,25 Likewise, in a recent prospective cross-
over clinical study, in 38 sedated septic patients, switching
from standard-of-care propofol to DEX (0.7 mg/kg per h)
significantly reduced norepinephrine requirements (0.7
0.6
to 0.3
0.2 mg/kg per min) to attain target blood pressure, an
effect maintained up to 8 hours after replacing DEX with
propofol.22 Even when given alone, DEX prevented the
further deterioration in MAP seen in the vehicle-time control
group, an effect associated with vasoconstriction as shown by
the reductions in total peripheral and renal vascular
conductance. We have previously reported similar findings in
ovine sepsis with the less selective a2-adrenergic agonist,
clonidine.25,34 We have also demonstrated that a2-adrenergic
agonists can restore pressor responsiveness to the non-
catecholamine vasopressor, angiotensin II, in ovine sepsis.25
In this regard, a2-adrenergic agonists may increase phos-
pholipase A activity,35 which can restore vascular adrenergic
and angiotensin receptor sensitivity.36 Moreover, a2-adren-
ergic agonists may directly inhibit the vascular adenosine
triphosphate–sensitive potassium channel pore-forming
Kir6.0 subunit,37 thus improving the ability of vascular
smooth muscle cells to repolarize and respond to endogenous
and exogenous vasoconstrictors.
A novel finding from this study was that addition of DEX
during resuscitation allowed norepinephrine doses to be
reduced, which attenuated renal medullary hypoperfusion
and hypoxia and improved creatinine clearance. Our findings
differ from those of a clinical trial in septic patients treated
with DEX that reported no overt beneficial effects on renal
functional variables.38 However, patients within that study
received a wide spectrum of sedatives and opioids (propofol,
midazolam, fentanyl), in addition to DEX, and more than
20% were on renal replacement therapy at randomization.38
The late intervention in this study makes it difficult to
delineate the renal effects of DEX when given earlier in the
course of sepsis.39 In contrast, in rodent models of sepsis,
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YR Lankadeva et al.: DEX and norepinephrine therapy in sepsis basic research

Figure 2 | Renal functional responses to dexmedetomidine and norepinephrine treatment in bacteremia-induced acute kidney
injury. Urine flow (a,b), creatinine clearance (c,d), plasma creatinine (e,f), and fractional sodium excretion (g,h) during infusion of Escherichia
coli from 0 to 30 hours and subsequent treatment with norepinephrine þ dexmedetomidine (n ¼ 8, red circles), norepinephrine þ saline
(n ¼ 8, open circles), dexmedetomidine þ saline (n ¼ 8, blue squares), or vehicle-saline (n ¼ 8, open squares) in conscious sheep.
Drug infusions and statistical analyses are as detailed in Figure 1.

pretreatment with DEX protected against AKI.27–30 The Consistent with the notion of reduced inflammation, we
nephroprotective mechanism of DEX in sepsis has been found a reduction in proinflammatory IL-6 levels and a
attributed to its antioxidant and anti-inflammatory proper- preservation of anti-inflammatory IL-10 levels with DEX
ties,27–29,40 leading to improved microcirculatory perfusion.41 administration in septic sheep with established AKI. However,

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Figure 3 | Global renal hemodynamic and oxygen handling during dexmedetomidine and norepinephrine treatment in bacteremia-
induced acute kidney injury. Renal blood flow (a,b), renal vascular conductance (c,d), renal oxygen delivery (e,f), and renal oxygen
consumption (g,h) during infusion of Escherichia coli from 0 to 30 hours and subsequent treatment with norepinephrine þ dexmedetomidine
(n ¼ 8, red circles), norepinephrine þ saline (n ¼ 8, open circles), dexmedetomidine þ saline (n ¼ 8, blue squares), or vehicle-saline (n ¼ 8,
open squares) in conscious sheep. Drug infusions and statistical analyses are as detailed in Figure 1.

the enhanced diuretic and natriuretic responses to DEX are collecting ducts, which are independent of changes in
likely due to both the central inhibition of antidiuretic hor- glomerular filtration rate.42,43
mone release and a direct effect to inhibit renal tubular so- Our study has several strengths. We controlled for co-
dium reabsorption in the medullary loop of Henle and founders such as amount of parenteral fluids, use of

