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Am J Physiol Renal Physiol 296: F257-F265, 2009. First published November 12, 2008; doi:10.1152/ajprenal.90493.2008
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Activation of renal renin-angiotensin system in upstream stimulatory factor 2 transgenic mice

Lihua Shi,1,* Dejan Nikolic,1,* Shu Liu,1,* Hong Lu,2 and Shuxia Wang1

1Graduate Center for Nutritional Sciences and 2Cardiovascular Research Center, University of Kentucky, Lexington, Kentucky

Submitted 15 August 2008 ; accepted in final form 9 November 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previously we demonstrated that upstream stimulatory factor 2 (USF2) transgenic (Tg) mice developed nephropathy including albuminuria and glomerular hypertrophy, accompanied by increased transforming growth factor (TGF)-β and fibronectin accumulation in the glomeruli. However, the mechanisms by which overexpression of USF2 induces kidney injury are unknown. USF has been shown to regulate renin expression. Moreover, the renin-angiotensin system (RAS) plays important roles in renal diseases. Therefore, in the present studies the effects of USF2 on the regulation of RAS in the kidney as well as in mesangial cells from USF2 (Tg) mice were examined. The role of USF2-mediated regulation of RAS in TGF-β production in mesangial cells was also determined. Our data demonstrate that USF2 (Tg) mice exhibit increased renin and angiotensin (ANG) II levels in the kidney. In contrast, renal expression of other components of RAS such as renin receptor, angiotensinogen, angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1a (AT1a) receptor, and AT2 receptor was not altered in USF2 (Tg) mice. Similarly, mesangial cells isolated from USF2 (Tg) mice had increased renin and ANG II levels. Mesangial cells overexpressing USF2 also had increased TGF-β production, which was blocked by small interfering RNA-mediated renin gene knockdown or RAS blockade (enalapril or losartan). Collectively, these results suggest that USF2 promotes renal renin expression and stimulates ANG II generation, leading to activation of the intrarenal RAS. In addition, renin-dependent ANG II generation mediates the effect of USF2 on TGF-β production in mesangial cells, which may contribute to the development of nephropathy in USF2 (Tg) mice.

transforming growth factor-β

THE RENIN-ANGIOTENSIN SYSTEM (RAS) is a multistep enzymatic cascade to produce a family of angiotensin peptides that control blood pressure and fluid homeostasis. The components of the RAS, including angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE), are present systemically for endocrine production of angiotensin (ANG) II. AGT, the only known precursor to ANG II, is constitutively secreted from hepatocytes as well as other cell types and generally is considered to be non-rate-limiting in the production of systemic ANG II (28). AGT is cleaved by the aspartyl protease renin to generate ANG I. For systemic production of ANG II, renin synthesis and release from juxtaglomerular (JG) cells of the afferent arteriole is the rate-limiting step. Hydrolysis of the COOH-terminal amino acids of ANG I by ACE results in the formation of ANG II, the primary peptide of the RAS with biological activity. A homolog of ACE, termed ACE2, has been recently discovered, which hydrolyzes ANG I to ANG(1–9) and ANG II to ANG(1–7) (8). ANG II exhibits the majority of its well-known physiological effects via the angiotensin type 1 (AT1) receptor. In addition to the systemic RAS, each RAS component has been localized to kidney (3, 20, 21). Interestingly, it has been demonstrated that glomerular mesangial cells express mRNA for renin, AGT, and ACE (1). Moreover, mesangial cells in culture are able to synthesize and secrete renin, prorenin, and ANG II and contain both the AT1 receptor and the prorenin/renin receptor (1, 20, 22, 23). This cell type has been shown to produce extracellular matrix and contributes to progression of glomerulosclerosis (10).

