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Am J Physiol Endocrinol Metab 295: E1234-E1242, 2008. First published September 16, 2008; doi:10.1152/ajpendo.90672.2008
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TRANSLATIONAL PHYSIOLOGY

Diabetes-induced upregulation of urotensin II and its receptor plays an important role in TGF-β1-mediated renal fibrosis and dysfunction

Lin Tian,1,2 Cai Li,1 Jiping Qi,2 Peng Fu,3 Xiaoyan Yu,1 Xiaokun Li,4 and Lu Cai4,5

1Department of Experimental Pharmacology and Toxicology, School of Pharmacy, Jilin University, Changchun; 2Department of Pathology, The First Clinical College of Harbin Medical University, Haerbin; 3Department of Nuclear Medicine, The Fourth Affiliated Hospital of Harbin Medical University, Harbin; 4Chinese-American Research Institute for Diabetic Complications, Wenzhou Medical College, Wenzhou, China; 5Departments of Medicine and Radiation Oncology, the University of Louisville, Louisville, Kentucky

Submitted 6 August 2008 ; accepted in final form 9 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Urotensin II (UII) was identified as the ligand for a novel G protein-coupled receptor, GPR14. UII was found not only to have a potent vasoconstrictive action but also to have profibrotic effects in the heart. The present study was to define whether UII and GPR14 also play important roles in diabetes-induced renal fibrosis and dysfunction. Diabetic rats were induced using streptozotocin, and the rat proximal tubular epithelial cells (NRK-52E) were used for the in vitro mechanism study. Results showed that expression of UII and GPR14 was significantly upregulated at both mRNA and protein levels in the diabetic kidneys compared with controls. The upregulated expressions of UII and GPR14 in the kidney were accompanied by significant increases in the renal profibrotic factor transforming growth factor (TGF)-β1 expression, the renal extracellular matrix (fibronectin and collagen IV) accumulation, and the renal dysfunction (increases in urinal N-acetyl-β-D-glucosaminidase content, 24-h urinary retinol-binding protein excretion rate, and decrease in creatinine clearance rate). Exposure of NRK-52E cells to 10–8 mol/l UII for 48 h caused a significant increase of TGF-β1, but not ANG II, production that was GPR14- and calcium-dependent, since GPR14 small-interfering RNA and calcium channel blocker nimodipine or calcium chelator EDTA all could abolish the induction of TGF- β1 by UII. Furthermore, exposure of NRK-52E cells to TGF-β1 or ANG II also increased UII and GPR14 mRNA expressions. These results suggested that diabetes-induced upregulation of UII and GPR14, most likely through autocrine and/or paracrine mechanisms, plays an important role in TGF-β1-mediated renal fibrosis and dysfunction.

diabetic nephropathy; G protein-coupled receptor 14; renal extracellular matrix accumulation; streptozotocin diabetic rats; angiotensin II; transforming growth factor-β1

DIABETIC NEPHROPATHY (DN) is one of the major microvascular complications and also the leading cause of the end-stage renal disease (ESRD). Therefore, to develop an effective approach to delay or halt the pathogenic progression toward ESRD in patients with overt DN is urgent (8, 17, 19). To understand the mechanisms responsible for the initiation and development of the diabetes-induced renal pathogenesis will help us to develop the effectively preventive or therapeutic approaches for DN (19). Many factors play roles in the pathogenesis of DN; for instance, both transforming growth factor β1 (TGF-β1) and ANG II are important factors to promote the development of DN (10, 17, 19, 27).

Urotensin II (UII), an 11-amino acid vasoactive peptide, was identified as the ligand for a novel G protein-coupled receptor, GPR14 (also named as urotensin receptor) (13). UII was found to be a powerful vasoconstrictor with potency greater than that of endothelin-1 (1, 9). Experimental and clinical studies have revealed the increased expression of UII and GPR14 in animals with experimentally induced myocardial infarction and heart failure, and in patients with hypertension, atherosclerosis, and diabetes (25, 26, 30, 33). With the UII specific receptor antagonist, UII was found likely to become a new target for the prevention and treatment of the above pathogeneses (18, 30). However, because UII was originally discovered for its vascular activity, the most of previous studies have focused on the hemodynamic effect of UII (1, 9, 25, 26, 33). Recent studies revealed that UII also promoted cell proliferation and stimulated extracellular matrix (ECM) accumulation (35, 38).