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Figure 4 | Intrarenal perfusion and oxygenation during dexmedetomidine and norepinephrine treatment in ovine gram-negative
bacteremia. Renal cortical tissue perfusion (a,b), cortical oxygen tension (pO2) (c,d), medullary tissue perfusion (e,f), and medullary pO2
(g,h) during infusion of Escherichia coli from 0 to 30 hours and subsequent treatment with norepinephrine þ dexmedetomidine (n ¼ 8,
red circles), norepinephrine þ saline (n ¼ 8, open circles), dexmedetomidine þ saline (n ¼ 8, blue squares), or vehicle-saline (n ¼ 8,
open squares) in conscious sheep. Drug infusions and statistical analyses are as detailed in Figure 1.

norepinephrine, timing of sepsis, animal age, size, and sex. baseline central venous pressure and a hyperdynamic cir-
The study was randomized and placebo controlled. Ani- culatory state.10 Animals were resuscitated with clinically
mals were adequately fluid resuscitated with w2000 ml of relevant doses of norepinephrine and DEX at a clinically
crystalloids infused over 24 hours of sepsis to maintain relevant time, when the early stages of AKI were evident.

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Figure 5 | Pro- and anti-inflammatory cytokines responses to dexmedetomidine and norepinephrine treatment in ovine bacteremia-
induced acute kidney injury. Plasma levels of interleukin-6 (a,b) and interleukin-10 (c,d) during infusion of Escherichia coli from 0 to 32 hours
and subsequent treatment with norepinephrine þ dexmedetomidine (n ¼ 8, red circles), norepinephrine þ saline (n ¼ 8, open circles),
dexmedetomidine þ saline (n ¼ 8, blue squares), or vehicle-saline (n ¼ 8, open squares) in conscious sheep. Drug infusions and
statistical analyses are as detailed in Figure 1.

We studied unanesthetized sheep to remove the con-
founding effects of general anesthesia,44 although
acknowledge that this limits our ability to draw inferences
about the likely effects of DEX in anesthetized patients.
Our study has other limitations. Gram-negative sepsis-
induced changes may not replicate changes that occur with
other organisms. Renal histopathological studies were not
performed; however, a lack of histological changes has been
previously reported for this model.45,46 We only assessed a
single dose of DEX, but we chose a dose similar to that used
in clinical practice. Our study was confined to the assess-
ment of systemic and renal hemodynamics, intrarenal
perfusion and oxygenation, and renal function in response
to DEX over a 6-hour interventional period. Thus, we
cannot comment on any longer-term renal functional ef-
fects of treatment with DEX and the associated improve-
ment in medullary oxygenation. Further studies with
longer observational periods are warranted to better un-
derstand whether the putative advantages associated with
DEX are sustained. We used creatinine clearance as a
marker to estimate glomerular filtration rate, but these
measurements can be influenced by abrupt changes in
diuresis or dead space in the urinary tract or both, despite
the use of a bladder catheter. Moreover, enhanced renal
tubular secretion of creatinine in progressive renal disease
may also lead to overestimation of creatinine clearance,
which should be considered when interpreting these
results.
CONCLUSIONS
In a large animal model of gram-negative sepsis-induced AKI,
we found that administration of DEX as adjunct therapy
lowered the dose of norepinephrine required to restore
baseline MAP, alleviated renal medullary hypoperfusion and
hypoxia, and resulted in improved creatinine clearance over a
6-hour interventional period. DEX may therefore be a useful
catecholamine-sparing strategy offering renoprotection in
septic AKI. Additionally, this combination therapy did not
aggravate sepsis-induced systemic hemodynamic abnormal-
ities and it significantly reduced circulating plasma levels of
IL-6 while preserving levels of IL-10.
METHODS
Animal preparation
Experiments were approved by the Florey Institute of Neuroscience
and Mental Health Animal Ethics Committee under the guidelines
laid down by the National Health and Medical Research Council of
Australia.
Forty Merino ewes (35–45 kg) were instrumented in 2 operations
under general anesthesia to enable the continuous measurement in