Numerous studies suggest that RAS plays an important role in the development of nephropathy. However, the mechanisms by which this occurs and the mechanisms of RAS activation under disease conditions remain incompletely delineated. It has been shown that upstream stimulating factor (USF)1/2 binds to the mouse renin promoter and regulates renin gene expression in a kidney-derived renin-expressing cell line (As4.1) (27). However, whether USF2 regulates the intrarenal RAS has not been defined. USF2 belongs to the myc family of transcription factors characterized by a basic helix-loop-helix leucine zipper domain responsible for dimerization and DNA binding. Through binding to E-boxes of target genes, USF2 has been demonstrated to regulate expression of many genes in addition to renin (2, 5, 6, 15, 25, 29, 31, 36, 42). In our previous studies (38), we demonstrated that high glucose exposure stimulated USF2 accumulation, resulting in induction of thrombospondin1 gene expression and transforming growth factor (TGF)-β activity in glomerular mesangial cells and contributing to the development of diabetic nephropathy. To define the role of USF2 in vivo, we generated USF2 transgenic (Tg) mice. Importantly, we found that these mice developed nephropathy at ~6 mo of age. This included the occurrence of albuminuria and glomerular hypertrophy, accompanied by increased TGF-β and fibronectin accumulation in the glomeruli even under nondiabetic conditions (18). Whether overexpression of USF2 upregulates renin expression and ANG II production in the kidney and contributes to USF2-mediated nephropathy is unknown.

In the present studies, we determined the effects of overexpression of USF2 on the regulation of renal RAS in USF2 (Tg) mice. Since TGF-β is a well-known profibrotic factor and plays an important role in renal fibrosis (14, 26, 44), the effect of USF2 on RAS in mesangial cells isolated from USF2 (Tg) mice and its role in TGF-β production in these cells were also determined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. USF2 (Tg) mice generated by our laboratory have been described previously (18). Eight-week-old male USF2 (Tg) mice and age- and sex-matched littermate control mice were used in the studies. These mice were cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols were approved by the Institutional Animal Care and Use Committee of the University of Kentucky.

Kidney renin activity measurement. Kidneys were rapidly removed from mice, weighed, and homogenized in cold PBS buffer with protease inhibitors including PMSF, EDTA, and enalapril. Renin activity in kidney homogenates was measured as described previously (4) in the absence of exogenous AGT.

Real-time PCR. Total RNA (2 µg) extracted from kidneys of USF2 (Tg) mice and littermate control mice was converted to cDNA with murine leukemia virus reverse transcriptase (Promega). The validity of primers and appropriate melting temperature for real-time PCR were determined to ensure that only one band was amplified by the PCR reaction. Primers were synthesized by Integrated DNA Technologies, with sequences as follows: 1) AGT: 5'-GTACAGACAGCACCCTACTT-3' and 5'-CACGTCACGGAGAAGTTGTT-3'; 2) renin: 5'-TCTGGGCACTCTTGTTGCTC-3' and 5'-GGGGGAGGTAAGATTGGTCAA-3'; 3) ACE: 5'-GGAGTACTTCCAACCGGTCA-3' and 5'-GCCTTGGCTTCATCAGTCTC-3'; 4) ACE2: 5'-GGATACCTACCCTTCCTACATCAGC-3' and 5'-CTACCCCACATATCACCAAGCA-3'; 5) AT1 receptor: 5'-TCGCTACCTGGCCATTGTC-3' and 5'-TGACTTTGGCCACCAGCAT-3'; 6) AT2 receptor: 5'-CCTTCTTGGATGCTCTGACC-3' and 5'-GCGGTTTCCAACAAAACAAT-3'; 7) β-actin; sense 5'-CACACTGTGCCCATCTACG-3' and antisense 5'-GCCATCTCTTGCTCGAAGTC-3'. Real-time PCR analyses were performed with the SYBR Green PCR Master Mix kit and a MyiQ real-time PCR thermal cycler (Bio-Rad). For each target gene, a standard curve was established by a series of threefold dilutions of the gene of interest. All reactions were performed in triplicate in a final volume of 25 µl. Dissociation curves were run to detect nonspecific amplification, and we confirmed that single products were amplified in each reaction. The quantities of each test gene and internal control β-actin were then determined from the standard curve with the MyiQ system software, and mRNA expression levels of test genes were normalized to β-actin levels.

