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Increased urinary C-type natriuretic peptide excretion may be an early marker of renal tubulointerstitial fibrosis
Peptides 37 (2012) 98–105
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Peptides journal homepage: www.elsevier.com/locate/peptides
Increased urinary C-type natriuretic peptide excretion may be an early marker of renal tubulointerstitial fibrosis Peng Hu a,∗ , Jing Wang a , Bo Hu a , Ling Lu a , Qiang Xuan b , Yuan Han Qin c a
Department of Pediatrics, the First Affiliated Hospital of Anhui Medical University, No. 218 Ji-Xi Road, Hefei 230022, PR China Department of Urology, Anhui Provincial Hospital, Anhui Medical University, No. 17 Lu-Jiang Road, Hefei 230001, PR China c Department of Pediatrics, the First Affiliated Hospital of Guangxi Medical University, No. 6 Shuang-Yong Road, Nanning 530021, PR China b
a r t i c l e
i n f o
Article history: Received 10 May 2012 Received in revised form 18 June 2012 Accepted 18 June 2012 Available online 26 June 2012 Keywords: C-type natriuretic peptide Radioimmunoassay Tubulointerstitial fibrosis Unilateral ureteral obstruction Wistar rats
a b s t r a c t Although recent major advances have developed a much better understanding of the pathophysiological pathways, tubulointerstitial fibrosis (TIF) is still currently incurable. Therefore, early detection may mean that the condition is more manageable than it was in the past. C-type natriuretic peptide (CNP) has been found to be a potent vasodilator but a weak natriuretic factor. In addition, CNP has also been believed to be produced in tubular cells and presented as a local modulator with anti-inflammatory and anti-proliferative effects. Elimination of CNP occurs by three main mechanisms, neutral endopeptidase, natriuretic peptide receptor-C and urinary excretion. Among them, the status of urinary CNP excretion in nephropathies is not yet fully elucidated. In the present study, subgroups of rats were subjected to unilateral ureteral obstruction (UUO) or sham operation and observed for 24 h to 3 months. Urinary CNP excretion was significantly enhanced in UUO rats from 24 h to 1 month post-ligation compared to sham-operated rats. Urinary CNP excretion was also markedly higher than CNP concentrations both in abdominal aorta and in renal vein, and almost identical concentrations in these two vessels excluded major renal extraction of circulating CNP of systemic origin. Urinary CNP excretion was negatively correlated with urinary protein concentration, blood urea nitrogen and creatinine, while positively correlated with albumin. In conclusion, the increased urinary CNP excretion is strongly associated with TIF progression, and may serve as an early marker of TIF. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Tubulointerstitial fibrosis (TIF) is a final common pathway leading to end-stage renal failure (ESRF), characterized by excess accumulation of extracellular matrix (ECM) and irreversible loss of renal function [18]. According to an epidemiological survey of 2,000,000 residents in Nanjing, China, TIF is the third-leading cause of ESRF [14]. Recent major advances have developed a much better understanding of the pathophysiological pathways involved in the initiation of TIF. Proteinuria, hypoxia, oxidative stress and many other factors which are induced during pathological conditions can stimulate pro-inflammatory and pro-fibrotic signaling in tubular cells [10,17]. However, TIF is still currently incurable, except for renal replacement. Therefore, early detection may mean that the condition is more manageable than it was in the past. C-type natriuretic peptide (CNP), primarily isolated from central nervous tissues and endothelial cells, has only moderate natriuretic actions compared with the other natriuretic peptides and
∗ Corresponding author. Tel.: +86 551 2922058. E-mail address: [email protected] (P. Hu). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.06.009
acts mainly as a vasodilating agent [24]. Currently, CNP is believed to be produced locally in tubular cells and glomeruli of normal human kidney [29]. CNP specifically binds to the transmembrane natriuretic peptide receptor-B, resulting in the synthesis of intracellular cyclic guanosine monophosphate. Elimination of CNP occurs by three main mechanisms, neutral endopeptidase, natriuretic peptide receptor-C and urinary excretion [13]. Among them, the status of urinary CNP excretion in nephropathies is not yet fully elucidated. 2. Materials and methods 2.1. Animals and treatment Male Wistar rats weighting 190–250 g were used in the present study. All animal experimentation was performed at the animal facility within the Preclinical Medicine Institute of Anhui Medical University. The procedures and protocols were approved by the Institutional Animal Care and Use Committee. 96 rats were separated into 16 experimental groups: 8 groups undergoing left proximal unilateral ureteral obstruction (UUO) (n = 6) and 8 groups with sham-operated rats (SOR) (n = 6). The fasted animals were
P. Hu et al. / Peptides 37 (2012) 98–105
operated under intraperitoneal pentobarbital anesthesia (60 mg/kg body weight) and sterile conditions. UUO rats underwent left proximal ureteral ligation with 4–0 silk at the junction of the upper with the two lower thirds of its length. The ureter was cut between the ligatures to prevent retrograde urinary tract infection. SOR underwent a sham laparotomy with ureteric manipulation through a midline incision. No antibiotics were given. Animals were anesthetized by intraperitoneal pentobarbital injection and sacrificed by heart puncture at 24 h, 72 h, 1 w, 2 w, 3 w, 1 m, 2 m and 3 m postligation, respectively. The obstructed kidneys of UUO rats and the left kidneys of SOR were harvested. 2.2. Renal morphology At harvest, each kidney was washed with saline, blotted dry on gauze, and weighed. Whole kidney weight was expressed as a percentage of body weight determined at the time when rats were euthanized. Estimation of cortical thickness was done by measuring the distance from renal capsule to corticomedullary junction. Midcoronal kidney sections were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections (4 m thick) were stained with hematoxylin and eosin, and examined independently by two pathologists blinded to the experimental design. 