endotoxemia frequently occurs during critical phases of clinical diseases , including trauma and infectious diseases , and inevitably causes shock and organ damage contributing to poor survival rate [ 1 , 2 ] .
acute kidney injury ( aki ) is a common complication following endotoxic shock and is associated with a high morbidity rate of up to 64% . despite significant advances in control of endotoxemia , there remains an urgent need to promptly prevent the occurrence of aki or restore impaired renal function associated with sepsis . in sepsis
, renal blood flow is frequently reduced with progressive development of renal inflammation and oxidative stress , thereby contributing to the genesis of aki [ 5 , 6 ] .
early resuscitation using crystal or colloid fluids to improve tissue perfusion and inhibit oxidative and inflammatory response may provide a meaningful degree of renal protection [ 7 , 8 ] .
however , infusion of large - volume fluids is always limited to critically ill patients , although caution must be exercised in patients with heart dysfunction , in whom small - volume resuscitation is typically used .
the potential protective effects of small - volume fluid resuscitation on kidney injury induced by endotoxemia are currently unknown .
fortunately , small - volume resuscitation with hypertonic solutions has been successfully applied in clinical rescue of hemorrhagic and septic shock [ 1012 ]
. however , the debate persists regarding which solution ( crystalloids or colloids ) is most beneficial [ 13 , 14 ] . for instance , it is reported that appropriate administration of colloids seemed to be associated with reduced mortality , while others consider that resuscitation with balanced crystalloids was associated with a lower risk of in - hospital mortality [ 15 , 16 ] . in the present study
, we compared the effects of small - volume resuscitation with saline , 7.5% hypertonic saline ( hts ) , hydroxyethyl starch 130/0.4 ( he s ) , and hypertonic saline hydroxyethyl starch 40 ( hsh ) at a dose of 4  ml / kg on acute kidney injury induced by endotoxemia .
the potential mechanism of the hypothesized protection was further explored , focusing on renal perfusion and variations of oxidative and inflammatory mediators .
the study was reviewed and approved by the research ethics committee of the third affiliated hospital of sun yat - sen university , guangzhou , china .
thirty adult male sprague - dawley ( sd ) rats ( 180250  g body weight ) were provided by the experimental animal center of sun yat - sen medical school and were treated in accordance with both international and institutional guidelines .
experimental procedures on rats were performed as previously described . briefly , after anesthesia , animals were ventilated with 4050% oxygen ( rodent ventilator 638 , harvard apparatus , boston , ma , usa ) , with proper tidal volume ( 7  ml / kg ) and respiratory rate ( approximately 30 breaths / min ) .
a phased - array , low - frequency probe ( frequency 2.05.0  mhz ) was placed in the left renal door cross - sectional level for kidney color velocity imaging ( cvi ) detection .
all animals were given intravenous administration of 1  mg lps / kg through the right internal jugular vein to establish the endotoxemia model .
sodium pentobarbital in saline was obtained from sigma chemical co. ( st . louis , mo , usa ) .
lps was isolated from e. coli 0111:b4 and purchased from sigma chemical co. the experimental solutions were 0.9% saline ( zhejiang chimin pharmaceutical co. , ltd . , taizhou , china ) , 7.5% hypertonic saline
( hts , the third affiliated hospital of sun yat - sen university , guangdong , china ) , hydroxyethyl starch ( he s , fresenius kabi pharmaceutical co. , ltd . , bad homburg , germany ) , and hypertonic saline hydroxyethyl starch ( hsh , shanghai huayuan chang fu co. , ltd . ,
rats were randomized into five groups , including control and four resuscitation groups ( n = 6 per group ) .
the groups were designated as follows : ( 1 ) group c ( control group ) : 4  ml 0.9% saline / kg ; ( 2 ) group s ( saline group ) : lps 1  mg / kg + 4  ml 0.9% saline / kg ; ( 3 ) group hts : lps 1  mg / kg + 4  ml 7.5% hts / kg ; ( 4 ) group he s : lps 1  mg / kg + 4  ml hes / kg ; and ( 5 ) group hsh : lps 1  mg / kg + 4  ml hsh / kg . four kinds of resuscitation solutions at a dose of 4  ml / kg were administered at 30  min after intravenous injection of lps . and fluids were administered within 5  min according to . after performing systemic hemodynamic and renal blood flow measurements , rats were euthanized , blood was collected by cardiac puncture , and the kidneys were harvested .