Immunohistochemistry. Paraffin-fixed renal tissues were cut into 4- to 5-µm sections and placed onto slides. Sections were deparaffinized in xylene and rehydrated in graded mixtures of ethanol-water. Endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol for 30 min at room temperature. The slides were blocked, and the chicken IgY against ANG II was applied for 1 h at room temperature. This antibody cross-reacts with ANG II precursors and ANG II products (7). Negative control was included by omitting the primary antibody and substituting preimmune IgY. Additional negative control was included by adding primary antibody that was preincubated with ANG II peptide. After washing with PBS, biotinylated secondary antibody was applied for 30 min. After another 15-min washing, avidin-biotin-peroxidase complex was applied to the slides for 30 min. The slides were washed once again with PBS before color development with diaminobenzidine.

Mesangial cell isolation. Glomerular mesangial cells were isolated with standardized techniques (19, 40). First, glomeruli were isolated through gradual sieving as described previously (19, 32) from the kidneys from USF2 (Tg) mice or control littermates between the ages of 6 and 8 wk. The isolated glomeruli were decapsulated and digested in a solution containing Worthington type I collagenase at 37°C for 20 min. After brief centrifugation, the pellet was resuspended in RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum and 5 mM D-glucose. The suspension was plated in six-well plates and cultured. After 7–10 days, the primary culture was subcultured and split at a ratio of 1:3. The cultured mesangial cells were characterized as described previously (19). Because of the lack of a specific marker for mesangial cell identification, a combination of some criteria was used to characterize the isolated mesangial cells, including cell morphology, positive staining for smooth muscle {alpha}-actin and desmin, and negative staining for CD45 and factor VIII. Mesangial cells between passages 3 and 6 were used.

Transfection and luciferase assay. Mesangial cells were seeded onto six-well plates at a density of 3 x 104 cells/ml for 1 day and then were transiently transfected with Effectene transfection reagent (Qiagen) with (1 µg) mouse renin promoter luciferase reporter plasmid [generously provided by Dr. Curt D. Sigmund, University of Iowa (33)]. pRL-SV40 (0.02 µg; Promega) was used as an internal control. After 24 h of transfection, luciferase activities were assayed with a dual-luciferase assay kit (Promega) according to the manufacturer's directions. Transcriptional activity was analyzed and normalized to Renilla luciferase activity.

Immunoblot analysis. Expression of renin and renin receptor in kidney homogenate or expression of USF2 in mesangial cell nuclear extracts was assessed by Western blot analysis. The nuclear extracts from mesangial cells were prepared as described previously (38). Thirty micrograms of protein was subjected to SDS-PAGE under reducing condition and transferred onto polyvinylidene difluoride membrane. After blocking in Tris-buffered saline (TBS) buffer with 5% BSA, the membrane was incubated with polyclonal anti-USF2 antibody (Santa Cruz; 1:1,000 dilution), mouse anti-rat renin antibody (catalog no. RDI-rtreninabm, Research Diagnostics, Concord, MA) at 1:3,000 dilution in TBS buffer with 3.5% BSA, and chicken anti-renin receptor antibody (7) for 1 h at room temperature. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoLab). The reaction was visualized with an enhanced chemiluminescence system (Pierce).

ANG II measurement. ANG II concentrations in kidney or mesangial cell extracts were determined with commercial ELISA kits (Peninsula Laboratories) as described previously (34). The specificity of ANG II ELISA for ANG II was tested previously (35). For kidney extract preparation, kidneys from wild-type (WT) or USF2 (Tg) mice were rapidly removed, homogenized in cold PBS buffer containing protease inhibitors as described previously (11, 17, 32), and centrifuged at 13,000 g for 20 min at 4°C. The supernatant was collected, and ANG II concentrations were measured by ELISA.