2.3. Laboratory analysis All rats were placed in metabolic cages and urine was collected for 24 h. Blood samples were taken from abdominal aorta on the same time points. Urinary protein concentration (UP) from 24 h urine sample was determined by biuret colorimetric method (Boehringer Mannheim, Italy). Serum total protein (TP), albumin (Alb), blood urea nitrogen (BUN), and creatinine (Cr) were measured by standard enzymatic method (Randox, UK). 2.4. Radioimmunoassay CNP immunoreactivities from samples of urine, abdominal aorta and renal vein were determined by a radioimmunoassay (cross reactivity to human ANP, BNP and DNP <1%; Phoenix Pharmaceuticals, USA) after extraction as previously described [6]. Blood samples were collected in disodium EDTA vacutainers containing aprotinin (500 KIU/mL of blood) and centrifuged at 1600 × g for 15 min at 4 ◦ C. Plasma and urine samples were stored at −80 ◦ C until assayed for CNP. 2 mL plasma or 5 mL urine were passed through Sep-Pack C18 cartridges and eluted with 5 mL 60% acetonitrile containing 0.1% trifluoracetic acid. The eluate was lyophilized and reconstituted for radioimmunoassay. Duplicate samples were tested for radioimmunoassay. Serial dilutions (1/1, 1/2, 1/4, 1/8, 1/16) of plasma and urine samples were subjected to CNP immunoreactivities. The correlation coefficients between the levels of CNP immunoreactivities and the degree of dilution were r = 0.98 and r = 0.99. The intra- and interassay coefficients of variation were 5.0% and 10.0% for plasma CNP immunoreactivities, and 6.0% and 11.0% for urine CNP immunoreactivities. The sensitivity and specificity were found to be 97.2% and 96.5%, respectively. 2.5. Statistical analyses All values are expressed as mean ± SEM. Comparison of mean values between groups was made using one way ANOVA, and post hoc analysis was calculated using the Student–Newman–Keuls test. Correlations between variables were assessed by linear regression. A value of P < 0.05 was considered significant. Statistical analysis was performed using the statistical package for social studies SPSS version 11.5.
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3. Results Representative histology images of the kidneys obtained from SOR and UUO rats are presented in Fig. 1. All morphological lesions observed in the obstructed kidneys became more aggravated in time. After 24 h of UUO, glomerular and tubulointerstitial morphologies were almost normal. After 72 h of UUO, renal damage was limited to a reduction of peritubular capillaries, tubular atrophy and widen interstitial space, accompanied by an inflammatory cell infiltration in the interstitium. At 1, 2 and 3 weeks post-obstruction, all lesions observed at earlier time point worsened and increased ECM deposition became a prominent feature. Atrophic tubules were surrounded by a thickened, wrinkled basement membrane. Glomerular damage was limited to thickening of Bowman’s capsule. At 1, 2 and 3 months after UUO, ECM deposition increased further and the interstitial space of the obstructed kidneys was populated by numerous fibroblasts. However, section derived from sham-operated kidneys had a normal appearance. Measurement of renal anatomy is shown in Fig. 2. There were two intersections of the ratio of kidney weight to body weight (KW/BW) between SOR and UUO rats. KW/BW was significantly raised in UUO rats at 24 h, 72 h, 2 months, and 3 months postligation (P < 0.05), whereas decreased in UUO rats at 2 weeks post-ligation (P < 0.05), in comparison to their sham-operated counterparts. As compared with rats at 24 h after UUO, KW/BW was significantly lower in UUO rats at 1 week, 2 weeks, 3 weeks, and 1 month post-ligation respectively (P < 0.05). UUO rats exhibited significantly lower cortical thickness than those in SOR at 1 week, 2 weeks, 3 weeks, 1 month, 2 months, and 3 months post-ligation respectively (P < 0.05). In addition, the cortical thickness decreased time-dependently in the obstructed kidneys, and these differences reached statistical significance from 1 week to 3 months post-obstruction when comparing with 24 h after UUO (P < 0.05). Biochemical parameters in all groups are shown in Table 1. At the time of sacrifice, UP levels were raised gradually, but significantly, in UUO rats from 72 h to 3 months post-ligation in comparison to their sham-operated counterparts (P < 0.05). Although no significant differences in serum TP were observed between SOR and UUO rats at all time points (P > 0.05), Alb was obviously decreased in UUO rats at 3 weeks, 1 month, 2 months, and 3 months post-ligation respectively (P < 0.05). UUO rats exhibited significant higher BUN than those in SOR from 2 weeks to 3 months post-ligation (P < 0.05), and significant higher Cr than those in SOR from 1 week to 3 months post-ligation (P < 0.05). The mean CNP concentrations from urine, abdominal aorta and renal vein of SOR were 1.13 pmol/L, 1.11 pmol/L and 1.17 pmol/L at 24 h post-ligation, respectively. In comparison to SOR, urinary CNP excretion was significantly raised in UUO rats at 24 h, 72 h, 1 week, 2 weeks, 3 weeks and 1 month post-ligation (P < 0.05). CNP immunoreactivities from urine, abdominal aorta and renal vein of UUO rats are shown in Fig. 3. The mean urinary CNP excretion was markedly increased during the period from 24 h to 1 month postligation compared to CNP concentrations both in abdominal aorta and in renal vein (P < 0.05). At 24 h post-ligation, the mean urinary CNP excretion was significantly higher in UUO rats than that in SOR (3.96 pmol/L vs. 1.13 pmol/L, P < 0.05). In addition, urinary CNP excretion of UUO rats progressively declined over time and initially reached significance at 1 week after UUO (P < 0.05). To further investigate the origin of increased urinary CNP excretion, simultaneous determinations of CNP concentrations in abdominal aorta and renal vein were performed. Almost identical concentrations in these two vessels excluded major renal extraction of circulating CNP of systemic origin (P > 0.05). Urinary CNP excretion also significantly enhanced in UUO rats from 24 h to 1 month post-ligation compared to their sham-operated counterparts (data not shown,
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P. Hu et al. / Peptides 37 (2012) 98–105
Fig. 1. Representative histology images of the kidneys obtained from SOR and UUO rats at 24 h, 72 h, 1 w, 2 w, 3 w, 1 m, 2 m and 3 m post-ligation.