In this regard, a few recent studies have demonstrated its critical role in cardiac fibrogenesis through increased collagen synthesis in cardiac fibroblasts and cardiac hypertrophy (15, 35, 38). Upexpression of UII and its receptor in the kidney was also observed mainly in the epithelial cells of distal tubule, proximal tubule, and collecting tube of the epithelial cells (2, 14, 22), implying that the upregulated UII is also likely to take part in the DN development process. However, the direct role of and the mechanisms for UII in DN have not been addressed.

In the present study, therefore, expression of UII and its receptor in diabetic kidney and its association with functional and pathological changes were investigated using the streptozotocin (STZ)-induced diabetic rat model. To dissect the direct role of UII in the fibrotic effect and its signaling pathways, cultured rat proximal renal tubular epithelial cells (NRK-52E) were used in combination with pharmacological and genetic approaches to manipulate gene expressions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal treatments. Thirty male Wistar rats (180~220 g) were purchased from Jilin University Laboratory Animal Center. The animals were fasted overnight before induction of diabetes but had free access to drinking water. Diabetes was induced by a single intraperitoneal injection of 55 mg/kg body wt STZ (Sigma, St. Louis, MO) that was freshly dissolved in 0.1 mol/l citrate buffer at pH 4.5. The plasma glucose (PG) in all diagnosed diabetic rats, examined 72 h after STZ injection, was higher than 16.7 mmol/l. Control rats accepted an equal volume of 0.1 mol/l citrate buffer. Diabetic rats were randomly killed at 2, 4, 8, and 12 wk after the onset of diabetes. The University Animal Care and Use Committee have approved all these procedures.

Assessments of renal function. The animals were placed in individual metabolic cages for 24 h to collect urine samples under the condition of only access to tap water on the day before the rats were killed. Total urinary volumes were measured, and the urinary D-glucosaminidase (NAG) contents and retinol-binding protein (RBP) were detected using an assay kit (Boster Biological Technology, Wuhan, China). When animals were killed, blood samples were collected to measure PG, glycosylated serum proteins (GSP) using corresponding assay kits (Jiancheng Biological Technology Co, Nanjing, China), and creatinine concentration in serum and urine using automatic analyzers (Hitach-7150) to calculate the creatinine clearance rate (Ccr) as before (39).

Electron microscope examination. One-fourth kidneys were immersion-fixed in 10% buffered formalin and embedded in paraffin for a light microscopic study. For electron microscope examination, the renal cortex was cut into small pieces, prefixed in 2.5% glutaraldehyde (0.2 mol/l cacodylate buffer, pH 7.4) for 4 h, postfixed in 1% buffered sodium tetroxide for 1 h, and embedded in Epon 812. Ultrathin sections were examined using a JEM-1200 EX electron microscope.

Immunohistochemical staining. Renal tissue sections at 4 µm were used to perform immunohistochemical staining for UII, GPR14, TGF-β1, fibronectin, and collagen IV (Col IV) with the following specific antibodies: polyclonal goat anti-rat UII and anti-rat GPR14 antibodies, polyclonal rabbit anti-rat TGF-β1 and anti-rat fibronectin antibodies, and monoclonal mouse anti-rat Col IV antibody (Sigma). Color was developed by incubating with diaminobenzidine and counterstaining with hematoxylin. Controls were obtained by replacing the primary antibody with PBS. For semiquantitative analysis, 10 high-power microscope fields were randomly selected, and the pathological image analysis system was used to calculate the positive-staining signal percentage.

RT-PCR. The total RNA was extracted from the renal medulla tissues and cultured NRK-52E cells using TRIzol reagent (GIBCO). Primers for UII, GPR14, TGF-β1, ANG II receptor (AT1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed and synthesized by Shanghai Biological Engineering (Shanghai, China). The sequences for these primers are presented in Table 1.


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  		Table 1. Upstream and downstream of UII, GPR14, GAPDH, TGF-β1, and AT1 primers

 
Total RNA (0.5 µg) was amplified using the Titan TM One Tube RT-PCR kit (Boehringer-Mannheim, Shanghai, China). Twenty-five cycles of replication were used. The products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Bands were digitized using a Tanon-1000 Gel Image System (Shanghai). The ratios of UII, GPR14, TGF-β1, or AT1 band density to GAPDH band density in various groups are presented.