Small interfering RNA-mediated renin and renin receptor gene knockdown. Primary mouse mesangial cells were cultured, and renin or renin receptor levels in these cells were knocked down by small interfering RNA (siRNA)-renin (Dhamacon) and siRNA-renin receptor (Santa Cruz). Renin or renin receptor siRNA (10 nM) and control siRNA were transfected into mesangial cells isolated from control WT mice with transfection reagents according to the instruction manual. After 48-h incubation, conditioned media were collected to analyze total TGF-β protein levels by plasminogen activator inhibitor (PAI)-1/luciferase bioassay. Cells were harvested to determine the extent of inhibition of renin or renin or renin receptor levels by immunoblotting.

TGF-β assay (PAI-1/luciferase bioassay). Total TGF-β levels in conditioned media were assayed with the PAI-1/luciferase assay as described previously (37). Briefly, mink lung epithelial cells were plated onto 24-well tissue culture plates and incubated for 3–4 h for optimal attachment. To measure total TGF-β, conditioned medium samples were heat activated for 3 min at 100°C and then added to the attached cells. After overnight incubation, cells were lysed and analyzed for luciferase activity with a luminometer. The mean values of triplicate samples were converted into concentrations of TGF-β (pM) with a standard curve obtained with human recombinant TGF-β1. Specificity of the assay was proven by neutralization of the TGF-β activity in conditioned media with an anti-TGF-β antibody or an ELISA kit from R&D Systems.

Statistical analysis. Data are expressed as means ± SE. ANOVA was used to analyze variation within groups. Student's t-tests were used to compare variation between groups. Statistical significance was accepted at a value of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Kidney RAS is activated in USF2 (Tg) mice. Our previous studies (18) demonstrated that USF2 (Tg) mice developed nephropathy, including albuminuria and mesangial matrix expansion, at 6 mo of age under nondiabetic conditions. USF2 has been shown to regulate renin expression (27). Therefore, we determined whether renal RAS is activated in USF2 (Tg) mice before the development of nephropathy in these mice. As shown in Fig. 1, A and B, renal renin mRNA and protein levels were upregulated in USF2 (Tg) mice compared with littermate control mice. Elevations in renin mRNA and protein were accompanied by increased renin activity in kidneys from USF2 (Tg) mice (Fig. 1C). Since renin is the rate-limiting step for ANG II generation, we determined whether increased renin activity promoted elevated ANG II expression in kidneys from USF2 (Tg) mice. ANG II immunoreactivity was moderate in a few tubules in kidney sections from control mice (Fig. 2C). In contrast, kidney sections from USF2 (Tg) mice displayed strong ANG II staining in glomeruli as well as in tubules (Fig. 2D). In addition, ANG II concentrations in kidney extracts were measured by ELISA. As shown in Fig. 2E, ANG II levels in the kidney of USF2 (Tg) mice were significantly increased compared with WT control mice.


Figure 1
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  		Fig. 1. Kidney renin mRNA, protein, and activity increased in upstream stimulatory factor (USF)2 transgenic (Tg) mice. Kidneys from USF2 (Tg) mice and control littermates were harvested. A: total RNA from kidneys were extracted, and real-time PCR was utilized to analyze renin mRNA abundance. Renin protein (B) and renin activity (C) from kidney homogenates were analyzed by immunoblotting and radioimmunoassay, respectively. Data shown are representative of 3 experiments. Relative renin protein levels were determined by scanning densitometry of blots. Data are means ± SE (n = 4 mice/group). *P < 0.05, USF2 (Tg) vs. wild type (WT). MM, molecular mass.

 

Figure 2
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  		Fig. 2. Kidney angiotensin (ANG) II immunostaining increased in USF2 (Tg) mice. A–D: representative light micrographs of ANG II immunostaining in kidney sections from USF2 (Tg) mice and littermate control mice. Original magnification: x40. A: no staining was obtained in control kidney sections when preimmune IgY was used. B: no staining was obtained in control kidney sections when anti-ANG II antibody that was preincubated with ANG II peptides was used. C: kidney sections from littermate controls displayed modest ANG II staining in tubules. D: kidney sections from USF2 (Tg) mice displayed strong ANG II staining in glomeruli and tubules. E: ANG II concentrations in kidney extracts were measured by ELISA. Data are means ± SE (n = 4 mice/group). *P < 0.05, USF2 (Tg) vs. WT.