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Fig. 1. (Continued ).
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Fig. 2. Measurement of renal anatomy. *P < 0.05, significantly different from the corresponding SOR group.
P < 0.05 compared with rats at 24 h after UUO.
Table 1 Biochemical parameters at time of sacrifice of SOR and UUO rats. UP (mg/L)
TP (g/L)
Alb (g/L)
BUN (mmol/L)
UUO rats 24 h 72 h 1 week 2 weeks 3 weeks 1 month 2 months 3 months
476.55 567.30 676.24 684.01 821.73 853.54 880.14 1079.63
± ± ± ± ± ± ± ±
57.20 74.70* 79.12* 56.61* 55.10* 50.49* 86.14* 94.31*
66.92 64.71 63.55 59.22 61.65 59.45 64.75 66.41
± ± ± ± ± ± ± ±
5.80 2.07 4.33 4.79 9.00 5.76 2.55 2.50
32.02 31.76 30.43 28.12 24.08 22.03 20.55 20.17
± ± ± ± ± ± ± ±
4.63 5.34 2.34 1.97 6.99* 4.17* 3.08* 2.33*
SOR 24 h 72 h 1 week 2 weeks 3 weeks 1 month 2 months 3 months
462.72 477.99 477.98 497.10 442.14 458.59 441.81 499.55
± ± ± ± ± ± ± ±
72.78 73.71 95.60 86.36 51.48 53.82 63.39 74.15
68.38 59.30 64.88 62.04 60.50 61.95 65.50 65.29
± ± ± ± ± ± ± ±
4.15 1.66 4.12 3.25 4.66 2.28 2.97 2.49
33.33 29.80 33.45 30.60 32.64 32.48 33.97 31.98
± ± ± ± ± ± ± ±
4.56 3.71 4.85 4.60 2.79 2.57 2.28 1.56
*
Cr (mol/L)
8.70 8.95 10.20 13.61 15.63 19.38 20.22 26.11
± ± ± ± ± ± ± ±
2.43 2.47 2.43 1.65* 2.04* 1.74* 2.12* 3.29*
29.44 28.60 35.00 45.13 47.56 59.00 59.67 60.25
± ± ± ± ± ± ± ±
4.14 4.08 4.00* 5.94* 3.73* 7.49* 7.70* 9.54*
8.28 7.78 8.02 7.46 7.93 7.84 8.61 7.95
± ± ± ± ± ± ± ±
2.38 2.71 3.65 1.96 1.29 1.54 1.96 1.92
25.00 21.33 24.67 24.20 20.80 24.00 21.50 29.27
± ± ± ± ± ± ± ±
4.56 3.33 4.80 5.71 5.77 4.62 3.26 4.90
P < 0.05, significantly different from the corresponding SOR group.
Fig. 3. CNP immunoreactivities from urine, abdominal aorta and renal vein of UUO rats. *P < 0.05, significantly different from both abdominal aorta and renal vein. compared with rats at 24 h after UUO.
P < 0.05
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Fig. 4. Relationship between biochemical parameters and urinary CNP excretion in UUO rats.
P < 0.05). In SOR, no significant differences in CNP immunoreactivities were observed among urine, abdominal aorta and renal vein at all time points (data not shown, P > 0.05). Relationship between biochemical parameters and urinary CNP excretion in UUO rats is given in Fig. 4. Urinary CNP excretion
was negatively correlated with UP (r = −0.94, P < 0.05, Fig. 4a), BUN (r = −0.91, P < 0.05, Fig. 4d) and Cr (r = −0.92, P < 0.05, Fig. 4e), while positively correlated with Alb (r = +0.86, P < 0.05, Fig. 4c). Furthermore, there was no correlation between urinary CNP excretion and TP (r = 0.135, P = 0.361, Fig. 4b).
