Urotensin II in the development and progression of chronic kidney disease following ⅚ nephrectomy in the rat

New Findings What is the central question of this study? Urotensin II is upregulated in patients in the later stages of chronic kidney disease (CKD), particularly in individuals requiring dialysis. Could treatment with a urotensin II receptor antagonist slow progression of renal disease? What is the main finding and its importance? In the rat, expression of urotensin II and its receptor increased, extending into cortical structures as CKD progressed towards end‐stage renal failure. Subchronic treatment with a urotensin receptor antagonist slowed but did not prevent progression of CKD. This suggests that urotensin II contributes to the decline in renal function in CKD. Abstract Elevated serum and urine urotensin II (UII) concentrations have been reported in patients with end‐stage chronic kidney disease (CKD). Similar increases in UII and its receptor, UT, have been reported in animal models of CKD, but only at much earlier stages of renal dysfunction. The aim of this study was to characterize urotensin system expression as renal disease progresses to end‐stage failure in a ⅚ subtotal nephrectomy (SNx) rat model. Male Sprague–Dawley rats underwent SNx or sham surgery and were killed at 8 weeks postsurgery [early (E)] or immediately before end‐stage renal failure [30 ± 3 weeks postsurgery; late (L)]. Systolic blood pressure, urinary albumin:creatinine ratio and glomerulosclerosis index were all increased in SNx‐E rats compared with sham‐E by 8 weeks postsurgery. These changes were associated with an increase in renal immunoreactive UII staining but little change in UT expression. As CKD progressed to end‐stage disease in the SNx‐L group, markers of renal function deteriorated further, in association with a marked increase in immunoreactive UII and UT staining. Subchronic administration of a UT antagonist, SB‐611812, at 30 mg kg−1 day−1 for 13 weeks, in a separate group of SNx rats resulted in a 2 week delay in the increase in both systolic blood pressure and urinary albumin:creatinine ratio observed in vehicle‐treated SNx but did not prevent the progression of renal dysfunction. The urotensin system is upregulated as renal function deteriorates in the rat; UT antagonism can slow but not prevent disease progression, suggesting that UII plays a role in CKD.


INTRODUCTION
The mammalian urotensin system comprises two structurally homologous peptides, urotensin II (UII) and urotensin II-related peptide (URP), and a single G q/11 -linked receptor, UT. Despite the similarity of the mature proteins, UII and URP are the products of two different genes, Uts2 (Coulouarn, Jegou, Tostivint, Vaudry, & Lihrmann, 1999) and Uts2b (Sugo & Mori, 2008) respectively. Urotensin II was shown initially to be a potent vasoconstrictor in mammals (Ames et al., 1999), but it is now known to exert a wide range of influences, including a regulatory role in the kidney (Forty & Ashton, 2012;Song et al., 2006). Indeed, the kidney is a major source of UII in both humans (Shenouda, Douglas, Ohlstein, & Giaid, 2002) and rats (Song et al., 2006); in contrast, the primary source of URP is the brain (Sugo et al., This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. , although it is also found in the kidney (Song et al., 2006). UT receptors have been localized to the human (Matsushita et al., 2001) and rat (Abdel-Razik, Forty, Balment, & Ashton, 2008b;Forty & Ashton, 2012;Song et al., 2006) kidney, with greater abundance in the medulla compared with the cortex (Abdel-Razik et al., 2008b;Song et al., 2006).