 
To determine whether these effects were specific to renin, we measured other RAS components in kidneys from both WT and USF2 (Tg) mice. Renin receptor protein levels in kidney homogenates were not changed in USF2 (Tg) mice compared with littermate control mice (Fig. 3). In addition, mRNA abundance of other components of the RAS (AGT, ACE, ACE2, AT1a and AT2 receptors) in kidneys was not different between USF2 (Tg) mice and littermate control mice (Fig. 4). Together, these data suggest that USF2 promotes renin expression to enhance ANG II synthesis, leading to the activation of renal RAS in USF2 (Tg) mice.


Figure 3
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  		Fig. 3. Kidney renin receptor levels were unchanged in USF2 (Tg) mice compared with littermate control mice. Protein levels of renin receptor in kidney homogenates from USF2 (Tg) mice and WT littermate control mice were determined by Western blotting. Experiments were repeated 3 times, and a representative result is shown. Data are means ± SE of 3 replicates.

 

Figure 4
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  		Fig. 4. mRNA of renin-angiotensin system (RAS) components in kidneys from USF2 (Tg) and littermate control mice. Total RNA was extracted from kidneys of USF2 (Tg) mice and littermate control mice. mRNA levels of angiotensinogen (A), angiotensin-converting enzyme (ACE; B), ACE2 (C), angiotensin type 1a (AT1a) receptor (D), and AT2 receptor (E) were determined by real-time PCR. Results are means ± SE (n = 3).

 
RAS is activated in mesangial cells isolated from USF2 (Tg) mice. It has been reported that mesangial cells contain all the components of the RAS (1, 20, 22, 23), and mesangial cell hypertrophy is a contributing factor in diabetic nephropathy. Thus we determined whether overexpression of USF2 upregulates RAS in mesangial cells. As expected, mesangial cells isolated from USF2 (Tg) mice had increased protein levels of USF2 compared with littermate control mice (Fig. 5). Moreover, overexpression of USF2 increased renin mRNA and protein levels (Fig. 6, B and C). In cultured mesangial cells transiently transfected with mouse renin promoter luciferase construct (–4.1 kb) [provided by Dr. Curt D. Sigmund (33)], renin promoter activity (normalized to Renilla luciferase activity) was increased (Fig. 6A).


Figure 5
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  		Fig. 5. USF2 protein levels increased in mesangial cells isolated from USF2 (Tg) mice. Mesangial cells (passage 4) isolated from USF2 (Tg) mice and littermate control mice were cultured. Top: protein levels of USF2 in the nuclear extracts were analyzed by immunoblotting. TATA binding protein (TBP) was used as an internal control. Data shown are representative of 3 experiments. Bottom: relative USF2 levels were determined by scanning densitometry of blots. Data are means ± SE. *P < 0.05, USF2 (Tg) vs. WT.

 

Figure 6
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  		Fig. 6. Renin expression (promoter activity, mRNA and protein levels) was upregulated in mesangial cells isolated from USF2 (Tg) mice. A: mesangial cells (passage 3) isolated from USF2 (Tg) and control mice were cultured and transiently transfected with renin promoter-luciferase construct (1 µg) for 24 h. Then luciferase activity in the cell lysate was measured and normalized to Renilla luciferase activity. Fold change relative to WT group is shown. B: total RNA was extracted from mesangial cells for analysis of renin mRNA levels by real-time PCR as described in MATERIALS AND METHODS. C: renin protein levels in cell lysates were analyzed by Western blotting. Experiments were repeated 3 times. Data are means ± SE of 3 replicates. *P < 0.05, USF2 (Tg) vs. WT.