Cell cultures of rat NRK-52E cells and treatments. The NRK-52E cells, purchased from American Type Culture Collection, were resuspended in DMEM supplemented with 20% FBS and 100 U/ml antibiotics. The NRK-52E cell suspensions were plated on tissue flasks and incubated at 37°C in 5% CO2. After trypsinization, cells were then incubated in DMEM with 0.5% FBS for 24 h and then treated with UII for 48 h. Cell lysates and conditioned media were harvested up to different posttreatment time points. For UII treatment alone, NRK-52E cells were incubated with 10–8 mol/l UII for 48 h under 0.5% FBS medium. For cotreatment of UII with nimodipine (a calcium channel blocker) or EDTA (a calcium chelator), NRK-52E cells were preincubated with 10–5 mol/l nimodipine for 5 min or with 2x10–3 mol/l EDTA for 30 min under 0.5% FBS medium and then incubated with UII at 10–8 mol/l UII for 48 h. For ANG II treatment, NRK-52E cells were treated with ANG II at 10–8, 10–7, and 10–6 mol/l for 48 h. For measurement of ANG II contents in the cultured medium, NRK-52E cells were exposed to 5 ng/ml TGF-β1 for 48 h with and without TGF-β1 neutralizing antibody at 10 µg/ml for preincubation for 3 min and then at 5 ng/ml for coincubation for 48 h.

Measurements of ANG II by RIA. ANG II contents in media were detected by liquid competition RIA based on published methods (21) and following the instruction provided by the assay kit (Boster Biological Technology).

Preparation and transfection of small-interfering RNA. Small interfering RNA (siRNA) targeting to rat GPR14 was designed according to GenBank (serial no. NM-020537). Four selected sequences specifically targeting to GPR14 and one nonspecific to GPR14 as control siRNA were designed by Dharmacon. The sequences are provided in Table 2. These sequences of siRNA duplexes synthesized by Dharmacon were checked with the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/BLAST/) to ensure the specificity targeting to GPR14 gene.


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  		Table 2. Upstream and downstream of GPR14 siRNA

 
NRK-52E cells were plated at a density of 3 x 105 cells/well followed by incubation for at least 24 h and then transfected with 50 nmol/l siRNA using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. GPR14 siRNA was detected by RT-PCR assay at different times after trasfection to ensure the transfection efficiency with GPR14 siRNA. At the time point that siRNA can efficiently inhibit GPR14 mRNA expression, NRK-52E cells were exposed to 10–8 mol/l UII for another 48 h, and then RNA was extracted from these cells for analysis of TGF-β1 and GAPDH mRNA by the RT-PCR method.

Statistical analysis. Data were presented as means ± SD from six samples (rats) for in vivo study and at least five samples for in vitro experiments. One-way ANOVA was used for statistical analysis. Differences were considered to be significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Renal dysfunction and morphological change. STZ-induced diabetic rats showed persistent high levels of PG (Fig. 1A), in parallel with an increased GSP (Fig. 1B). A mild renal dysfunction was observed, shown by time-dependent increases in 24-h urinary protein contents (Fig. 1C), RBP excretion rate (Fig. 1D), and NAG contents (Fig. 1E), and also a time-dependent decrease in Ccr (Fig. 1F). The decrease in Ccr suggests the glomerular dysfunction, whereas increases in RBP excretion rate and NAG contents suggest the existence of renal tubular lesion. Renal tubule morphological changes were assessed by transmission electron microscope. The tubule cells in the control group show a round nucleus with normally distributed chromatin and a closed arrangement of mitochondria and microvilli (Fig. 1G). In contrast, diabetic renal tubule cells show nucleus chromatin margination with some mitochondria swelling, broken mitochondrial cristae, disturbed microvilli, and increased lysosomes at 2 wk (Fig. 1H), with increased lysosomes around nuclei and disturbed microvilli at 4 wk (Fig. 1I). Diabetic renal tubule cells show severe nucleus chromatin margination with matrix agglutination and amorphous electron-dense deposit in mitochondria at 8 wk (Fig. 1J), with more mitochondrial swelling and cristolysis at 12 wk after the onset of diabetes (Fig. 1K).