1158 Kidney International (2019) 96, 1150–1161

YR Lankadeva et al.: DEX and norepinephrine therapy in sepsis
conscious sheep of MAP, heart rate, CO, RBF, intrarenal tissue
perfusion and pO2, and core temperature, as previously
described.47,48 In addition, a cannula was implanted in the left renal
vein for blood sampling, a carotid artery for measurement of MAP
and collection of arterial blood, and a jugular vein for infusion of
E. coli, fluids, and drugs. A bladder catheter was inserted for mea-
surement of urine flow and collection of urine. Pre- and postsurgical
analgesia and antibiotics were given, as previously described.10–12
After 4 days’ recovery from the second operation, the baseline
period was started and analog signals for cardiovascular and renal
variables were continuously recorded.10–12 Total peripheral
conductance, renal vascular conductance, creatinine clearance,
fractional sodium excretion, RDO2, and renal oxygen consumption
were calculated using standard formulae.10–12
Experimental protocol
After 20 hours of baseline measurements, gram-negative sepsis was
induced in conscious sheep by intravenous infusion of live E. coli
(2.8  109 colony-forming units over 30 minutes, followed by 1.26 
109 colony-forming units per hour for 30 hours). In the first 24
hours of bacteremia, all sheep received 0.9% wt/vol sodium chloride
(2 ml/kg per h; Baxter, Australia) as fluid replacement. At 24 hours of
sepsis, sheep were randomized (ratio 1:1:1:1) to receive an intrave-
nous infusion of norepinephrine-saline, norepinephrine-DEX, DEX-
saline, or vehicle-saline as time control (n ¼ 8 per group). DEX (0.5
mg/kg per h) was infused from 24 to 30 hours of sepsis. The choice of
this dose of DEX was based on pilot studies that demonstrated it
returned the sepsis-induced high level of renal sympathetic nerve
activity49 to baseline (premorbid) levels, in a similar manner to our
previous finding of a similar effect with the a2-adrenergic receptor
agonist, clonidine.25 Norepinephrine (0.1–1.0 mg/kg per min) was
infused from 24 to 30 hours of sepsis, with the dose being titrated
every 10 minutes to restore MAP to premorbid levels. Antibiotics
were not administered. Following cessation of experiments, animals
were euthanized with pentobarbital (100 mg/kg, i.v.) and the posi-
tion of the renal fiber optic probes were confirmed at autopsy.
Sample collection
Arterial and renal venous blood were collected at predefined time
intervals for measurement of blood gases (ABL Systems-625;
Advanced BioScience Laboratories, Denmark), creatinine, and so-
dium. Plasma IL-6 and IL-10 were also measured in arterial blood
using enzyme-linked immunosorbent assays.11,12 Urine was collected
for measurement of urine flow and concentration of creatinine and
sodium.
Statistical analysis
All variables passed tests for normality (D’Agostino and Pearson
Omnibus test, confirmed by a Shapiro-Wilk test).11 Data are re-
ported as mean
SD. RBF and functional variables were corrected
for body weight. Specific time-point comparisons within-groups
between baseline and 24 hours of gram-negative sepsis were per-
formed using Student’s paired t test. The interventional period, 24 to
30 hours of sepsis, was analyzed using 2-way repeated measures
analysis of variance with factors treatment (Ptreatment) and time
(Ptime) and their interaction (PTreatment  Time) (GraphPad PRISM
6.0; GraphPad Inc., San Diego, CA). P values from within-subjects’
factors were conservatively adjusted using the Greenhouse-Geisser
method.50 Two-sided P # 0.05 was considered statistically
significant.
Kidney International (2019) 96, 1150–1161
basic research
DISCLOSURE
All the authors declared no competing interests.
ACKNOWLEDGMENTS
This study was supported by a grant from the National Health and
Medical Research Council of Australia (ID APP1050672) and by
funding from the Victorian Government Operational Infrastructure
Support Grant. YRL was supported by a Future-Leader Postdoctoral
Fellowship by the National Heart Foundation of Australia (ID 101853).
We would like to sincerely thank Tom Vale and Tony Dornom for
their excellent technical assistance.
SUPPLEMENTARY MATERIAL
Table S1. Changes in systemic hemodynamics, global and regional
kidney perfusion, oxygenation, renal function, and plasma cytokines
from baseline (premorbid) to 24 hours of gram-negative sepsis in
conscious sheep. Values are between-animal mean
SD. Systemic
variables include mean arterial pressure (MAP), heart rate (HR), car-
diac output (CO), total peripheral conductance (TPC), and plasma