 
To define whether USF2-mediated increases in renin promote ANG II synthesis in mesangial cells, we measured ANG II concentrations in mesangial cells isolated from littermate control mice and USF2 (Tg) mice. The results demonstrate that ANG II concentrations in mesangial cell lysates from USF2 (Tg) mice were significantly increased compared with control mesangial cells (Fig. 7). In addition, we determined the mRNA abundance of other components of the RAS including AGT, ACE, and AT1 and AT2 receptors by real-time PCR, which did not reveal a change in any of these components in mesangial cells from USF2 (Tg) compared with control cells (data not shown).


Figure 7
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  		Fig. 7. ANG II levels increased in mesangial cells from USF2 (Tg) mice. Mesangial cells (passage 4) isolated from USF2 (Tg) mice and control littermates were cultured, and cell lysates were prepared. ANG II concentrations in cell lysates were measured by ELISA. Values are means ± SE of 3 experiments. *P < 0.05, USF2 (Tg) vs. WT.

 
Mesangial cells isolated from USF2 (Tg) mice have increased TGF-β levels, which are blocked by siRNA-mediated renin gene knockdown or RAS blockade. It has been shown that USF2 regulates TGF-β expression (15, 30, 39, 42). Thus we determined whether overexpression of USF2 stimulates TGF-β production in mesangial cells. As shown in Fig. 8, total TGF-β secretion in mesangial cells isolated from USF2 (Tg) mice was increased compared with mesangial cells isolated from littermate control mice. Recent studies reported that renin increases mesangial cell TGF-β levels through receptor-mediated, ANG II-independent mechanisms (13). Thus we determined whether USF2-induced renin expression is involved in TGF-β production in mesangial cells from USF2 (Tg) mice and further determined whether an ANG II-dependent or -independent mechanism is involved. As shown in Fig. 9, A and B, renin siRNA significantly knocked down renin gene expression in both USF2 (Tg) and WT mesangial cells. In control mesangial cells, total TGF-β levels were only slightly reduced by renin gene knockdown. However, in USF2 (Tg) mesangial cells, siRNA mediated-renin gene knockdown inhibited the increase in total TGF-β levels. To determine whether renin receptors are involved in the above effects of USF2/renin on TGF-β production in mesangial cells, siRNA-mediated renin receptor gene knockdown was utilized. Mesangial cells isolated from USF2 (Tg) mice had levels of renin receptor similar to those from WT mice. Renin receptor siRNA significantly reduced renin receptor gene expression in both USF2 (Tg) and WT mesangial cells (Fig. 9C). However, siRNA-mediated renin receptor gene knockdown had no effect on TGF-β levels in control or USF2 (Tg) mesangial cells (Fig. 9D). To determine whether the above effects of renin to mediate USF2-induced TGF-β production in mesangial cells are ANG II dependent, mesangial cells isolated from control or USF2 (Tg) mice were treated with enalapril (5 µM, Sigma) or losartan (1 µM, Merck) for 24 h. In conditioned medium from USF2 (Tg) mesangial cells, enalapril or losartan treatment significantly reduced total TGF-β levels (Fig. 9E).


Figure 8
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  		Fig. 8. Total transforming growth factor (TGF)-β increased in conditioned media from mesangial cells isolated from USF2 (Tg) mice. Mesangial cells (passage 4) isolated from USF2 (Tg) mice and control littermates were cultured in normal culture medium (RPMI medium with 20% serum and 5 mM glucose) until 85–90% confluence. Then serum-free media were added to the cells. After 24 h, conditioned media were collected and used for measuring total TGF-β levels by plasminogen activator inhibitor (PAI)-1/luciferase assay. Data are means ± SE of 3 replicates. *P < 0.05, USF2 (Tg) vs. WT.