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4. Discussion Approximately 90% of the kidney is composed of tubulointerstitium [10]. Activated tubulointerstitial cells play a pivotal role in many forms of renal disease both in experimental animal models and in patients [15,26]. During renal injury, these activated tubulointerstitial cells participate in the initiation of fibrogenic processes which eventually may lead to TIF and ESRF. UUO rats have been identified as one of the most classical animal models providing a valid analog for human TIF [4]. In the present study, the histological changes in tubulointerstitium began to emerge since 72 h after UUO, characterized by reduced peritubular capillary, notable tubular atrophy, widen interstitial space and inflammatory cell infiltration. When the kidney damage developed into chronic progressive stage, the excessive ECM deposition and numerous fibroblasts became the most prominent feature. The origin of fibroblasts in the kidney is not completely understood, but may derive from resident fibroblasts, from the circulating fibroblast population or from hematopoietic progenitor or stromal cells derived from the bone marrow [21]. In this study, the gross histological changes in the obstructed kidneys also exhibited periodical variation. There were two intersections of KW/BW between SOR and UUO rats, 1 week and 3 weeks post-ligation respectively. In the early stages of obstructive nephropathy, the elevation in KW/BW may be mainly attributed to tubulointerstitial congestion and edema [12]. During the period from 72 h to 2 weeks post-ligation, KW/BW then gradually declined in UUO rats. According to the studies conducted by Erkan and Tanaka [8,27], tubulointerstitial cells subjected to massive proteinuria and prolonged hypoxia–ischemia can induce mitochondrial deficits and persistent energy exhausts, subsequently causing them to undergo apoptosis. Thus, the gross histological features of apoptosis in this study could be reflected through the declined KW/BW of the obstructed kidneys. However, during the period from 3 weeks to 3 months, KW/BW progressively increased in UUO rats again, which may be due to the excessive fibroblast activation and ECM deposition. Different from the variation trend of KW/BW, the cortical thickness decreased time-dependently in the obstructed kidneys throughout the study period, in accord with the previous observations by Park et al. [20]. UUO is associated with massive proteinuria, hypoalbuminema and a decline in renal function [1,7]. In the present study, the increased UP was the earliest biochemical abnormality in UUO rats, far preceding the elevation in Cr and BUN. Comparatively speaking, hypoalbuminema was the latest biochemical abnormality observed in UUO rats, because serum Alb levels were not obviously decreased until 3 weeks post-ligation. In fact, although most UUO rats have possessed massive proteinuria before they develop into chronic progressive stage, the reality is that the majority of them present with established tubulointerstitial damage, highlighting the need to identify novel and early biomarkers of TIF progression [10]. CNP, an endothelium-derived peptide, has been found to be a potent vasodilator but a weak natriuretic factor in various species of laboratory animals [3,30]. In addition, CNP has also been believed to be produced in tubular cells and presented as a local modulator with anti-inflammatory and anti-proliferative effects in pathological conditions [11]. Both plasma levels and renal tissue expression of CNP may change to some extent in nephropathies. A historical cohort study carried out by Cataliotti et al. [2] showed that plasma CNP concentrations were significantly higher in patients with nephrotic syndrome compared to normal subjects, which could be partially offset by a low-protein diet. In our latest research, we established a UUO model in Wistar rats, and observed that the expressions of CNP mRNA and protein in the obstructed kidneys were significantly higher from 72 h to 3 months post-ligation compared to SOR, measured by real-time PCR and western blot analysis
(data not shown). However, the status of urinary CNP excretion in nephropathies is not yet fully elucidated. The present study demonstrated that urinary CNP excretion significantly enhanced in UUO rats from 24 h to 1 month post-ligation compared to SOR. Subsequently, to identify the origin of increased urinary CNP excretion in UUO rats, we also simultaneously determined CNP concentrations in abdominal aorta and renal vein, and found that urinary CNP excretion was markedly higher than CNP concentrations both in abdominal aorta and in renal vein, and almost identical concentrations in these two vessels excluded major renal extraction of circulating CNP of systemic origin. Therefore, the results of our study indirectly support that the increased urinary CNP excretion in UUO rats mainly comes from the up-regulated renal expression. Shin et al. [23] established a diabetic nephropathy model in Wistar rats by a single peritoneal injection of 55 mg/kg streptozotocin, and found that urinary CNP excretion was significantly higher in rats with diabetic nephropathy compared to control rats after day 7. Conformably, Gulberg et al. [9] studied 20 cirrhotic patients with impaired renal function, and also found that their urinary CNP excretion was higher compared to patients with normal renal function and healthy controls. What is more, the authors did not observe a renal arteriovenous concentration gradient for CNP. More persuasively, a recent study published in Hypertension revealed a negative renal arteriovenous concentration gradient for CNP existed in 120 subjects investigated for cardiovascular disorders, suggesting regional release of CNP in the human kidneys [19]. An expansion of extracellular fluid volume may account for the increased CNP expression [5]. In addition, several inflammatory factors, such as transforming growth factor-, tumor necrosis factor-␣ and interleukin-1, can also stimulate its expression in local renal tissue [16,22,25]. Very interestingly, although our study showed that urinary CNP excretion was markedly increased in UUO rats, it is still progressively declined accompanied by TIF progression, which may be attributed to the elevated expression of NPR-C in the obstructed kidneys [28]. The last but most important objective of this study is to clarify whether the increased urinary CNP excretion can serve as an early marker of TIF. The following four points may help answer this question. (1) Urinary CNP excretion was significantly increased at 24 h after UUO, far earlier than the observed changes in UP, Alb, BUN and Cr concentrations. (2) The elevation of urinary CNP excretion after UUO was greater than the reduction in UP and Alb, consistent with the study undertaken by Cataliotti et al. [2]. (3) Urinary CNP excretion was negatively correlated with UP, BUN and Cr, while positively correlated with Alb. (4) It is a noninvasive biomarker for urinary CNP excretion in comparison to serum levels of Alb, BUN and Cr. Together, here we report for the first time that the increased urinary CNP excretion is strongly associated with TIF progression.
Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81000306) and the Post-Doctoral Foundation of Anhui Medical University (No. 2009KJ02).
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Increased urinary C-type natriuretic peptide excretion may be an early marker of renal tubulointerstitial fibrosis Peng Hu a,∗ , Jing Wang a , Bo Hu a , Ling Lu a , Qiang Xuan b , Yuan Han Qin c a
Department of Pediatrics, the First Affiliated Hospital of Anhui Medical University, No. 218 Ji-Xi Road, Hefei 230022, PR China Department of Urology, Anhui Provincial Hospital, Anhui Medical University, No. 17 Lu-Jiang Road, Hefei 230001, PR China c Department of Pediatrics, the First Affiliated Hospital of Guangxi Medical University, No. 6 Shuang-Yong Road, Nanning 530021, PR China b
a r t i c l e
i n f o
Article history: Received 10 May 2012 Received in revised form 18 June 2012 Accepted 18 June 2012 Available online 26 June 2012 Keywords: C-type natriuretic peptide Radioimmunoassay Tubulointerstitial fibrosis Unilateral ureteral obstruction Wistar rats
a b s t r a c t Although recent major advances have developed a much better understanding of the pathophysiological pathways, tubulointerstitial fibrosis (TIF) is still currently incurable. Therefore, early detection may mean that the condition is more manageable than it was in the past. C-type natriuretic peptide (CNP) has been found to be a potent vasodilator but a weak natriuretic factor. In addition, CNP has also been believed to be produced in tubular cells and presented as a local modulator with anti-inflammatory and anti-proliferative effects. Elimination of CNP occurs by three main mechanisms, neutral endopeptidase, natriuretic peptide receptor-C and urinary excretion. Among them, the status of urinary CNP excretion in nephropathies is not yet fully elucidated. In the present study, subgroups of rats were subjected to unilateral ureteral obstruction (UUO) or sham operation and observed for 24 h to 3 months. Urinary CNP excretion was significantly enhanced in UUO rats from 24 h to 1 month post-ligation compared to sham-operated rats. Urinary CNP excretion was also markedly higher than CNP concentrations both in abdominal aorta and in renal vein, and almost identical concentrations in these two vessels excluded major renal extraction of circulating CNP of systemic origin. Urinary CNP excretion was negatively correlated with urinary protein concentration, blood urea nitrogen and creatinine, while positively correlated with albumin. In conclusion, the increased urinary CNP excretion is strongly associated with TIF progression, and may serve as an early marker of TIF. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Tubulointerstitial fibrosis (TIF) is a final common pathway leading to end-stage renal failure (ESRF), characterized by excess accumulation of extracellular matrix (ECM) and irreversible loss of renal function [18]. According to an epidemiological survey of 2,000,000 residents in Nanjing, China, TIF is the third-leading cause of ESRF [14]. Recent major advances have developed a much better understanding of the pathophysiological pathways involved in the initiation of TIF. Proteinuria, hypoxia, oxidative stress and many other factors which are induced during pathological conditions can stimulate pro-inflammatory and pro-fibrotic signaling in tubular cells [10,17]. However, TIF is still currently incurable, except for renal replacement. Therefore, early detection may mean that the condition is more manageable than it was in the past. C-type natriuretic peptide (CNP), primarily isolated from central nervous tissues and endothelial cells, has only moderate natriuretic actions compared with the other natriuretic peptides and
∗ Corresponding author. Tel.: +86 551 2922058. E-mail address: [email protected] (P. Hu). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.06.009
acts mainly as a vasodilating agent [24]. Currently, CNP is believed to be produced locally in tubular cells and glomeruli of normal human kidney [29]. CNP specifically binds to the transmembrane natriuretic peptide receptor-B, resulting in the synthesis of intracellular cyclic guanosine monophosphate. Elimination of CNP occurs by three main mechanisms, neutral endopeptidase, natriuretic peptide receptor-C and urinary excretion [13]. Among them, the status of urinary CNP excretion in nephropathies is not yet fully elucidated. 2. Materials and methods 2.1. Animals and treatment Male Wistar rats weighting 190–250 g were used in the present study. All animal experimentation was performed at the animal facility within the Preclinical Medicine Institute of Anhui Medical University. The procedures and protocols were approved by the Institutional Animal Care and Use Committee. 96 rats were separated into 16 experimental groups: 8 groups undergoing left proximal unilateral ureteral obstruction (UUO) (n = 6) and 8 groups with sham-operated rats (SOR) (n = 6). The fasted animals were
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operated under intraperitoneal pentobarbital anesthesia (60 mg/kg body weight) and sterile conditions. UUO rats underwent left proximal ureteral ligation with 4–0 silk at the junction of the upper with the two lower thirds of its length. The ureter was cut between the ligatures to prevent retrograde urinary tract infection. SOR underwent a sham laparotomy with ureteric manipulation through a midline incision. No antibiotics were given. Animals were anesthetized by intraperitoneal pentobarbital injection and sacrificed by heart puncture at 24 h, 72 h, 1 w, 2 w, 3 w, 1 m, 2 m and 3 m postligation, respectively. The obstructed kidneys of UUO rats and the left kidneys of SOR were harvested. 2.2. Renal morphology At harvest, each kidney was washed with saline, blotted dry on gauze, and weighed. Whole kidney weight was expressed as a percentage of body weight determined at the time when rats were euthanized. Estimation of cortical thickness was done by measuring the distance from renal capsule to corticomedullary junction. Midcoronal kidney sections were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections (4 m thick) were stained with hematoxylin and eosin, and examined independently by two pathologists blinded to the experimental design. 