Elevated concentrations of UII have been reported in patients with
a variety of renal diseases, including IgA nephropathy and diabetic nephropathy (Balat, Karakok, Yilmaz, & Kibar, 2007;Garoufi et al., 2017;Langham et al., 2004;Mosenkis et al., 2011;Totsune et al., 2001Totsune et al., , 2004Woo et al., 2010). Serum or urinary UII concentrations are higher in patients with more severe renal dysfunction, which has led to the suggestion that UII plays a role in chronic kidney disease (CKD).
Chronic kidney disease is characterized by the development of glomerulosclerosis, which arises through enhanced activity of Experimental Physiology. 2019;104:421-433.
wileyonlinelibrary.com/journal/eph myofibroblasts, leading to an accumulation of abnormal extracellular matrix components (Liu, 2006). Urotensin II has been shown to promote fibrosis in the heart (Bousette et al., 2006;Chen et al., 2008;Dai et al., 2007;Tran et al., 2010;Tzanidis et al., 2003), blood vessels (Zhao et al., 2013), lungs (Onat et al., 2012) and liver (Kemp et al., 2009;Liu et al., 2010). In streptozotocin-induced diabetic rats, UII and UT were upregulated in association with an increase in renal fibrosis and the accumulation of extracellular matrix components (Tian et al., 2008). Furthermore, rats subject to ⅚ subtotal nephrectomy exhibited increased UII and UT mRNA expression in the remnant kidney at a time when plasma creatinine concentrations were increased fivefold, indicative of impaired glomerular filtration .
Although components of the urotensin system have been reported to be upregulated in rodent models of renal impairment, there is a mismatch between current experimental data and patient profiles in terms of disease progression. The clinical reports of elevated UII associated with renal disease have been based on patients with marked reductions in glomerular filtration rate or those requiring dialysis (CKD stages 4 and 5). In contrast, animal studies to date have been conducted at much earlier stages in renal disease progression (CKD stages 2 and 3). Although these animal studies have been able to provide some mechanistic insight, they are not representative of the CKD patient with marked impairment of renal function.
Accordingly, the aims of this study were to characterize urotensin system expression in the rat kidney as renal disease progressed from early dysfunction to end-stage renal failure and to determine whether antagonism of the UT receptor could slow or prevent the progression of CKD in a ⅚ subtotal nephrectomy model.

Ethical approval
The animal experiments were conducted under the authority of a licence (PPL 40/3438) granted in accordance with the UK Animals (Scientific Procedures) Act 1986 and received local ethical approval.

Subtotal nephrectomy procedure
Male Sprague-Dawley rats (8-10 weeks, 285 ± 28 g) were purchased from Charles River (Margate, UK) and held in the animal facility for 1 week before use. Animals had access to a standard diet (RM1, expanded pellet diet; Special Diet Services, Witham, UK) and water ad libitum at all times, and were held in a 12 h-12 h light-dark cycle with controlled temperature (22-24 • C) and relative humidity.
Subtotal nephrectomy (SNx) was carried out as a two-step surgical procedure to reduce renal mass. This approach was chosen instead of the alternative, which involves ligating branches of the renal artery, because the latter induces a marked increase in activity of the reninangiotensin system (Yang, Zuo, & Fogo, 2010). In the first stage, the upper and lower poles of the left kidney were removed under isoflurane anaesthesia (2% isoflurane in oxygen at 2 l min −1 ), leaving one-third of the left kidney intact. The cut faces were sealed with tissue glue [Histoacryl (n-butyl-2-cyanoacrylate); Braun Medical Ltd,

New Findings
• What is the central question of this study?
Urotensin II is upregulated in patients in the later stages of chronic kidney disease (CKD), particularly in individuals requiring dialysis. Could treatment with a urotensin II receptor antagonist slow progression of renal disease?
• What is the main finding and its importance?
In the rat, expression of urotensin II and its receptor increased, extending into cortical structures as CKD progressed towards end-stage renal failure. Subchronic treatment with a urotensin receptor antagonist slowed but did not prevent progression of CKD. This suggests that urotensin II contributes to the decline in renal function in CKD.
Sheffield, UK] in order to prevent bleeding. Buprenorphine analgesia (0.06 mg per procedure; Sogeval UK, Sheriff Hutton, York, UK) and fluid replacement (1 ml, 154 mM sodium chloride for injection; Braun Medical Ltd) were administered S.C. before recovery from the anaesthetic. One week later, the right kidney was removed completely under isoflurane anaesthesia; analgesia and fluid replacement were provided as described above. A separate group of sham-operated animals, in which the left and 1 week later the right kidney were exteriorized and returned to the peritoneal cavity, acted as controls.
All animals were monitored closely over the first 48 h postsurgery for signs of acute renal failure. A total of 54 rats underwent the SNx procedure and 31 rats underwent the sham procedure. Nine rats from the SNx group were culled owing to acute renal failure (n = 3) or for other reasons (n = 6); one sham-operated rat was culled because of over-grooming of the surgical wound.