systolic arterial pressure ( sap ) was monitored from catheterized internal carotid arteries with a hewlett - packard monitor .
renal flow indices were measured by doppler ultrasound using a technos mpx du8 instrument ( esaote biomedica , genoa , italy ) .
image - pro plus image analysis software was used to calculate the ratio of renal blood signal and the peak renal cross - section area .
sap and renal microcirculation indices ( renal blood flow signals ( cvi ) , peak - systolic velocity ( vmax ) , and end - diastolic velocity ( vmin ) ) were recorded before lps administration ( t0 , baseline ) , 30  min after lps administration ( t1 ) , and 10  min ( t2 ) , 30  min ( t3 ) , and 60  min ( t4 ) after small - volume resuscitation .
renal vascular resistance index ( rvri ) was calculated by the formula 
 ( 1)rvri = vmaxvminvmax. blood samples were obtained from the abdominal aorta 60  min after small - volume resuscitation .
renal function was determined by assaying serum for serum creatinine ( scr ) and blood urea nitrogen ( bun ) using the av800 chemistry analyzer ( hitachi , hitachi city , japan ) .
renal tissues were taken 60  min after small - volume resuscitation and fixed in 10% formaldehyde . after being processed in an autotechnicon ,
sections of 5  m thickness were cut with a microtome and stained with hematoxylin - eosin ( h&e ) .
separated tissue samples were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1  m pbs ( ph 7.4 ) at 4c for 1 day for electron microscopy . after being postfixed with 2% osmium tetroxide , tissues were dehydrated in graded ethanol and embedded into araldite .
the stained sections were examined using a leo 906e electron microscope . because the renal cortex is highly sensitive to lps challenge , renal cortical tissue was separated from the rest of the kidney for analyses .
levels of tnf- and no were quantified with specific elisa and no kits ( boster company , wuhan , china , and nanjing jiancheng bioengineering institute , nanjing , china , resp . ) .
renal cortex was homogenized for assessment of mda level and sod activity with kits based on the thiobarbituric acid reactive substance assay and xanthine oxidase method , respectively ( both by jiancheng bioengineering institute , nanjing , china ) .
statistical analysis of data was performed by using one - way analysis of variance ( anova ) , while repeated measurements of anova were used for cvi analyses .
small - volume resuscitation with he s and hsh markedly decreased the augmented scr and bun levels ( p = 0.028 versus group s ) due to lps exposure , while hts resuscitation was less effective ( figures 1(a ) and 1(b ) ) .
lps caused severe renal tubular injury , manifested as significant tubular degeneration , disintegration , edema , necrosis , and abscission of renal tubular epithelium , as well as cellular debris obstructing the collecting tubules and basal membrane fracture ( figures 1(d ) and 1(e ) ) .
small - volume resuscitation with colloid and hyperoncotic solutions markedly ameliorated the renal injury , showing that local epithelial and mitochondrial edema , cellular necrosis , and abscission were seen in the hts group , while kidneys in the he s and hsh groups appeared with only slight edema with occasional cellular necrosis and abscission .
these changes were correlated with the paller histology grading of lps - mediated kidney damage at 60  min after resuscitation ( figure 1(c ) ) .
sap was measured to evaluate the systemic circulatory state and renal perfusion was detected through doppler ultrasound by simultaneously monitoring renal blood flow signals ( cvi ) , vmax , vmin , and the calculated rvri .
based on the measurement of these parameters , the lps - induced deterioration of systemic and renal hemodynamics was assessed by comparisons among the five groups at the t0 time point , while improvement after small - volume resuscitation was evaluated by comparisons among the five groups at the t1 time point .
doppler ultrasound showed that renal blood flow signal ( cvi ) was strongly and uniformly distributed with adequate cortex perfusion before lps administration and then dramatically decreased with discrepant distribution and weak renal perfusion ( cvi ) under lps challenge .
small - volume resuscitation with he s and hsh markedly improved cvi , while saline and hts showed only slight improvement on renal blood flow ( figure 2 ) .
the time courses for measurements of sap , vmax , vmin , rvri , and cvi throughout the five sequential study time points are presented in figures 2 and 3 .
lps caused marked decreases in sap , vmax , and vmin , and increased rvri ( p = 0.012 versus t0 ) ; effects of lps continuously worsened over time in group s ( p = 0.028 versus t0 and t1 ) .