2.3. Laboratory analysis All rats were placed in metabolic cages and urine was collected for 24 h. Blood samples were taken from abdominal aorta on the same time points. Urinary protein concentration (UP) from 24 h urine sample was determined by biuret colorimetric method (Boehringer Mannheim, Italy). Serum total protein (TP), albumin (Alb), blood urea nitrogen (BUN), and creatinine (Cr) were measured by standard enzymatic method (Randox, UK). 2.4. Radioimmunoassay CNP immunoreactivities from samples of urine, abdominal aorta and renal vein were determined by a radioimmunoassay (cross reactivity to human ANP, BNP and DNP <1%; Phoenix Pharmaceuticals, USA) after extraction as previously described [6]. Blood samples were collected in disodium EDTA vacutainers containing aprotinin (500 KIU/mL of blood) and centrifuged at 1600 × g for 15 min at 4 ◦ C. Plasma and urine samples were stored at −80 ◦ C until assayed for CNP. 2 mL plasma or 5 mL urine were passed through Sep-Pack C18 cartridges and eluted with 5 mL 60% acetonitrile containing 0.1% trifluoracetic acid. The eluate was lyophilized and reconstituted for radioimmunoassay. Duplicate samples were tested for radioimmunoassay. Serial dilutions (1/1, 1/2, 1/4, 1/8, 1/16) of plasma and urine samples were subjected to CNP immunoreactivities. The correlation coefficients between the levels of CNP immunoreactivities and the degree of dilution were r = 0.98 and r = 0.99. The intra- and interassay coefficients of variation were 5.0% and 10.0% for plasma CNP immunoreactivities, and 6.0% and 11.0% for urine CNP immunoreactivities. The sensitivity and specificity were found to be 97.2% and 96.5%, respectively. 2.5. Statistical analyses All values are expressed as mean ± SEM. Comparison of mean values between groups was made using one way ANOVA, and post hoc analysis was calculated using the Student–Newman–Keuls test. Correlations between variables were assessed by linear regression. A value of P < 0.05 was considered significant. Statistical analysis was performed using the statistical package for social studies SPSS version 11.5.
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3. Results Representative histology images of the kidneys obtained from SOR and UUO rats are presented in Fig. 1. All morphological lesions observed in the obstructed kidneys became more aggravated in time. After 24 h of UUO, glomerular and tubulointerstitial morphologies were almost normal. After 72 h of UUO, renal damage was limited to a reduction of peritubular capillaries, tubular atrophy and widen interstitial space, accompanied by an inflammatory cell infiltration in the interstitium. At 1, 2 and 3 weeks post-obstruction, all lesions observed at earlier time point worsened and increased ECM deposition became a prominent feature. Atrophic tubules were surrounded by a thickened, wrinkled basement membrane. Glomerular damage was limited to thickening of Bowman’s capsule. At 1, 2 and 3 months after UUO, ECM deposition increased further and the interstitial space of the obstructed kidneys was populated by numerous fibroblasts. However, section derived from sham-operated kidneys had a normal appearance. Measurement of renal anatomy is shown in Fig. 2. There were two intersections of the ratio of kidney weight to body weight (KW/BW) between SOR and UUO rats. KW/BW was significantly raised in UUO rats at 24 h, 72 h, 2 months, and 3 months postligation (P < 0.05), whereas decreased in UUO rats at 2 weeks post-ligation (P < 0.05), in comparison to their sham-operated counterparts. As compared with rats at 24 h after UUO, KW/BW was significantly lower in UUO rats at 1 week, 2 weeks, 3 weeks, and 1 month post-ligation respectively (P < 0.05). UUO rats exhibited significantly lower cortical thickness than those in SOR at 1 week, 2 weeks, 3 weeks, 1 month, 2 months, and 3 months post-ligation respectively (P < 0.05). In addition, the cortical thickness decreased time-dependently in the obstructed kidneys, and these differences reached statistical significance from 1 week to 3 months post-obstruction when comparing with 24 h after UUO (P < 0.05). Biochemical parameters in all groups are shown in Table 1. At the time of sacrifice, UP levels were raised gradually, but significantly, in UUO rats from 72 h to 3 months post-ligation in comparison to their sham-operated counterparts (P < 0.05). Although no significant differences in serum TP were observed between SOR and UUO rats at all time points (P > 0.05), Alb was obviously decreased in UUO rats at 3 weeks, 1 month, 2 months, and 3 months post-ligation respectively (P < 0.05). UUO rats exhibited significant higher BUN than those in SOR from 2 weeks to 3 months post-ligation (P < 0.05), and significant higher Cr than those in SOR from 1 week to 3 months post-ligation (P < 0.05). The mean CNP concentrations from urine, abdominal aorta and renal vein of SOR were 1.13 pmol/L, 1.11 pmol/L and 1.17 pmol/L at 24 h post-ligation, respectively. In comparison to SOR, urinary CNP excretion was significantly raised in UUO rats at 24 h, 72 h, 1 week, 2 weeks, 3 weeks and 1 month post-ligation (P < 0.05). CNP immunoreactivities from urine, abdominal aorta and renal vein of UUO rats are shown in Fig. 3. The mean urinary CNP excretion was markedly increased during the period from 24 h to 1 month postligation compared to CNP concentrations both in abdominal aorta and in renal vein (P < 0.05). At 24 h post-ligation, the mean urinary CNP excretion was significantly higher in UUO rats than that in SOR (3.96 pmol/L vs. 1.13 pmol/L, P < 0.05). In addition, urinary CNP excretion of UUO rats progressively declined over time and initially reached significance at 1 week after UUO (P < 0.05). To further investigate the origin of increased urinary CNP excretion, simultaneous determinations of CNP concentrations in abdominal aorta and renal vein were performed. Almost identical concentrations in these two vessels excluded major renal extraction of circulating CNP of systemic origin (P > 0.05). Urinary CNP excretion also significantly enhanced in UUO rats from 24 h to 1 month post-ligation compared to their sham-operated counterparts (data not shown,
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Fig. 1. Representative histology images of the kidneys obtained from SOR and UUO rats at 24 h, 72 h, 1 w, 2 w, 3 w, 1 m, 2 m and 3 m post-ligation.