Progression to end-stage renal failure
In order to characterize urotensin system expression as renal disease progressed, separate groups of SNx rats and time-matched sham control animals were followed to the stage of renal dysfunction (early, 8 weeks postsurgery; SNx-E n = 12 and sham-E n = 6) or (near) endstage renal failure (late, 30 ± 3 weeks postsurgery; SNx-L n = 15 and sham-L n = 12). The humane endpoint for the late SNx group was determined by a combination of behavioural changes and a rapid loss of body weight (>15% compared with peak weight). Using these criteria, all SNx rats were culled before death occurred as a result of renal failure.
Systolic blood pressure (SBP) was measured every 2-4 weeks by Sigma). Blood was collected by cardiac puncture into a chilled, heparinized syringe, and plasma was separated by centrifugation at 2000g for 10 min at 4 • C and stored at −80 • C before analysis; urine was collected into a chilled syringe by direct puncture of the bladder and stored at −20 • C before analysis. In the SNx animals, the remnant kidney was either perfusion fixed with 4% paraformaldehyde or flash frozen in liquid nitrogen. In the sham animals, the left kidney was perfusion fixed and the right kidney was frozen.

The UT antagonist
The UT antagonist SB-611812 {2,6-dichloro-N-[4-chloro-3-[2-(dimethylamino) ethyloxy] phenyl]-4-(trifluoromethyl) benzene-sulfonamide} was chosen on the basis of a previous in vivo study, in which 100% bioavailability and a 4-5 h half-life after administration by gavage were demonstrated (Rakowski et al., 2005). The bioavailability and clearance profile of SB-611812 were also confirmed in our hands using a 24 h pharmacokinetic protocol ( Figure 1). Biological efficacy was demonstrated in the rat in vivo after gavage administration at a dose of 30 mg kg −1 day −1 (Rakowski et al., 2005). The antagonist was synthesized by Pharmaron Beijing Co. Ltd (Beijing, China).

Subchronic UT antagonist treatment
Seven days after undergoing SNx or sham surgery, rats began a 13 week treatment course with either the UT antagonist SB-611812 or vehicle [0.5% hydroxyl-propyl methylcellulose (Sigma) with 0.1% Tween20 (Sigma) in deionized water]. Antagonist-treated rats (SNx-A n = 10, sham-A n = 6) received 30 mg kg −1 day −1 of SB-611812 by gavage (P.O.) in a vehicle volume of 2.5 ml kg −1 ; vehicle-treated rats (SNx-V n = 8, sham-V n = 6) received 2.5 ml kg −1 of the vehicle alone (P.O.). Body weight was recorded every 2-4 days; systolic blood pressure was measured as described above every 2 weeks. Urine samples were collected as described above every 2 weeks for the measurement of albumin and creatinine. At the end of the treatment period (14 weeks postsurgery), the rats were anaesthetized with Inactin (100 mg kg −1 ) to enable the collection of terminal blood and urine samples and tissue harvesting as described above.

Immunohistochemistry
Urotensin peptides (UII and/or URP) were localized in 6-m-thick sections using a polyclonal rabbit anti-flounder UII antibody [1:100 dilution, raised against the conserved amino-acid sequence Cys-Phe-Trp-Lys-Tyr-Cys by Dr P. Ingleton, University of Sheffield, UK (Winter, Hubbard, McCrohan, & Balment, 1999), RRID:AB_2744516], followed by application of a secondary polyclonal goat anti-rabbit antibody (1:100; DakoCytomation, Ely, UK; catalogue no. P0448, RRID:AB_2617138). A polyclonal goat anti-rat UT receptor TA B L E 1 Systolic blood pressure, terminal urine and plasma analysis and glomerulosclerosis index scores in subtotal nephrectomized (SNx) and sham-operated rats Systolic blood pressure was measured at 7 weeks in the early (E) group and at 28 weeks postsurgery in the late (L) group. Terminal samples were collected at 8 weeks in the early group and when the SNx rats reached the defined humane endpoint (mean 30 ± 11.6 weeks postsurgery) in the late group; a time-matched sham animal was killed at the same point. The number of animals per group is indicated in square brackets. Data are shown as means ± SD for normally distributed continuous variables or medians (interquartile range) for ordinal data or those that were not normally distributed.