although resuscitation with he s and hsh failed to completely restore systemic and renal hemodynamics to baseline ( p = 0.017 , versus t0 ) , the detected associated parameters were immediately and persistently improved ( p = 0.027 , versus t1 ) , while hts only temporarily ameliorated these indices ( p = 0.039 ; t2 versus t1 ) .
figures 3(c ) and 3(d ) ( line graph ) illustrate date shown in figures 3(a ) and 3(b ) ( histogram ) in a different manner .
production of tnf- and production of no , two important inflammatory mediators , were both promoted by the lps treatment , demonstrating generation of an inflammatory response . in this study ,
the levels of renal cortical tnf- and no markedly increased after lps challenge ( p = 0.001 ; all versus group c ) and were influenced to different degrees by the four types of fluid resuscitation . in the he s and hsh groups , tnf- levels decreased ( p = 0.013 versus group s ) ( figure 4(a ) ) and no levels did not change , while in the hts group both tnf- and no were maintained at high levels ( figure 4(b ) ) .
mda , a by - product of lipid peroxidation , reflects the extent of oxidative damage , while the activity of sod , a critical antioxidant , can be used to represent the antioxidative capacity .
lps challenge increased renal cortical levels of mda and inhibited activity of sod ( p = 0.002 ; all versus group c ) .
small - volume resuscitation improved the renal oxidative stress state , with resuscitation with he s and hsh decreasing the augmented mda level and enhancing the sod activity ( p = 0.036 , versus group s or hts ) ( figures 4(c ) and 4(d ) ) .
severe infection causes endotoxemia and the kidney is one of the first organ systems affected by lps .
reports indicate that endotoxemia - induced renal hypoperfusion contributes to an ischemia - reperfusion insult that potentially leads to the activation of renal inflammation and oxidative stress .
fluid resuscitation has always been recognized as the primary resuscitation strategy for patients with aki after endotoxemia [ 7 , 11 , 12 , 20 ] .
although large amounts of fluid infusion could quickly improve renal perfusion , it is strictly limited to use in some critically ill patients suffering from endotoxemia and is contraindicated in patients with heart dysfunctions .
small - volume resuscitation with hypertonic fluids has been regarded as an effective strategy for hemorrhagic shock , but it is unclear how different kinds of fluids affect kidney injury induced by lps .
hence , our purpose here was to investigate the varying ameliorating effects of small - volume resuscitation with saline , 7.5% hypertonic saline , hydroxyethyl starch 130/0.4 , and hypertonic saline hydroxyethyl starch 40 on lps - induced aki .
based on the available reports , we used the intravenous resuscitation dose of 4  ml / kg as an effective and safe dose . to this end , we conducted assessments of pathology images , systemic blood pressure , and renal perfusion , as well as renal levels of tnf- , no , and mda and activity of sod .
our results showed that , as compared with saline , small - volume hydroxyethyl starch 130/0.4 and hypertonic saline hydroxyethyl starch 40 could rapidly ameliorate the severity or postpone the development of renal tubular injury in the rat model of endotoxemia . at the same time , hydroxyethyl starch 130/0.4 and hypertonic saline hydroxyethyl starch 40 immediately restored systemic and renal blood flow and even reduced the renal inflammatory response and oxidative stress .
in contrast , 7.5% hypertonic saline only temporarily improved systemic circulation and renal blood flow without having effects on cytokine release .
endotoxemia has been proven to be associated with progressive renal dysfunction , even in the presence of normal or elevated blood pressure and cardiac output [ 13 , 14 ] .
insight into renal microcirculation should provide more meaningful information about the underlying mechanisms of renal dysfunction .
doppler ultrasound is a precise method to evaluate renal blood flow . in the present study , we applied doppler ultrasound and showed that small - volume resuscitation with he s and hsh immediately and permanently improved the deceased renal blood flow that resulted from lps challenge , while hts showed only slight and temporary effects compared to those elicited by saline .
meanwhile , parameters for systemic blood pressure evolved in coincidence with the trend of renal blood flow and pathological damage .
these results suggest that the choice of different fluids is closely related to the improvement by small - volume resuscitation of renal perfusion .
accordingly , we conclude that infusion of colloids is better for clinical small - volume resuscitation for endotoxemia patients having complications with heart dysfunction . furthermore , because the small - volume resuscitation could not completely restore systemic circulation and renal blood flow , other strategies should be adopted to improve the protective effect , including resuscitation in combination with vasoactive or organ - protective drugs .