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Fig. 1. (Continued ).
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Fig. 2. Measurement of renal anatomy. *P < 0.05, significantly different from the corresponding SOR group.
P < 0.05 compared with rats at 24 h after UUO.
Table 1 Biochemical parameters at time of sacrifice of SOR and UUO rats. UP (mg/L)
TP (g/L)
Alb (g/L)
BUN (mmol/L)
UUO rats 24 h 72 h 1 week 2 weeks 3 weeks 1 month 2 months 3 months
476.55 567.30 676.24 684.01 821.73 853.54 880.14 1079.63
± ± ± ± ± ± ± ±
57.20 74.70* 79.12* 56.61* 55.10* 50.49* 86.14* 94.31*
66.92 64.71 63.55 59.22 61.65 59.45 64.75 66.41
± ± ± ± ± ± ± ±
5.80 2.07 4.33 4.79 9.00 5.76 2.55 2.50
32.02 31.76 30.43 28.12 24.08 22.03 20.55 20.17
± ± ± ± ± ± ± ±
4.63 5.34 2.34 1.97 6.99* 4.17* 3.08* 2.33*
SOR 24 h 72 h 1 week 2 weeks 3 weeks 1 month 2 months 3 months
462.72 477.99 477.98 497.10 442.14 458.59 441.81 499.55
± ± ± ± ± ± ± ±
72.78 73.71 95.60 86.36 51.48 53.82 63.39 74.15
68.38 59.30 64.88 62.04 60.50 61.95 65.50 65.29
± ± ± ± ± ± ± ±
4.15 1.66 4.12 3.25 4.66 2.28 2.97 2.49
33.33 29.80 33.45 30.60 32.64 32.48 33.97 31.98
± ± ± ± ± ± ± ±
4.56 3.71 4.85 4.60 2.79 2.57 2.28 1.56
*
Cr (mol/L)
8.70 8.95 10.20 13.61 15.63 19.38 20.22 26.11
± ± ± ± ± ± ± ±
2.43 2.47 2.43 1.65* 2.04* 1.74* 2.12* 3.29*
29.44 28.60 35.00 45.13 47.56 59.00 59.67 60.25
± ± ± ± ± ± ± ±
4.14 4.08 4.00* 5.94* 3.73* 7.49* 7.70* 9.54*
8.28 7.78 8.02 7.46 7.93 7.84 8.61 7.95
± ± ± ± ± ± ± ±
2.38 2.71 3.65 1.96 1.29 1.54 1.96 1.92
25.00 21.33 24.67 24.20 20.80 24.00 21.50 29.27
± ± ± ± ± ± ± ±
4.56 3.33 4.80 5.71 5.77 4.62 3.26 4.90
P < 0.05, significantly different from the corresponding SOR group.
Fig. 3. CNP immunoreactivities from urine, abdominal aorta and renal vein of UUO rats. *P < 0.05, significantly different from both abdominal aorta and renal vein. compared with rats at 24 h after UUO.
P < 0.05
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Fig. 4. Relationship between biochemical parameters and urinary CNP excretion in UUO rats.
P < 0.05). In SOR, no significant differences in CNP immunoreactivities were observed among urine, abdominal aorta and renal vein at all time points (data not shown, P > 0.05). Relationship between biochemical parameters and urinary CNP excretion in UUO rats is given in Fig. 4. Urinary CNP excretion
was negatively correlated with UP (r = −0.94, P < 0.05, Fig. 4a), BUN (r = −0.91, P < 0.05, Fig. 4d) and Cr (r = −0.92, P < 0.05, Fig. 4e), while positively correlated with Alb (r = +0.86, P < 0.05, Fig. 4c). Furthermore, there was no correlation between urinary CNP excretion and TP (r = 0.135, P = 0.361, Fig. 4b).