 

Figure 9
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  		Fig. 9. Effects of renin, renin receptor gene knockdown, and RAS blockade on total TGF-β levels in conditioned media from mesangial cells isolated from USF2 (Tg) mice. Control or USF2 (Tg) mesangial cells (passage 4) were transiently transfected with mouse renin small interfering RNA (siRNA, 10 nM; A) or renin receptor siRNA (10 nM; C) and control siRNAs using siRNA transfection reagent for 48 h. Conditioned media were collected, and total TGF-β levels were measured by PAI-1/luciferase assay (B and D). Renin or renin receptor levels in cell lysates were determined by immunoblotting (A and C). E: mesangial cells were made quiescent in serum-free medium and treated with an ACE inhibitor (5 µM enalapril; Sigma) or an AT1 receptor antagonist (1 µM losartan; Merck) for 24 h. Then conditioned media were collected and total TGF-β levels were measured. Data are means ± SE of 3 replicates.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study demonstrates that USF2 stimulates renin expression, which enhances ANG II synthesis in the kidney, leading to the activation of renal RAS in USF2 (Tg) mice. In addition, RAS is also activated in mesangial cells isolated from USF2 (Tg) mice. Moreover, the renin-dependent ANG II generation mediates the effect of USF2 on TGF-β production in mesangial cells.

In the present study, we showed that kidney renin mRNA and protein levels are upregulated in USF2 (Tg) mice, demonstrating that overexpression of USF2 stimulates renal renin synthesis. However, at this time, the exact cell sources for the increased renin expression in the kidney from USF2 (Tg) mice are not clear. Kidney renin mRNA could be derived from multiple sources. It is known that renin is classically expressed by the JG apparatus, where it is stored in secretary granules (16). Aside from JG cells, renin can also be expressed by a variety of other cells in the kidney, including mesangial cells, proximal tubular cells, and podocytes (1, 9, 41, 43). All these cell types may contribute to the increased renin production in kidneys of USF2 (Tg) mice. Glomerular mesangial cells have been shown to produce excessive amounts of extracellular matrix proteins and contribute to progressive glomerulosclerosis (10). Our previous studies (38) have shown that mesangial cells exhibit increased USF2 expression after exposure to high glucose concentrations and play a role in diabetic renal fibrosis. Moreover, it has been reported that mesangial cells contain all the components of the RAS (1, 20, 22, 23). Therefore, we isolated mesangial cells from USF2 (Tg) mice and determined whether USF2 expression is increased in these cells and whether these cells have increased renin expression. As expected, mesangial cells isolated from USF2 (Tg) mice had increased USF2 levels. These cells also exhibited increased renin promoter activity and mRNA and protein levels compared with control cells, contributing to increased renal renin expression from USF2 (Tg) mice. To our knowledge, this is the first report demonstrating USF2-mediated upregulation of renin gene expression in mesangial cells.

It is known that renin synthesis and release is the rate-limiting step for generation of the physiologically active peptide ANG II. Increased renin expression in the kidney could lead to stimulation of AGT conversion to ANG I, which may be converted to ANG II via ACE. Therefore, we determined ANG II levels in the kidneys from USF2 (Tg) mice by immunostaining the kidney sections with anti-ANG II IgY (7). Kidney sections from USF2 (Tg) mice exhibited strong positive immunostaining compared with those from littermate control mice. In addition, ANG II levels in the cell lysates of mesangial cells isolated from USF2 (Tg) mice were also increased. In addition to the increase in renin expression, which contributes to the increased ANG II production, we further determined whether USF2 affected the expression of other components of RAS, which might play a role in the increased ANG II generation. Our results demonstrate that levels of AGT, the only known precursor for ANG II, were not altered in kidney or mesangial cells from USF2 (Tg) mice. In addition, ACE, ACE2, AT1, and AT2 expression were not altered in kidney or mesangial cells from USF2 (Tg) mice. Renin/prorenin receptor has been cloned recently and is a 350-amino acid membrane-associated polypeptide (24). This receptor has been identified in mesangial cells, which mediates cell surface ANG II generation and also mediates renin effects independent of ANG II (12, 24). Therefore, we determined renin receptor protein levels in kidney homogenates or mesangial cell lysates and found no changes of renin receptor levels in USF2 (Tg) mice compared with control littermates. Together, these results suggest that renin is an important target of USF2 in the kidney, which stimulates ANG II generation and leads to activation of the intrarenal RAS in USF2 (Tg) mice. Our present studies provide new information on the mechanisms of activation of the intrarenal RAS system in USF2 (Tg) mice. Whether this pathway (USF2-renin-intrarenal RAS activation) contributes to the development of nephropathy in USF2 (Tg) mice in vivo needs to be further investigated.