Progression of CKD in the SNx rat
The SNx-E rats started to develop increased SBP compared with  Table 1). The mean survival time in SNx rats allowed to progress to end-stage renal failure was 30 ± 11.6 weeks.  (Table 1).
Although terminal plasma creatinine in the SNx-E group was not statistically different from that of the sham-E control group (P = 0.52), blood urea nitrogen was significantly higher in SNx-E animals (P = 0.014; Table 1). In the SNx-L group, both plasma creatinine (P < 0.001) and blood urea nitrogen (P < 0.001) were significantly higher than in the sham-L group.
The glomerulosclerosis index was significantly higher in both SNx-E (P < 0.001) and SNx-L (P < 0.001) compared with their respective controls (Table 1). Glomerular basement membrane marker mRNAs Col4a1 (P = 0.95), Lamb1 (P = 0.99) and Fn1 (P = 0.27) were not significantly different in the renal cortex of SNx-E rats compared with sham-E. However, expression of these markers was significantly upregulated in the SNx-L animals compared with time-matched shams (P < 0.001; Figure 2).

Renal UII and UT protein expression increases as CKD progresses
In common with all commercially available antibodies, the UII antibody used for immunohistochemistry is unable to distinguish between UII and URP owing to their structural homology. Hence, UII immunoreactivity represents cross-reactivity with both UII and URP. In

Subchronic UT antagonism induces a modest delay in CKD progression
The SBP in the vehicle-and antagonist-treated sham rats remained stable and similar throughout the 13 week period (Figure 5a). At the end of the experiment, SBP was 135.1 ± 5.9 mmHg in the sham-V group and 134.3 ± 7.8 mmHg in the sham-A group. In contrast, SBP increased in the SNx rats throughout the duration of the experiment (P time*SNx < 0.001); as a result, the SNx rats had significantly higher SBP compared with the sham animals (P SNx < 0.001). Overall, antagonisttreated rats had significantly lower SBP than their vehicle-treated counterparts (P UT-A = 0.032); this effect was attributable to the SNx-A animals having significantly lower SBP than the SNx-V rats (mean difference 5.9 ± 1.4 mmHg, P SNx*UT-A = 0.05). Closer inspection of the  F I G U R E 3 Immunolocalization of urotensin system proteins in the renal cortex as chronic kidney disease progresses. Urotensin II (UII; a-d) and urotensin receptor (UT; e-h) proteins in the cortex of sham and SNx rats during the early and late stages of renal disease. Urotensin II immunoreactivity was generally diffuse and localized to the cytoplasm of cells in the proximal tubules (P), with some staining also seen in the distal tubules (D) in the sham groups (a and b). Similar staining was seen in the early SNx group (c). In the late SNx group (d), staining was seen in the tubular epithelia (arrows), with further diffuse staining within the glomeruli and in areas of peritubular hypercellularity (red star). Diffuse UT immunoreactivity was observed in the sham groups (e and f) and in the early SNx group (g). Staining in the late SNx cohort (h) was particularly abundant in areas of peritubular hypercellularity (arrowheads). (i-p) Representative negative controls (primary antibody omitted) across each group. All images are representative and were captured at ×40 magnification with scale bars representing 50 m (d, f, n), ×20 magnification with scale bars representing 100 m (a-c, e, h; i-m, p) or ×10 magnification with scale bars representing 200 m (g, o) data revealed that SBP in the SNx-V group diverged from that in the sham-V rats after 10 weeks (sham-V 137.1 ± 9.8 mmHg, n = 6 versus SNx-V 154.4 ± 23.1 mmHg, n = 9, P < 0.001), whereas that in the SNx-A rats did not increase compared with sham-A rats until 12 weeks (sham-A 134.8 ± 9.6 mmHg, n = 6 versus SNx-V 151.5 ± 34.8 mmHg, n = 10, P < 0.001). Thus, subchronic treatment with the UT antagonist SB-611812 delayed the increase in SBP in SNx rats by 2 weeks. (a) SNx-E Sham-L Sham-E F I G U R E 4 Immunolocalization of urotensin system proteins in the renal medulla as chronic kidney disease progresses. Urotensin II (a-d) and UT (e-h) proteins in the medulla of sham and SNx rats during the early and late stages of renal disease. Typically, UII immunoreactivity was localized within the cytoplasm of collecting duct epithelial cells of sham-E (a) and sham-L rats (b). In the SNx-E (c) and SNx-L (d) groups, the staining was localized in a similar manner, with additional faint staining visible in the surrounding interstitium. Expression of UT was localized within the collecting ducts (arrows). Expression of UT in SNx-E (g) was comparable with that in sham-E animals (e). In the SNx-L group (h), the staining was localized in a pattern similar to that of the time-matched shams (f) but with a pronounced apical distribution, and additional faint staining was visible in the surrounding cells. (i-p) Representative negative controls (primary antibody omitted) across each group. All images are representative and were captured at ×40 magnification with scale bars representing 50 m (e-h, n, o), ×20 magnification with scale bars representing 100 m (a-d, i-m) or ×10 magnification with scale bar representing 200 m (p)

