Impact of aortocaval shunt flow on cardiac and renal function in unilateral nephrectomized rats

Abstract

We previously reported significantly enhanced cardiac remodeling post aortocaval fistula (AV) in unilateral nephrectomized (UNX) rats. However, the relationship between the size of the AV and the cardiorenal effects in UNX rats remains unknown. In the present study, AV was induced by 20, 18 and 16 gauge needles in UNX rats to see if larger shunt would definitely induce heavier cardiac and renal damage in UNX rats. Our results demonstrated that bigger shunt size is linked with proportional more significant cardiorenal remodeling and dysfunction in UNX rats. Expression of inflammatory biomarkers including CRP, TNF-α, IL-6, IL-1β, TGF-β and MCP-1 in left kidney and heart was significantly increased in all UNX + AV groups compared to Sham rats. Inflammation might thus participate in the worsening cardiorenal functions and remodeling processes in this model.

on cardiac and renal function in unilateral nephrectomized rats

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JieWu, Zhong Cheng, Mingjing Zhang, Pengfei Zhu & Ye Gu

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Previously, we reported that cardiac remodeling post aortocaval stula (AV) was signicantly enhanced in unilateral nephrectomized (UNX) rats compared to rats without UNX1. According to our results, healthy living donors might face increased cardiac and renal dysfunction risk in case of future chronic volume overload insult, as in the case of pregnancy and other high output heart failure situations as in the case of hyperthyroidism, anemia and in patients with arteriovenous stula1. However, the relationship between dierent degrees of volume overload and adverse cardiorenal remodeling in UNX rats is still not fully understood. In the previous report, AV was established by 18-gauge needle puncture. In the present study, we performed new experiments by using 20, 18 and 16 gauge needles to induce AV in UNX rats. The main objective was to explore if larger shunt would denitely induce heavier cardiac/renal dysfunction in UNX rats.

Results

Three rats died during the 8 weeks post operation, one died on the rst day post AV in group AV18 (AV produced by 18 gauge needle) and post mortem examination revealed massive bleeding around the puncture site, and the death might thus be caused by failure of sealing the puncture site by cyanoacrylate; one died on the 14th day post operation in group AV20 (AV produced by 20 gauge needle) and another died on the 12th day post operation in group AV16 (AV produced by 16 gauge needle). Post mortem examination visualized hypertrophied heart, congested liver and lung, presence of ascites and pleural eusion, as well as edema of the limbs, suggesting that overt congestive heart failure might be the cause of death in these rats died around two weeks aer AV operation. Food and water intake did not dier between the groups, and ascites and pleural eusion were not evidenced in the rats survived to study end. Compared to the UNX alone group of previous study1, UNX+ AV increased perioperative mortality (8% vs. 0%).

Body weight and organ weights are shown in Fig.1. Eight weeks post various procedures, body weight was similar among groups. Heart weight and heart/ body weight ratio, LVW (mg), LVW/BW (mg/g), RVW (mg) and RVW/BW (mg/g) in group AV20, AV18 and AV16 tended to be higher than in Sham group with the highest value in group AV16. Wet lung weight and wet lung/BW ratio, liver wet weight and liver wet weight/BW ratio, le kidney wet weight and le kidney weight/BW also tended to be higher in group AV20, AV18 and AV16 compared to Sham group and the highest value was still seen in group AV16. Compared to the UNX alone group from the previous study1, the le kidney wet weight/ body weight ratio (4.31 1.63mg/g in UNX) was similar while heart weight/body weight ratio (2.560.17mg/g in UNX), le ventricular weight/body weight ratio (1.93 0.11 mg/g in UNX), right ventricular weight/body weight ratio (0.45 0.07mg/g in UNX) and lung wet weight/body weight ratio (3.34 0.44mg/g in UNX) tended

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Figure 1. Changes in body weight and organ weights in response to three dierent shunt size in UNX+AV rats. Values are mean SD. BW, Body weight (a); HW, Heart weight (b); HW/BW (c); LVW, le ventricular weight (d); LVW/BW (e); RVW, right ventricular weight (f); RVW/BW (g); Lung Wt, Lung wet weight (h); Lung Wt/BW (i); Liver Wt (j); Liver Wt/BW (k); LK Wt, le kidney Wt (l); LK Wt/BW (m). *p<0.05.