TGF-β is a profibrotic factor. Accumulating evidence suggests that TGF-β plays an important role in renal fibrosis (14, 26, 44). In agreement with our previous studies (38), we showed in this study that overexpression of USF2 stimulated TGF-β secretion in mesangial cells. However, the molecular mechanisms are not clear. Previous studies from both Weigert et al. (39) and Zhu et al. (42) demonstrated that USF1/2 bound to TGF-β1 promoter in mesangial cells by electrophoretic mobility shift assay, an assay to determine protein-DNA binding in vitro. Moreover, Zhu et al. (42) demonstrated that USF1 but not USF2 bound to TGF-β1 promoter in vivo by chromatin immunoprecipitation assay, an important approach to identify a specific protein that associates with a control element in the gene promoter in the context of an endogenous allele. This result suggests that USF2 may not directly regulate TGF-β1 gene expression in vivo. In this study, we demonstrate that USF2 indirectly regulates TGF-β production in mesangial cells through stimulation of renin. Our results demonstrate that siRNA-mediated renin gene knockdown blocked increased TGF-β production in mesangial cells from USF2 (Tg) mice, suggesting that USF2-induced renin expression is involved in increased TGF-β production in mesangial cells overexpressing USF2. Since renin elicits its cellular functions through enzymatic (ANG II generation and AT1 or AT2 receptor-dependent action) or nonenzymatic (binding to renin receptor) action, we further defined by which action renin regulates TGF-β production. We demonstrated that an ACE inhibitor or an AT1 receptor antagonist blocks the increase in TGF-β production in mesangial cells from USF2 (Tg) mice. Furthermore, renin receptor siRNA was utilized but had no effect on TGF-β production in mesangial cells isolated from USF2 (Tg) mice. Together, these data suggest that USF2-induced renin-dependent ANG II production mediates USF2-stimulated TGF-β production. This result differs from the results from Huang et al. (13). They showed that recombinant renin treatment increased rat/human mesangial cell TGF-β and matrix proteins through receptor-mediated, ANG II-independent mechanisms. Species-specific regulation of USF2-induced renin in TGF-β expression in mesangial cells might contribute to differences in experimental findings.

In conclusion, we demonstrated that overexpression of USF2 promotes renin expression and enhances renin activity in kidney, which stimulates ANG II generation and leads to activation of intrarenal RAS in USF2 (Tg) mice. In addition, renin gene expression is also upregulated in glomerular mesangial cells from USF2 (Tg) mice and leads to increased ANG II production. Moreover, the renin-dependent ANG II generation mediates the effect of USF2 on TGF-β production in mesangial cells, which may contribute to USF2-mediated renal injury.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a Scientist Development Grant from the American Heart Association (to S. Wang) and a Center of Biomedical Research Excellence pilot grant from the University of Kentucky (to S. Wang).


   ACKNOWLEDGMENTS
 
We thank Drs. Lisa Cassis (University of Kentucky) and Alan Daugherty (University of Kentucky) for careful review of the manuscript. We also thank Dr. Lisa Cassis for measuring renal renin activity and Dr. Alan Daugherty for providing renin, renin receptor, and ANG II antibodies. We thank Dr. Curt. D. Sigmund (University of Iowa) for providing mouse renin promoter luciferase constructs.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Wang, Graduate Center for Nutritional Sciences, Univ. of Kentucky, Wethington Bldg. Rm. 517, 900 S. Limestone St., Lexington, KY 40536-0200 (e-mail: swang7{at}uky.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.

* L. Shi, D. Nikolic, and S. Liu contributed equally to this research. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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