Expression of UT is unaffected by subchronic UT antagonism, but expression of UII is diminished in the renal cortex
Urotensin II-immunoreactive staining was mainly associated with the proximal tubules in the renal cortex of both sham-V ( Figure 7a) and sham-A rats (Figure 7b). A similar staining pattern was evident in sections from SNx rats receiving vehicle (Figure 7c
with a UT antagonist for 13 weeks delayed the progression of CKD, albeit for a modest 2 week period. Collectively, these data suggest that upregulation of the urotensin system contributes to the progressive decline in renal function in CKD.
Immunohistochemistry revealed parallel increases in UII immunoreactivity and UT expression in the kidneys of SNx rats as renal function declined. These observations confirm a report in SNx rats at 8 weeks postsurgery ) and now show that urotensin system expression continues to increase as renal function declines. The pattern of UII-immunoreactive staining in the cortex and medulla of SNx rats was broadly similar to that of time-matched sham-operated rats and our previous observations in both 'normal' (Song et al., 2006) and hypertensive rats (Abdel-Razik, Balment, & Ashton, 2008a).
However, as disease progressed UII-positive staining became apparent both in the glomeruli and in areas undergoing proliferative expansion.
In addition, there was profound upregulation in the expression of UT in the cortex of SNx rats. In control conditions, we have observed only occasional staining for UT in the cortex, specifically in the glomerular F I G U R E 6 Collagen IV 1 (Col4a1; a), laminin-1 (Lamb1; b) and fibronectin 1 (Fn1; c) mRNA expression normalized to B2m in the renal cortex of SNx and sham rats after subchronic treatment with the UT antagonist SB-611812 (30 mg kg −1 day −1 ) or vehicle. Compared with the SNx-V group, 13 weeks of UT antagonist treatment in SNx rats had no effect on Col4a1 (P = 0.49), Lamb1 (P > 0.99) or Fn1 (P = 0.83) mRNA expression. Data are shown as a scatter plot with median; statistical comparisons were by two-way ANOVA of log 10 -transformed data followed by Tukey's post hoc test arterioles and macula densa; the majority of UT staining is found in the medulla (Song et al., 2006). However, in the late SNx animals abundant UT-positive staining was observed throughout the cortex.
The UT expression pattern in the medulla also appeared to change as SNx rats entered the later stages of renal disease. Compared with diffuse staining for UT in the cytoplasm of the collecting ducts in early SNx and sham rats, there was a pronounced apical distribution of UT in the late SNx animals. We did not measure urinary electrolyte excretion in these animals; however, UII is known to alter renal ion transport (Abdel-Razik et al., 2008b;Song et al., 2006), and this translocation of UT receptors may therefore reflect a change in the regulation of electrolyte excretion in the later stages of renal disease.
Increased UII and UT expression have been reported in the kidneys of streptozotocin-induced diabetic rats, in association with the accumulation of extracellular matrix components, leading to the conclusion that UII might play a causal role in renal injury and fibrosis in diabetes (Tian et al., 2008). The present study shows that increased UII and UT expression are also associated with renal dysfunction and the accumulation of extracellular matrix components in non-diabetic CKD.
It is worth noting that although protein expression for urotensin system components was high, particularly in the kidneys from the SNx-L group that were killed immediately before renal failure, attempts to quantify Uts2, Uts2b and Uts2r (UT receptor) mRNA were unsuccessful.
Relative to the housekeeping gene Ywhaz, expression levels of Uts2, Uts2b and Uts2r were very low. Others have experienced similar difficulties in detecting mRNA for the urotensin system genes in rat kidneys. Sugo et al. (2003) were not able to detect Uts2 or Uts2b transcripts in rat kidney using qPCR, which is consistent with the earlier work of Coulouarn et al. (1999); interestingly, both groups detected transcripts in human kidney (Coulouarn et al., 1998;Sugo et al., 2003). Tal et al. (1995) describe an absence of mRNA for Uts2r by RT-PCR and Northern blot. Mori et al. (2009) F I G U R E 7 Immunolocalization of urotensin system proteins in the renal cortex of SNx