Figure 1
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  		Fig. 1. Renal dysfunction and histological changes in streptozotocin (STZ)-induced diabetic rats. Diabetic animals were randomly divided into the following 5 groups: 2, 4, 8, and 12 wk after the onset of diabetes. Plasma glucose (PG, A), glycosylated serum proteins (GSP, B), 24-h urinary protein excretion (C), retinol-binding protein (RBP) excretion rate (D), urinary D-glucosaminidase (NAG) contents (E), and creatinine clearance (Ccr, F) were measured for diabetic model and renal function, as described in MATERIALS AND METHODS. Ultrastructural changes of the renal tubules of control (G) and diabetic 2 (H), 4 (I), 8 (J), and 12 (K) wk rats were representatively given from each group. *P < 0.05 vs. control.

 
Diabetic increases in UII and GPR14 expressions were accompanied with an increased fibrotic response in the kidney. Examination of UII and GPR14 proteins by immunohistochemical staining indicated that both UII and GPR14 protein expressions are predominant in the cytoplasm of renal tubular epithelial cells, in particular in the epithelial cells of the distal and collecting tubes, and slight in the cytoplasm of certain proximal tubular epithelial cells, in the kidneys of normal rats (representative images of each group are presented in Fig. 2A). Semiquantitative analysis of the intensity and area of UII and GPR14 staining showed that diabetes significantly increased both UII and GPR14 protein expressions in a time-dependent manner from 2 to 12 wk (Table 3). Furthermore, the diabetic increase of both UII and GPR14 protein expressions in the renal medulla was confirmed at the mRNA level by RT-PCR assay, showing significant increases of UII and GPR14 mRNA expressions in a time-dependent manner from 2 to 12 wk (Fig. 3).


Figure 2
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  		Fig. 2. Immunohistochemical staining. Animal grouping is same as described in Fig. 1. Renal tissues were immunohistochemically stained for urotensin II (UII), G protein-coupled receptor 14 (GPR14; A), transforming growth factor (TGF)-β1, fibronectin (FN), and collagen IV (Col IV) (B). In B, representative images of each staining are presented for certain groups. Others are not shown. Semiquantitative data for these proteins are summarized in Table 3.

 

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  		Table 3. The expression (%) of U II, TGF-β1, FN and Col IV proteins in renal tubules (mean ± SD)

 

Figure 3
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  		Fig. 3. Diabetes-induced upregulation of renal UII and GPR14 mRNA expression. Renal medullas of different groups were collected at indicated postdiabetes times to analyze UII (A) and GPR14 (B) mRNA expression by RT-PCR, as described in MATERIALS AND METHODS. *P < 0.05 vs. control.

 
TGF-β1 expression was lightly observed in the normal renal tubular epithelial cells, and glomeruli. Compared with controls, TGF-β1 expression was also significantly increased in glomeruli, with the highest expression at 12 wk (Fig. 2B and Table 3). In line with the increase in TGF-β1 expression, both Col IV and fibronectin expressions were also significantly, time-dependently increased in the glomeruli and the renal tubules of diabetic rats compared with controls (Fig. 2 showing the representative staining images from control, 2 wk, and 12 wk diabetic kidneys; semiquantitative results are summarized in Table 3).

UII directly induces upregulation of TGF-β1 via GPR14- and calcium-dependent pathways. Because both UII and TGF-β1 proteins were upregulated in the renal tubules of diabetic rats, along with significant upregulation of fibrotic response, the next experiment was to investigate the role of UII in upregulating TGF-β1 expression using rat proximal tubular NRK-52E cells. UII has been found to stimulate SW-13 cell and renal epithelial cell proliferation in a dose-dependent manner within 10–9 to 10–7 mol/l (24, 32). Therefore, we exposed NRK-52E cells to UII at 10–8 mol/l for 48 h, which significantly increased TGF-β1 mRNA expression (Fig. 4A). The UII-induced upregulation of TGF-β1 mRNA expression was almost completely prevented by coincubation with the calcium channel blocker nimodipine or calcium chelating agent EDTA (Fig. 4A). This suggests that UII-induced TGF-β1 production is mostly calcium dependent. Because diabetes often increases systemic and renal ANG II levels, we also examined whether UII can directly increase ANG II production. Exposure of NRK-52 cells to UII did not increase either ANG II secretion in culture medium (Fig. 4B) or AT1 mRNA expression (Fig. 4A).