Sham(n=10) AV20(n=11) AV18(n=11) AV16(n=11)

LVEDD(mm) 6.4180.305 6.2960.516 7.6180.41 8.851.13*

LVESD(mm) 3.3330.396 3.3340.576 4.4040.576 4.9881.165*

EF(%) 84.334.50 83.405.32 81.202.39 79.387.05

FS(%) 48.005.55 47.006.04 44.401.34 44.257.13

Table 1. Echocardiographic Parameters. Values are mean SD. LVEDD, le ventricular end-diastolic dimension; LVESD, le ventricular end-systolic dimension; EF, ejection fraction; FS, fractional shortening. *p< 0.05 vs. Sham; p< 0.05 vs. AV20; p< 0.05 vs. AV18.

Sham(n=10) AV20(n=11) AV18(n=11) AV16(n=11)

MRAP(mmHg) 104.39.4 92.81.4 87.89.8* 79.97.5* RVP(mmHg) 3.11.3 5.90.9* 8.91.8* 11.42.1*

RPP(mmHg) 101.23.3 86.91.1 78.91.8* 68.58.7*

Table 2. The parameters of renal hemodynamics. Values are mean SD. MRAP, mean renal artery pressure; RVP, renal vein pressure; RPP, renal perfusion pressure. *p< 0.05 vs. Sham; p< 0.05 vs. AV20; p<0.05 vs.

AV18.

to be higher in all UNX + AV groups with the highest value in group AV16. The liver wet weight/body weight ratio was similar among AV20, AV18 and UNX alone (28.96 3.81 mg/g) groups in the previous study1 while signicantly increased in AV16 compared UNX alone group in the previous study1.

As shown in Table1, LVEDD and LVESD tended to be higher while LVEF and LVFS values tended to be lower in group AV18 and AV16 than in Sham group and group AV20. There is a trend of more severe LV remodeling and dysfunction with the increased stula sizes. Compared to the UNX alone group from the previous study1, LVEDD (5.960 0.659 mm in UNX) and LVESD (3.162 0.686 mm in UNX) tended to be higher while LVEF (86.44 4.22% in UNX) and LVFS (50.78 5.74% in UNX) values tended to be lower in all UNX+AV groups.

As shown in Table2, The UNX+ AV operation decreased the mean renal artery pressure and renal perfusion pressure and increased the renal vein pressure. These changes were more signicant in proportion to larger stula size. Compared to the UNX alone group from the previous study1, the mean renal artery pressure (126.4 15.4 mmHg in UNX) was lower and the renal vein pressure (5.1 0.9 mmHg in UNX) was higher in all UNX+ AV groups. These changes were more apparent in AV16 group.

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Figure 2. Changes in biochemistry and renal function parameters in response to three dierent shunt sizein UNX+AV rats. Values are mean SD. BNP-45, rat brain natriuretic peptide-45 (a); hs-CRP, hypersensitive C-reactive protein (b); Cys-c, Cystatin C (c); sCr, plasma creatinine (d); 24h Albumiuria (e); GFR, glomerular ltration rate (f); ERPF, eective renal plasma ow (g); RBF, renal blood ow (h); RVR, renal vascular resistance (i); FENa, fractional excretion of sodium (j); FGS, focal segmental glomerulosclerosis quantication (k). *p<0.05.

As shown in Fig.2, rat brain natriuretic peptide-45 tended to be slightly higher post UNX+ AV operations compared to Sham group, but the value was similar among group AV20, AV18 and AV16. Plasma Hs-CRP levels in group AV20, AV18 and AV16 tended to be higher than in Sham group, with the highest value in group AV20. Plasma creatinine and cystatin C levels in group AV20, AV18 and AV16 also tended to be higher than in Sham group, with the highest value in group AV16. GFR was signicantly and equally reduced in group AV18 and AV16 compared to Sham group, ERPF and RBF were signicantly decreased while RVR and FENa were signicantly increased in group AV20, AV18 and AV16 compared to Sham group. These changes were more signicant in proportion to larger stula sizes. 24hours albuminuria was higher in group AV18 and AV16 than in Sham and AV20 group. Focal segmental glomerulosclerosis quantication showed FGS was signicantly increased in group AV20, AV18 and AV16, and the highest FGS value was seen in group AV16. Compared to the UNX alone group from the previous study1, rat brain natriuretic (0.170.03ng/ml in UNX) tended to be higher in all UNX+ AV groups and there was a trend for more apparent changes in AV16 group. In proportion to AV size, especially in group AV18 and AV16, GFR (0.0450.015 ml/min/kg in UNX), ERPF (0.871 0.712ml/min/kg in UNX), RBF (1.468 1.200ml/min/kg in UNX) further decreased and FENa (0.120 0.075 in UNX), 24 h Albumiuria (113.35 127.44 ug in UNX) and focal segmental glomerulosclerosis quantication (0.620 0.030 in UNX) further increased compared to the UNX alone group from the previous study1.