and sham rats after subchronic treatment with the UT antagonist SB-611812 (30 mg kg −1 day −1 ) or vehicle. Urotensin II (a-d) and UT (e-h) proteins in the cortex of sham and SNx rats receiving the UT antagonist or vehicle. Urotensin II immunoreactivity was diffuse in both sham (a, b) and SNx (c, d) groups, with the most prominent staining localized to the proximal tubules (P) (compare with distal tubules (D) and glomeruli (G)). There was limited immunostaining for UT in cortical sections from both sham groups (e, f). In SNx rats receiving vehicle (g) or UT antagonist (h), staining was observed primarily within areas of hypercellular expansion (arrowheads) rather than within intact cortical structures; some additional staining was seen in the parietal layer (arrows). (i-p) Representative negative controls (primary antibody omitted) across each group. All images are representative and were captured at ×20 magnification with scale bars representing 100 m which upregulation of the urotensin II system results in albuminuria and hypertension is likely to involve both direct and indirect effects.
Urotensin II is a potent vasoconstrictor (Ashton, 2006) and has been shown to increase fibrosis and the accumulation of extracellular matrix components in the kidney (Tian et al., 2008). Furthermore, urotensin II acts synergistically with the renin-angiotensin system, itself a major contributor to the progression of CKD (Takahashi et al., 2009), to stimulate fibrosis (Song et al., 2012) and contraction of the thoracic aorta . Hence, administration of SB-611812 could on SBP and uACR are unclear. The preliminary pharmacokinetic profile after oral dosing at 5 mg kg −1 showed good bioavailability and an acceptable clearance profile. The drug dose used in the subsequent experiment, 30 mg kg −1 day −1 , has been shown previously to be effective in preventing lesion of the carotid artery intima in rats after balloon angioplasty (Rakowski et al., 2005). The drug was given by gavage in both studies, although the treatment period was longer, at 28 days, in the earlier report by Rakowski et al. (2005). SB-611812 is reported to be a competitive antagonist, with a negative log concentration of antagonist required to produce an agonist dose ratio of 2 (pA 2 ) of 6.60 and 6.59 in HEK293 cells transfected with rUT and in isolated rat aorta, respectively. Given that both UII immunoreactivity and UT expression increased in the kidney as CKD progressed, it is possible that the antagonist was outcompeted by increasing SNx-V Sham-A Sham-V F I G U R E 8 Immunolocalization of urotensin system proteins in the renal medulla of SNx and sham rats after subchronic treatment with the UT antagonist SB-611812 (30 mg kg −1 day −1 ) or vehicle. Urotensin II (a-d) and UT (e-h) proteins in the medulla of sham and SNx rats receiving the UT antagonist or vehicle. In the medulla of sham rats receiving vehicle (sham-V; a) or UT antagonist (sham-A; b), immunohistochemical staining for urotensin II was localized primarily within the collecting duct epithelium (arrows, panels a-d). This pattern was also seen in SNx rats receiving vehicle (SNx-V; c) or UT antagonist (SNx-A; d). Expression of the UT receptor was consistent across sham-V (e), sham-A (f), SNx-V (g) and SNx-A (h) groups. Staining was most obvious in the collecting ducts (arrowheads, all panels); however, staining was also seen in the thin limbs of the loop of Henle (arrows in panels e and h). (i-p) Representative negative controls (primary antibody omitted) across each group. All images are representative and were captured at ×20 magnification with scale bars representing 100 m Overall, the present study has shown that the urotensin system is upregulated in non-diabetic CKD and that expression increases as renal function declines. In particular, there is a marked increase in both UII immunoreactivity and UT expression in the renal cortex in the later stages of renal disease. These observations are consistent with stimulatory effects of UII on the deposition of extracellular matrix components and fibrosis. Subchronic treatment with a UT antagonist resulted in a modest but significant delay in disease progression in SNx rats; however, it did not prevent glomerulosclerosis. Taken together, these data suggest that the urotensin system contributes to the progression of chronic kidney disease in the rat.