Figure 4
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  		Fig. 4. Effect of UII on TGF-β1 and ANG II receptor (AT1) mRNA expression and ANG II secretion in medium from cells. TGF-β1 and AT1 mRNA (A) in the cells and ANG II concentration in the media (B) were measured after NRK-52E cells were directly exposed to 10–8 mol/l UII for 48 h. In the nimodipine/UII group, NRK-52E cells were pretreated with nimodipine (10–5 mol/l) for 5 min and then coincubated with 10–8 mol/l UII for 48 h. In the EDTA/UII group, NRK-52E cells were pretreated with EDTA (2x10–3 mol/l) for 30 min and then coincubated with 10–8 mol/l UII for 48 h. P < 0.05 vs. control (*) and vs. UII (#).

 
To determine whether UII-increased TGF-β1 mRNA expression is mediated by its interaction with GPR14, GPR14 mRNA expression was silenced by GPR14 siRNA that was found to significantly inhibit GPR14 mRNA expression at 6 h after treatment (Fig. 5A). Next, we found that, if the cultured NRK-52E cells were preincubated with GPR14 siRNA for 6 h and then coexposed to UII at 10–8 mol/l for 48 h, UII-increased TGF-β1 mRNA expression was significantly abolished (Fig. 5B).


Figure 5
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  		Fig. 5. Effect of silencing GPR14 mRNA expression on UII-induced TGF-β1 mRNA expression. NRK-52E cells were treated with small-interfering RNA (siRNA) for 6 h, and GPR14 mRNA expression, analyzed by RT-PCR as described in MATERIALS AND METHODS, was almost completely inhibited (A). Therefore, NRK-52E cells were pretransfected with GPR14 siRNA 6 h before exposure to 10–8 mol/l UII for 48 h, and then mRNA expression of TGF-β1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed by RT-PCR (B). P < 0.05 vs. control (*) and vs. UII (#).

 
Effects of TGF-β1 and ANG II on UII and GPR14 mRNA expressions in NRK-52E cells. It is known that diabetic increases in systemic and renal levels of ANG II and TGF-β1 play critical roles in diabetic renal pathogenesis (3, 28). Other studies also demonstrated the increase in UII levels along with ANG II and TGF-β1 in the subjects with various pathogeneses (3, 6, 15, 36, 37). Although we did not find the increased ANG II secretion in medium (Fig. 4B) from, and AT1 expression (Fig. 4A) in, UII-exposed NRK-52E cells, we further questioned whether ANG II and TGF-β1 affect UII and GPR14 expressions. Exposure of NRK-52E cells to different concentrations of ANG II caused a dose-dependent upregulation of both UII and GPR14 mRNA expressions within a dose range of 10–8 to 10–6 mol/l (Fig. 6). Similarly, both UII and GPR14 mRNA expressions were significantly increased in the NRK-52E cells exposed to 5 ng/ml TGF-β1 for 48 h (Fig. 7). The upregulation of UII and GPR14 mRNA expressions by exposure to TGF-β1 could be completely attenuated by TGF-β1 neutralization with its specific antibody (Fig. 7).


Figure 6
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  		Fig. 6. Effect of ANG II on expression of UII and GPR14 mRNA expressions. UII and GPR14 mRNA expressions were measured by RT-PCR assay for the NRK-52E cells directly exposed to ANG II at the indicated concentrations for 48 h. *P < 0.05 vs. control.

 

Figure 7
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  		Fig. 7. Effect of TGF-β1 on expression of UII and GPR14 mRNA expressions. UII and GPR14 mRNA expressions were measured by RT-PCR assay for the NRK-52E cells directly exposed to 5 ng/ml TGF-β1 for 48 h with and without TGF-β1 neutralizing antibody. For the group with neutralizing antibody, the cells were exposed to TGF-β1 neutralizing antibody at 10 µg/ml for 3 min and then coincubated with 5 ng/ml TGF-β1 for 48 h. P < 0.05 vs. control (*) and vs. TGF-β1 (#).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
UII was first recognized 30 years ago as an important teleost fish hormone (4), and consequently has been identified as a vasoconstrictive peptide in mammals, which functions via binding to its receptor GPR14 (1, 9, 13). Furthermore, recent studies have demonstrated that UII plays multiple roles in cardiovascular diseases (14, 15, 22, 35, 38), some of which are blood pressure independent, such as its trophic and mitogenic actions. Indeed, UII has been found to play a critical role in the cardiac fibrosis under several pathological conditions (15, 22).