As shown in Fig.3, the kidney injury biomarkers such as Cys-c, NGAL and -SMA expressions in le kidney tended to be higher in group AV20, AV18 and AV16 compared with Sham group. The highest expression was seen in group AV18. Inammation biomarkers such as IL-6, IL-1, TGF- and MCP-1 expressions in le kidney also tended to be higher in group AV20, AV18 and AV16 compared with Sham group and the highest expression was seen in group AV18. CRP and TNF- expression tended to be higher in group AV20, AV18 and AV16 compared to Sham group. As shown in Figs4 and 5, mRNA expression of BNP, CRP, TNF-, IL-6, IL-1, TGF- and MCP-1 in le and right ventricle also tended to be higher in group AV20, AV18 and AV16 compared to Sham group. For most parameters in le ventricle, the highest expression was seen in AV18 group. On the other hand, for most parameters in right ventricle, the highest expression was seen in AV20 group.

Discussion

As described in our previous report, although UNX only induces minor renal dysfunction, additional chronic volume overload placement during the adaptation phase of the remaining kidney is associated with aggravated cardiorenal dysfunction and remodeling in UNX rats1. In 2012, Martin et al. reported that UNX induced mild CKD could result in early cardiac brosis in case of mild diastolic impairment and preserved systolic function, the cardiac hypertrophy and impairment of heart function progressed over time2. This kidney-heart interaction seems to be independent of increase in blood pressure, sodium, or water retention, or activation of aldosterone2.

In this study, we observed if the shunt volume would be a critical determinant for above changes, i.e. the larger

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Figure 3. Changes in mRNA expression in le kidney in response to three dierent shunt size in UNX+AV rats. Values are mean SD. Cys-c, Cystatin C (a); NGAL, neutrophil gelatinaseassociated lipocalin (b); -SMA, -smooth muscle actin (c); CRP, C-reaction protein (d); TNF-, tumor necrosis factor- (e); IL-6, interleukin-6 (f); IL-1, interleukin-1 (g); TGF-, transforming growth factor- (h); MCP-1, monocyte chemotactic protein 1 (i). *p<0.05.

the shunt, the bigger the harm. So we used the previous experimental settings and induced various AV shunt ow by using 3 needle sizes. The major ndings of present study are as follows: (1) LV dilation is proportional to AV shunt ow. The extent of hypertrophy was greater in RV than that of the LV. (2) Renal function was signicantly deteriorated in this model in proportion to AV ow. (3) Above changes are concomitant with upregulated mRNA expression of inammatory and tissue injury biomarkers in the le kidney and heart.

In our model, the shunt size was controlled by using dierent sizes of needles. As mentioned in previous studies3, the dierently sized stulas were useful in creating dierent degrees of congestive heart failure. This is exemplied by a distinct dierence between the remodeling and functional parameters seen in the AV groups produced using the 20, 18 and 16 gauge needle in this study. Rats in AV16 group underwent transition to decompensated heart failure (reected by the signicantly greater right ventricular weight, LV dilatation, increased lung and liver weight), whereas rats in AV18 group were still in the compensatory phase of remodeling and rats in AV20 group only showed signs of myocardial and renal hypertrophy. The criteria for making above determinations has been well characterized in the previous aortocaval stula induced heart failure rat models by several authors4,5. Some researchers have pointed out the hemodynamic challenges to the LV and RV post AV are different. RV faced both volume and pressure overload challenges, thus exhibited a more rapid and greater hyper-trophic response6, in fact, proportional RV hypertrophy was observed with increasing AV shunt ow (Fig.1). For the LV, there is a trend of aggravation in cardiac remodeling and dysfunction with increasing AV shunt ow (Fig.1), the reason for absence of signicant dierences between the three shunt-groups (AV20, AV18 and AV16) could be that we performed the AV at 1 week post UNX, the time interval between the two operations as well as the observation time might be too short to validate the cardiac performance dierences in the LV. So, future studies with various experimental settings are required to determine the impacts of various gauge size, the various timing of AV operation post UNX surgery and post AV observation period of this model on cardiac and renal outcome as well as the eects of various pharmaceutical interventions in this model.