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4. Discussion Approximately 90% of the kidney is composed of tubulointerstitium [10]. Activated tubulointerstitial cells play a pivotal role in many forms of renal disease both in experimental animal models and in patients [15,26]. During renal injury, these activated tubulointerstitial cells participate in the initiation of fibrogenic processes which eventually may lead to TIF and ESRF. UUO rats have been identified as one of the most classical animal models providing a valid analog for human TIF [4]. In the present study, the histological changes in tubulointerstitium began to emerge since 72 h after UUO, characterized by reduced peritubular capillary, notable tubular atrophy, widen interstitial space and inflammatory cell infiltration. When the kidney damage developed into chronic progressive stage, the excessive ECM deposition and numerous fibroblasts became the most prominent feature. The origin of fibroblasts in the kidney is not completely understood, but may derive from resident fibroblasts, from the circulating fibroblast population or from hematopoietic progenitor or stromal cells derived from the bone marrow [21]. In this study, the gross histological changes in the obstructed kidneys also exhibited periodical variation. There were two intersections of KW/BW between SOR and UUO rats, 1 week and 3 weeks post-ligation respectively. In the early stages of obstructive nephropathy, the elevation in KW/BW may be mainly attributed to tubulointerstitial congestion and edema [12]. During the period from 72 h to 2 weeks post-ligation, KW/BW then gradually declined in UUO rats. According to the studies conducted by Erkan and Tanaka [8,27], tubulointerstitial cells subjected to massive proteinuria and prolonged hypoxia–ischemia can induce mitochondrial deficits and persistent energy exhausts, subsequently causing them to undergo apoptosis. Thus, the gross histological features of apoptosis in this study could be reflected through the declined KW/BW of the obstructed kidneys. However, during the period from 3 weeks to 3 months, KW/BW progressively increased in UUO rats again, which may be due to the excessive fibroblast activation and ECM deposition. Different from the variation trend of KW/BW, the cortical thickness decreased time-dependently in the obstructed kidneys throughout the study period, in accord with the previous observations by Park et al. [20]. UUO is associated with massive proteinuria, hypoalbuminema and a decline in renal function [1,7]. In the present study, the increased UP was the earliest biochemical abnormality in UUO rats, far preceding the elevation in Cr and BUN. Comparatively speaking, hypoalbuminema was the latest biochemical abnormality observed in UUO rats, because serum Alb levels were not obviously decreased until 3 weeks post-ligation. In fact, although most UUO rats have possessed massive proteinuria before they develop into chronic progressive stage, the reality is that the majority of them present with established tubulointerstitial damage, highlighting the need to identify novel and early biomarkers of TIF progression [10]. CNP, an endothelium-derived peptide, has been found to be a potent vasodilator but a weak natriuretic factor in various species of laboratory animals [3,30]. In addition, CNP has also been believed to be produced in tubular cells and presented as a local modulator with anti-inflammatory and anti-proliferative effects in pathological conditions [11]. Both plasma levels and renal tissue expression of CNP may change to some extent in nephropathies. A historical cohort study carried out by Cataliotti et al. [2] showed that plasma CNP concentrations were significantly higher in patients with nephrotic syndrome compared to normal subjects, which could be partially offset by a low-protein diet. In our latest research, we established a UUO model in Wistar rats, and observed that the expressions of CNP mRNA and protein in the obstructed kidneys were significantly higher from 72 h to 3 months post-ligation compared to SOR, measured by real-time PCR and western blot analysis
(data not shown). However, the status of urinary CNP excretion in nephropathies is not yet fully elucidated. The present study demonstrated that urinary CNP excretion significantly enhanced in UUO rats from 24 h to 1 month post-ligation compared to SOR. Subsequently, to identify the origin of increased urinary CNP excretion in UUO rats, we also simultaneously determined CNP concentrations in abdominal aorta and renal vein, and found that urinary CNP excretion was markedly higher than CNP concentrations both in abdominal aorta and in renal vein, and almost identical concentrations in these two vessels excluded major renal extraction of circulating CNP of systemic origin. Therefore, the results of our study indirectly support that the increased urinary CNP excretion in UUO rats mainly comes from the up-regulated renal expression. Shin et al. [23] established a diabetic nephropathy model in Wistar rats by a single peritoneal injection of 55 mg/kg streptozotocin, and found that urinary CNP excretion was significantly higher in rats with diabetic nephropathy compared to control rats after day 7. Conformably, Gulberg et al. [9] studied 20 cirrhotic patients with impaired renal function, and also found that their urinary CNP excretion was higher compared to patients with normal renal function and healthy controls. What is more, the authors did not observe a renal arteriovenous concentration gradient for CNP. More persuasively, a recent study published in Hypertension revealed a negative renal arteriovenous concentration gradient for CNP existed in 120 subjects investigated for cardiovascular disorders, suggesting regional release of CNP in the human kidneys [19]. An expansion of extracellular fluid volume may account for the increased CNP expression [5]. In addition, several inflammatory factors, such as transforming growth factor-, tumor necrosis factor-␣ and interleukin-1, can also stimulate its expression in local renal tissue [16,22,25]. Very interestingly, although our study showed that urinary CNP excretion was markedly increased in UUO rats, it is still progressively declined accompanied by TIF progression, which may be attributed to the elevated expression of NPR-C in the obstructed kidneys [28]. The last but most important objective of this study is to clarify whether the increased urinary CNP excretion can serve as an early marker of TIF. The following four points may help answer this question. (1) Urinary CNP excretion was significantly increased at 24 h after UUO, far earlier than the observed changes in UP, Alb, BUN and Cr concentrations. (2) The elevation of urinary CNP excretion after UUO was greater than the reduction in UP and Alb, consistent with the study undertaken by Cataliotti et al. [2]. (3) Urinary CNP excretion was negatively correlated with UP, BUN and Cr, while positively correlated with Alb. (4) It is a noninvasive biomarker for urinary CNP excretion in comparison to serum levels of Alb, BUN and Cr. Together, here we report for the first time that the increased urinary CNP excretion is strongly associated with TIF progression.
Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81000306) and the Post-Doctoral Foundation of Anhui Medical University (No. 2009KJ02).
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