Although UII and GPR14 levels were found to be increased in the blood of diabetic patients with nephropathy (33) and the kidneys of diabetic (22) and nondiabetic (16, 24, 25, 29, 33) subjects, their roles in the renal fibrogenesis under diabetic conditions remain lack of direct evidence. We provide here the first evidence for the increased expression of UII mRNA and protein in the glomeruli and tubular epithelial cells of STZ-induced diabetic rats. Diabetic upregulation of UII predominant in the epithelial cells of the distal and collecting tubes, observed in the presents study (Fig. 2A), is also consistent with the early study using hypertensive rats, indicating that UII and GPR14 mRNA expression in renal medulla was significantly higher than that of renal cortex (31). The most important finding of the present study is that increased expression of both UII and GPR14 mRNA and protein in the kidney medulla of diabetic rats was coincident with the increase of renal ECM (fibronectin and Col IV, Fig. 2B) accumulation, the induction of renal morphological changes (Fig. 1, G–K), and the mild renal dysfunction (Fig. 1, C-F), suggesting a potential role of UII in the renal fibrosis and dysfunction under diabetic conditions.

The critical role of TGF-β1 in diabetes-induced renal cell hypertrophy and renal ECM accumulation, leading to DN, has been extensively addressed (10, 19, 20, 27). Whether diabetic upregulation of renal UII and its receptor is also involved in renal upregulation of TGF-β1, leading to renal fibrosis and even to DN, remains unclear. However, fibrotic effects of UII in the heart and kidney under various pathogenic conditions have been documented (15, 22, 24, 25, 31, 35, 38). For instance, UII has recently been found to play an important role in adverse cardiac remodeling and fibrosis by upregulating TGF-β1 expression via its interaction with GPR14 (15). Activation of the UII receptor GPR14 was found to increase calcium contents in the cultured human aortic endothelial cells (5, 7, 23), suggesting the possible involvement of calcium in the UII intracellular signaling. Consistent with these studies, the present study shows, for the first time, that UII can directly upregulate TGF-β1 mRNA expression via its receptor GPR14, since RNA interference decreases UII-induced TGF-β1 production (Fig. 5). As outlined in Fig. 8, the UII-upregulated TGF-β1 mRNA expression is calcium dependent, since calcium channel blocker nimodipine or calcium chelator agent EDTA significantly prevented UII-induced TGF-β1 production (Fig. 4).


Figure 8
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  		Fig. 8. Schematic illustration for the important role of UII in diabetes-induced TGF-β1-mediated extracellular matrix (ECM) accumulation via GPR14- and calcium-dependent pathways.

 
The autocrine and paracrine regulation of UII in the renal tubule cells is another interesting finding of the present study. Exposure of NRK-52E cells to either ANG II (Fig. 6) or TGF-β1 (Fig. 7) significantly upregulated UII and its receptor expression. Furthermore, exposure of NRK-52E cells to UII also significantly increased TGF-β1 expression (Figs. 4 and 5) but not ANG II and AT1 expression (Fig. 4). That UII acted as a possible autocrine and paracrine growth factor for renal epithelial cells has been documented previously (24), which was further supported by the present study. Therefore, a schematic illustration for the interaction of UII and GPR14 with ANG II and TGF-β1 is proposed in Fig. 8. This suggests that ANG II that is increased systemically and also in the kidney under diabetic conditions stimulates NRK-52E cells to synthesize and express UII and its receptor, which in turn causes an increase in TGF-β1 production, leading to ECM synthesis and accumulation. The upregulated TGF-β1 can further enhance the upregulation of UII and its receptor in a feedback manner. Therefore, UII may be an important causative factor for renal injury and fibrosis under diabetic conditions. To support this notion, administration of UII specific antagonist (palosuran) significantly improved the survival rate of diabetes; lowered blood glucose, Hb A1c, and lipid levels; and delayed the progress of kidney damage (11, 12, 30, 34). Therefore, to prevent renal UII synthesis may become an efficiently alternative approach for the prevention of the development of DN.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported, in parts, by Grant N30570855 from the National Science Foundation of China (to C. Li and L. Cai) and Grant 05-07-CD-02 from the American Diabetes Association (to L. Cai).


   ACKNOWLEDGMENTS
 
We thank Y. S. Xu from the Department of Nuclear Medicine, the First Affiliated Hospital of Haerbin Medical University, for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Li, Dept. of Experimental Pharmacology and Toxicology, School of Pharmacy, Jilin Univ., Changchun, P.R.China (e-mail: lic{at}jlu.edu.cn or LOcai001{at}lovisvillle.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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