normal saline , an isotonic solution , is rapidly distributed around vessels , while hypertonic saline can transfer interstitial fluids into vessels to improve circulation based on its hypertonicity .
however , it has a short duration of action because of the rapid reestablishment of osmotic balance between intra- and extracellular fluids .
he s and hsh can elevate colloid osmotic pressure and stay longer in vessels to effectively maintain intravascular volume .
this may be the main reason why small - volume resuscitation with he s and hsh alleviates kidney injury by improving renal hemodynamics and microperfusion . during the initial period of endotoxemia
, it is the severity of the inflammatory response and oxidative stress that trigger microvascular dysfunction and early organ failure , and treatment during the first six to twenty - four hours is critical for patient outcome and survival [ 2628 ] .
in addition to cytokine storms ( e.g. , tnf- , ifn- , and il ) , no has been increasingly recognized as a biological mediator that plays an important role in the damage process . as to sepsis ,
both experimental and clinical investigations have revealed enhanced no in the plasma , which correlates inversely with arterial bp . here , we analyzed alterations to the levels of tnf- , no , and mda , as well as sod activity , and found that small - volume resuscitation with hsh and he s reduced renal cortical inflammatory response and oxidative stress , while hts did not .
several studies of fluid resuscitation have also verified that renal perfusion is closely related to local renal oxidative and inflammatory response in endotoxic and hemorrhagic shock models .
based on the previous and these current results , we conclude that hsh and he s were more effective in improving renal blood flow .
considerable controversy exists , however , about the safety and effectiveness of using colloid solutions in critical care units . of note
, one clinical trial indicated that application of he s is associated with increased rates of renal - replacement therapy .
indeed , patients in this trial repeatedly or predominantly received infusion with he s , resulting in collagen deposition in the perinephric space , contributing to the main cause of hes - associated renal injury .
similarly , another randomized controlled study found that he s provided significantly better lactate clearance and less renal injury than saline .
the authors concluded that resuscitation with hydroxyethyl starch improves renal function and lactate clearance in penetrating trauma . in the present study
, we showed that early single resuscitation with small - volume colloids or hyperoncotic solutions could effectively alleviate renal injury and was better than the use of crystalloids .
therefore , our results provide a rationale and valuable strategy for ameliorating renal injury in the early stage of endotoxemia using resuscitation with a single small volume of colloids or hyperoncotic solutions , especially for patients complicated with heart dysfunction .
the first is that we did not assess the long - term outcome of rats , including survival rate and mortality rate observation .
secondly , albumin is another kind of colloids being considered to infuse during the early resuscitation of patients with severe sepsis . in the future study , we will replenish the detected parameters and added group albumin to make the investigation more integrated . in conclusion
, our study provides the important finding that early small - volume resuscitation with single small volumes of hydroxyethyl starch 130/0.4 and hypertonic saline hydroxyethyl starch 40 leads to renal - protective effects from the damage due to lps exposure .
 acute kidney injury associated with renal hypoperfusion is a frequent and severe complication during sepsis . 
 fluid resuscitation is the main therapy . 
 however , heart failure is usually lethal for those patients receiving large volumes of fluids . we compared the effects of small - volume resuscitation using four different treatment regimens , involving saline , hypertonic saline ( hts ) , hydroxyethyl starch ( he s ) , or hypertonic saline hydroxyethyl starch ( hsh ) , on the kidneys of rats treated with lipopolysaccharide ( lps ) to induce endotoxemia . 
 lps injection caused reduced and progressively deteriorated systemic ( arterial blood pressure ) and renal hemodynamics ( renal blood flow and renal vascular resistance index ) over time . 
 this deterioration was accompanied by marked renal functional and pathological injury , as well as an oxidative and inflammatory response , manifesting as increased levels of tumor necrosis factor- , nitric oxide , and malondialdehyde and decreased activity of superoxide dismutase . 
 small - volume perfusion with saline failed to improve renal and systemic circulation . 
 however , small - volume perfusion with he s and hsh greatly improved the above parameters , while hts only transiently improved systemic and renal hemodynamics with obvious renal injury . 
 therefore , single small - volume resuscitation with he s and hsh could be valid therapeutic approaches to ameliorate kidney injury induced by endotoxemia , while hts transiently delays injury and saline shows no protective effects . 