We previously showed, UNX alone induced a statistically significant compensatory hypertrophy of the remaining kidney while the renal dysfunction was very mild1. Results from present study demonstrate that glomerulosclerosis worsened, proteinuria progressed, ERPF, RBF further decreased and RVR, FENa, plasma creati-nine and cystatin C level further increased while the kidney injury biomarkers such as Cys-c, NGAL and -SMA expressions in le kidney tended to be higher in proportion to AV size. Thus, renal remodeling and dysfunction are shunt ow-dependent processes in this model, suggesting the hemodynamic factor may be the most important determinant in AV induced renal remodeling and dysfunction. The two important hemodynamic variables

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Figure 4. Changes in mRNA expression in le ventricle in response to three dierent shunt size in UNX+AV rats. Values are mean SD. BNP, brain natriuretic peptide (a); CRP, C-reaction protein (b); TNF-, tumor necrosis factor- (c); IL-6, interleukin-6 (d); IL-1, interleukin-1 (e); TGF-, transforming growth factor- (f); MCP-1, monocyte chemotactic protein 1 (g). *p<0.05.

that linking the heart and kidneys are renal blood ow and renal perfusion pressure (RPP). RPP was usually dened as mean arterial pressure minus renal venous pressure7. Traditionally, cardiorenal syndrome (CRS) has been interpreted as a consequence of an insufficient renal perfusion7,8, as a result of forward heart failure. Recently, there was renewed interest with the concept of a backward transmission of central venous pressure (CVP) elevation leading to renal dysfunction7, a scenario of backward heart failure911. In our study, creation of AV stula results in an immediate and sustained decrease in mean arterial pressure together with a substantial increase in venous blood ow to the right heart as evidenced by increased RVP. The increase in pressure in the venous system in rats with larger stula shunt ow should be higher than that in rats with smaller stula shunt ow. Although an increase in RVP does not always correspond to increased venous blood ow, but rather results in congestive heart failure, as a consequence of increased back ow or stasis in the venous return. As shown in Table2, the RPP value was lowered to 80mm Hg in AV16 group, that is the threshold of kidney autoregulation, which could lead to the decrease in renal perfusion, and the latter might be responsible for the further decrease in glomerular ltration rate in AV rats with larger shunt ow. It is worth noting that the blood pressure was reported to remain unchanged or only slightly lower in rat AV model12, observed reductions in renal hemodynamics in our study might possibly an artifact of anesthesia. This should be acknowledged as a potential limitation.

In addition to the traditional intra- and extra- renal hemodynamics eects post AV, there has been a growing interest in inammatory and endothelial cell activation in the AV-induced heart failure pathogenesis. It has been proposed that in case of heart failure, the endothelium changes from a quiescent redox prole to an activated proinammatory, prooxidant, and provasoconstrictive state13. It was theorized that venous congestion might cause a biochemical signal to endothelial cells inducing changes on redox phenotype of reactive oxygen species, endothelin, interleukin-6, tumor necrosis factor alpha, and nitric oxide bioavailability14. Thus, volume overload and venous congestion could serve as a source of inammatory mediators. These biomarkers activated as part of the inammatory process and might serve as major contributors to renal and heart hypertrophy/brosis. In our study, expression of biomarker like TGF-, CRP, TNF-, IL-6, IL-1 and MCP-1 in le kidney and heart all tended to be higher in UNX+ AV groups compared to Sham group, thus suggesting a potential role of these cytokines in modulating the cardiac and renal remodeling in this model. It is to note, the highest levels of these cytokines were not seen in AV16 group. In le ventricle, prominent changes were seen in AV18 group. In right ventricle, prominent changes were seen in AV20 group. The underlying reason remains unclear now. It could be speculated that the dynamic inammatory changes might dier upon various degree of AV ow in this model. The reliance on mRNA may not reect the actual levels of the cytokines in kidney and heart. Future studies with a

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Figure 5. Changes in mRNA expression in right ventricle in response to three dierent shunt size in UNX+AV rats. Values are mean SD. BNP, brain natriuretic peptide (a); CRP, C-reaction protein (b); TNF-, tumor necrosis factor- (c); IL-6, interleukin-6 (d); IL-1, interleukin-1 (e); TGF-, transforming growth factor- (f); MCP-1, monocyte chemotactic protein 1 (g). *p<0.05.

more quantitative assessment of protein changes by Western blot analysis, as well as better characterization of the remodeling are needed to address the specic causal or mechanistic events underlying the observed dierences.