interleukin-6 and interleukin-10 levels. Renal variables include urine
flow, creatinine (Cr) clearance, plasma Cr, fractional sodium excretion
(FeNa), renal blood flow (RBF), renal vascular conductance (RVC), renal
oxygen delivery (RDO2), renal oxygen consumption (RVO2), renal
cortical and medullary tissue perfusion, and oxygen tension (pO2).
Renal flow variables are corrected for kilograms of body weight. *P <
0.05, **P < 0.01, and ***P < 0.001 indicate significant differences
between variables at baseline (premorbid) compared with variables
at 24 hours of gram-negative sepsis in the groups of sheep prior to
treatment with norepinephrine-saline, norepinephrine-dexmedeto-
midine, dexmedetomidine-saline, or vehicle-saline (time control). P
values were derived from Student’s 2-tailed paired t test.
Figure S1. Systemic hemodynamic responses in individual sheep to
dexmedetomidine and norepinephrine treatment in ovine gram-
negative bacteremia. Norepinephrine dose (A), mean arterial pressure
(B,C), heart rate (D,E), cardiac output (F,G), and total peripheral
conductance (H,I) during infusion of Escherichia coli from 0 to 30
hours and subsequent treatment with norepinephrine þ dexmede-
tomidine (n ¼ 8, red circles), norepinephrine þ saline (n ¼ 8, open
circles), dexmedetomidine þ saline (n ¼ 8, blue squares), or vehicle-
saline (n ¼ 8, open squares) in conscious sheep. Norepinephrine (0.1–
1.0 mg/kg per min) and dexmedetomidine (0.5 mg/kg per h) were
infused from 24 to 30 hours of sepsis. Time 0 is the mean of the 24th
hour of the baseline period and times 24 to 30 hours are means of 1-
hour periods. Data are within-animal mean
SD. P values represent
treatment-time interactions from a 2-way repeated-measures analysis
of variance from 24 to 30 hours of sepsis.
Figure S2. Renal functional responses in individual sheep to
dexmedetomidine and norepinephrine treatment in bacteremia-
induced acute kidney injury. Urine flow (A,B), creatinine clearance
(C,D), plasma creatinine (E,F), and fractional sodium excretion (G,H)
during infusion of Escherichia coli from 0 to 30 hours and subsequent
treatment with norepinephrine þ dexmedetomidine (n ¼ 8, red cir-
cles), norepinephrine þ saline (n ¼ 8, open circles),
dexmedetomidine þ saline (n ¼ 8, blue squares), or vehicle-saline
(n ¼ 8, open squares) in conscious sheep. Drug infusions and statis-
tical analyses are as detailed in Supplementary Figure S1.
Figure S3. Global renal hemodynamic and oxygen handling in
individual sheep during dexmedetomidine and norepinephrine
treatment in bacteremia-induced acute kidney injury. Renal blood
flow (A,B), renal vascular conductance (C,D), renal oxygen delivery
(E,F), and renal oxygen consumption (G,H) during infusion of
Escherichia coli from 0 to 30 hours and subsequent treatment with
norepinephrine þ dexmedetomidine (n ¼ 8, red circles),
1159
basic research
norepinephrine þ saline (n ¼ 8, open circles), dexmedetomidine þ
saline (n ¼ 8, blue squares), or vehicle-saline (n ¼ 8, open squares) in
conscious sheep. Drug infusions and statistical analyses are as
detailed in Supplementary Figure S1.
Figure S4. Intrarenal perfusion and oxygenation in individual sheep
during dexmedetomidine and norepinephrine treatment in ovine
gram-negative bacteremia. Renal cortical tissue perfusion (A,B),
cortical oxygen tension (pO2) (C,D), medullary tissue perfusion (E,F),
and medullary pO2 (G,H) during infusion of Escherichia coli from 0 to
30 hours and subsequent treatment with norepinephrine þ dexme-
detomidine (n ¼ 8, red circles), norepinephrine þ saline (n ¼ 8, open
circles), dexmedetomidine þ saline (n ¼ 8, blue squares), or vehicle-
saline (n ¼ 8, open squares) in conscious sheep. Drug infusions and
statistical analyses are as detailed in Supplementary Figure S1.
Figure S5. Pro- and anti-inflammatory cytokines responses in indi-
vidual sheep to dexmedetomidine and norepinephrine treatment in
ovine bacteremia-induced acute kidney injury. Plasma levels of
interleukin-6 (A,B) and interleukin-10 (C,D) during infusion of
Escherichia coli from 0 to 32 hours and subsequent treatment with
norepinephrine þ dexmedetomidine (n ¼ 8, red circles),
norepinephrine þ saline (n ¼ 8, open circles), dexmedetomidine þ
saline (n ¼ 8, blue squares), or vehicle-saline (n ¼ 8, open squares) in
conscious sheep. Drug infusions and statistical analyses are as
detailed in Supplementary Figure S1.
Supplementary material is linked to the online version of the paper at
www.kidney-international.org.
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