Taken together, our results show that AV induced cardiac and renal remodeling in UNX rats are AV ow dependent and inammatory cytokines might play potential role in volume overload induced cardiac and renal remodeling in this model.

Materials and Methods

Study protocol and experiments were approved by the Jianghan Medical College Council on the Animal Care Committee of Jianghan University (Wuhan, China). Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). All surgery was performed under sodium pentobarbital anesthesia, and all eorts were made to minimize suering as described previously1. Male Sprague-Dawley (SD) rats (weighing 200 to 250g) were housed under standard conditions with free access to food and drinking water. Rats received a normal salt diet (0.3% NaCl) throughout the study.

Rats were randomly divided into Sham (n= 10), AV20 (AV by 20-gauge angiocath at one week aer UNX, n = 12), AV18 (AV by 18-gauge angiocath at one week aer UNX, n= 12) and AV16 (AV by 16-gauge angiocath at one week aer UNX, n= 12), respectively. We omit the nephrectomy only group, since the changes post nephrectomy were already described previously1 and obtained results were compared with data in nephrectomy only rats from the previous study1. At time (t0) = 0 wk , all rats were subjected to UNX or sham operation. At time (t1)= 1wk, rats were subjected to AV (AV established between the levels of renal arteries and iliac bifurcation) by 20, 18, and 16-gauge angiocath needles or sham operation. Rats were followed up to week 9. Aer eight weeks, survived rats were placed in individual metabolic cages and two consecutive 24-hour urine samples were collected aer 5 adaptation days. Echocardiography was performed two days aer the metabolic cage studies. Invasive hemodynamic and renal function measurements were applied to rats aer echocardiography examination. Blood sample was then collected from the vena cava. Finally, rats were sacriced under deep anesthesia (70mg/kg sodium pentobarbital intraperitoneally), and organs were removed, weighed, and processed for histological and molecular examinations.

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Forward/Reverse Sequence (5-3)

BNP F: 5-GACAAGAGAGAGCAGGACACCAT-3

R: 5-TAAGGAAAAGCAGGAGCAGAATCAT-3

TGF- F: 5-CTAATGGTGGACCGCAACAAC-3

R: 5-CACTGCTTCCCGAATGTCTGA -3

TNF- F: 5-AGCAAACCACCAAGCGGAGG-3

R: 5-CAGCCTTGTCCCTTGAAGAGAAC-3

MCP-1 F: 5-TCTGTGCTGACCCCAATAAGGAA-3

R:5-GAGGTGGTTGTGGAAAAGAGAGTG-3

Cys-C F: 5-CCACCAGGAGACAGTAAAGAAGC-3

R: 5-ATTGAGCAAGAGCAAGGTATGAC-3

NGAL F: 5-GGCTGTCGCTACTGGATCAGA-3

R: 5-GCTTGGTGGAATCATGGCTGG-3 -SMA F: 5-CTCCCAGCACCATGAAGATCAA-3

R: 5-GGGCGTGACTTAGAAGCATTTG-3

CRP F: 5-AAGCCTTCACTGTGTGTCTCTATGC-3

R: 5-TTCAGGCCCACCTACTGCAATA-3

IL-6 F: 5-AGTTGCCTTCTTGGGACTGATGT-3

R: 5-GGTCTGTTGTGGGTGGTATCCTC-3

IL-1 F: 5-ATGTATGCCTACTCATCGGGA-3

R: 5-CAACACAGGCTTGTCTTCTCC-3

GAPDH F: 5-CAACGGGAAACCCATCACCA-3

R: 5-ACGCCAGTAGACTCCACGACAT-3

Table 3. RT-PCR Forward/Reverse (F/R) Primers Sequences. BNP, brain natriuretic peptide; TGF-, transforming growth factor-; TNF-, tumor necrosis factor-; MCP-1, monocyte chemotactic protein1; Cys-c, Cystatin C; NGAL, neutrophil gelatinaseassociated lipocalin; -SMA, -smooth muscle actin; CRP, C-reaction protein; IL-6, interleukin-6; IL-1, interleukin-1; GAPDH, glyceraldehydes-3-phosphate dehydrogenase.

UNX operation was performed as previously described. Briey, the right kidney was carefully separated from the adrenal gland and the surrounding tissue15, then removed aer laparotomy under anesthesia with 1% pentobarbital sodium salt (40mg/kg intraperitoneal injection). AV was established on UNX rats at 1 week post right kidney removal with the method described by Garcia and Diebold16 with 20 or 18 or 16-gauge angiocath needles, respectively. Sham rats underwent similar surgical procedures as rats in UNX +AV group, but right kidney was not removed and AV was not established in sham rats.

Echocardiography examination was performed by an investigator blinded to the study protocol with an echocardiographic system (GE Vivid 7) equipped with a 11.4MHz transducer as previously described1. Le ventricular end-diastolic diameter (LVEDd), le ventricular end-systolic diameter (LVEDs), le ventricular ejection fraction (LVEF) and le ventricular fractional shortening (FS) were determined. Mean values of at least three consecutive cardiac cycles were used17.

in vivo Two days post echocardiography examination, le and right heart catheterization was made in pentobarbital sodium salt anesthetized (40 mg/kg, intraperitoneal injection) rats as previously described1. The mean renal artery pressure (RAP) and renal vein pressure were measured according the method described by Dong et al18 and the renal perfusion pressure (RPP) was dened as RAP minus RVP.

Renal function measurements were made as previously described1. Briey, the jugular vein was cannulated, intravenous inulin/para-aminohippurate (PAH) solution was injected as a bolus (2ml/kg), then continuously via an infusion pump (25l/min). One hour later, hemodynamics and renal function were determined for 90 min (three 30 min clearance periods). Urine was collected at 30 min intervals through the bladder catheter, arterial blood samples (500 l) were collected via the carotid artery catheter at the midpoint of each urine collection period into prechilled heparinized tubes for related measurements.

Aer blood sampling from vena cava, the rats were sacriced under additional deep anesthesia (70 mg/kg sodium pentobarbital intraperitoneally). The heart, le kidney, lung and liver were removed and immediately placed in ice-cold saline to wash out the blood. Total heart, right ventricular (RV), le ventricular (LV) weight, le kidney wet weight, lung wet weight and liver wet weight were measured aer the removal of connective tissue; the septum was included in the LV. One part of the kidney was xed in 40g/l formaldehyde, embedded in paraffin, cut into sections and stained by hematoxylin and eosin (H&E) and periodic acid-Schi (PAS) for light microscopy. The incidence of focal glomerulosclerosis (FGS) was determined as previously descried19. Gross glomerulosclerosis score was calculated as a summation of . The remaining kidney and heart tissues were snap-frozen in liquid nitrogen.

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24-hour urine albumin, plasma creatinine, plasma rBNP-45 concentrations, plasma cystatin C concentrations, plasma hs-CRP concentrations, inulin, plasma and urine PAH concentrations, GFR, clearance of inulin were determined as previously described1. Eective renal plasma ow (ERPF), renal blood ow, renal vascular resistance and fractional excretion of sodium was calculated as previously described in detail1.

Total RNA was extracted from renal cortical tissue and le/right ventricular free wall using Total RNA kit (Takara, Japan) according to the manufacturers instructions. Reverse transcription and cDNA synthesis were accomplished using RNA PCR kit (Takara, Japan). Real-time polymerase chain reaction was performed to detect the expression of various cytokines by Step One SYBR Green Mix Kit (Takara, Japan) and ABI Prism Sequence Detection System (Applied Biosystems, USA) according to the manufacturers instructions. The conditions of amplication reaction were 95 C for 30sec, 95C for 5 sec, 60 C for 30 sec, and PCR was done for 40 cycles. PCR primers are shown in Table3. Relative gene expression was calculated using the 2CT method.

All data are presented as mean SD. Dierences between groups in mean values with normal distribution were compared by one-way ANOVA followed by Tukey test, otherwise a kruskal-wallis test followed by Mann-Whitney U test with Bonferroni correction was used. P< 0.05 was considered statistically signicant.

References

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J.W., Z.C. and Y.G. conception and design of research, J.W. and P.Z. analyzed data, J.W., Z.C. and Y.G. interpreted results of experiments, J.W. draed manuscript, Z.C. and Y.G. edited and revised manuscript, J.W. and M.Z. performed experiments, J.W. and Y.G. approved nal version of manuscript. All authors reviewed the manuscript.

Competing nancial interests: The authors declare no competing nancial interests.

How to cite this article: Wu, J. et al. Impact of aortocaval shunt ow on cardiac and renal function in unilateral nephrectomized rats. Sci. Rep. 6, 27493; doi: 10.1038/srep27493 (2016).

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