Smad7 protects against chronic aristolochic acid nephropathy in mice.

Chronic Aristolochic Acid Nephropathy (AAN) is a progressive chronic kidney disease related to herb medicine. However, treatment for chronic AAN remains ineffective. We report here that Smad7 is protective and has therapeutic potential for chronic AAN. In a mouse model of chronic AAN, progressive renal injury was associated with a loss of renal Smad7 and disruption of Smad7 largely aggravated the severity of chronic AAN as demonstrated by a significant increase in levels of 24-hour urinary protein excretion, serum creatinine, and progressive renal ﬁbrosis and inflammation. In contrast, restored Smad7 locally in the kidneys of Smad7 knockout mice prevented the progression of chronic AAN. Further studies revealed that worsen chronic AAN in Smad7 knockout mice was associated with enhanced activation of TGF-β/Smad3 and NF-κB signaling pathways, which was reversed when renal Smad7 was restored. Importantly, we also found that overexpression of Smad7 locally in the kidneys with established chronic AAN was capable of attenuating progressive chronic AAN by inactivating TGF-β/Smad3-medated renal fibrosis and NF-κB-driven renal inflammation. In conclusion, Smad7 plays a protective role in the pathogenesis of chronic AAN and overexpression of Smad7 may represent a novel therapeutic potential for chronic AAN.


INtrODUctION
Aristolochic acid nephropathy (AAN) is initially called Chinese-herb nephropathy before aristolochic acid (AA) is known as a cause of AAN [1,2]. AAN is first reported in Belgium in patients with prolonged intake of Chinese herbs [3]. Since then, new cases of AAN are regularly reported because of the use of herbal therapy worldwide [4][5][6]. Clinically, patients with AAN exhibit a rapidly progressive interstitial nephritis leading to endstage renal disease [3,7,8]. A similar clinical course is also observed in experimental animals treated with AA [9]. Pathologically, chronic AAN is characterized by extensive tubulointerstitial fibrosis with atrophy and loss of the tubules in both patients and animal models of AAN [7][8][9].
However, the mechanisms of AAN remain largely unclear and no treatment for chronic AAN is yet available.
In the context of renal fibrosis, TGF-β1 acts by stimulating Smad3 to mediate fibrosis, which is negatively regulated by Smad7 [10,11]. In a mouse model of chronic AAN, deletion of Smad3 gene protects against progressive renal fibrosis, revealing a pathogenic role for Smad3 in the development of chronic AAN [12]. In addition, Smad7 is also known to be a negative regulator of NF-κB signaling [13]. Deletion of Smad7 enhances renal fibrosis and inflammation, whereas, overexpression of Smad7 attenuates renal fibrosis and inflammation by blocking the activation of both TGF-β/Smad and nuclear factor-κB (NF-κB) signaling pathway in a variety of animal models with kidney diseases [14][15][16][17][18][19][20][21][22]. However, role of Smad7 in www.impactjournals.com/oncotarget chronic AAN remains unexplored. Thus, in the current study, we investigated the mechanisms and potential role of Smad7 in a mouse model of chronic AAN induced in Smad7 gene knockout (KO) mice and by restoring Smad7 locally in the kidneys of Smad7 KO mice. Furthermore, we also showed that overexpression of Smad7 in the established chronic AAN was capable of attenuating the progression of chronic AAN.

Deletion of smad7 results in more severe kidney injury in a mouse model of chronic AAN
Compared with normal mice, after intraperitoneal injection of AA for 6 weeks, Smad7 WT mice developed chronic AAN as evidenced by severe tubulointerstitial fibrosis accompanied by dilated tubular lumens with bared tubular basement membrane and an increase Proteinuria (24-h) over the 42-day experimental time course. c: Serum creatinine at day 42 after induction of AAN. Note that disruption of Smad7 enhances AA-induced renal injury, including severe histological damage such as dilated and bared tubular basement membrane, higher levels of proteinuria and serum creatinine when compared with Smad7 WT mice. Data are expressed as mean ±SE for groups of 6 mice. *P < 0.05, ***P < 0.001 compared with saline control mice. # P < 0.05, ## P < 0.01 compared with Smad7 WT mice with chronic AAN mice. Magnifications: x200.
Enhanced activation of TGF-β/Smad3 and NF-κB signaling is a key mechanism by which deletion of smad7 promotes chronic AAN injury To investigate the mechanisms by which deletion of Smad7 promoted renal fibrosis and inflammation in mice with chronic AAN, we examined TGF-β/Smad and NF-κB signaling pathways since both pathways are critical in the development of renal fibrosis and inflammation in many pathological conditions [10]. Immunohistochemistry, real-time PCR and western blot analysis revealed that chronic AA injection significantly up-regulated renal TGF-β1 with over-activation of TGF-β/Smad3 signaling, which was associated with a significant reduction in renal Smad7 protein in Smad7 WT mice (  S3). Furthermore, disruption of Smad7 also enhanced activation of NF-κB signaling as demonstrated by higher levels of NF-κB/p65 phosphorylation and phosphorylated p65 nuclear translocation, which was associated with a marked reduction in the NF-κB inhibitor, IκBα, on the basis of increased IκBα degradation by phosphorylation ( Fig. 3C and 3D).

KO mice
To further confirm the protective role of Smad7 in chronic AAN, we locally delivered an inducible Smad7 gene into the kidneys of Smad7 KO mice via tail vein using an ultrasound-microbubble-mediated technique as previously described [14][15][16][17]. We found that restored renal Smad7 largely prevented AA-induced chronic AAN such as dilated and bared tubular basement membrane changes in tubulointerstitium and attenuated 24h proteinuria and serum creatinine when compared with those treated with or without empty vector control (Fig. 4). Immunohistochemistry, western blot, and realtime PCR also showed that restored renal Smad7 blocked AA-induced severe renal fibrosis such as upregulation of collagen I and α-SMA and inhibited renal inflammation including F4/80+ macrophages and CD3+ T cells and upregulation of MCP-1 and TNFα ( Fig. 5 and Figs. S4 and S5). Western blot analysis clearly revealed that the inhibitory effect of Smad7 on renal fibrosis and inflammation was associated with the restoration of exogenous Smad7, thereby inhibiting TGF-β/Smad signaling by suppressing the phosphorylation of Smad3 and upregulation of TGF-β1 ( Fig. 6A and 6B and Fig.  S6) and attenuating NF-κB signaling by lowering phosphorylated IκBα and NF-κB/p65 in the AAN kidney ( Fig. 6C and 6D).

smad7 has therapeutic potential for chronic AAN
To explore whether Smad7 has therapeutic effect on chronic AAN, we transferred Smad7 gene into the diseased kidney of Smad7 WT mice with established chronic AAN at day 14 after AA administration. Results showed that compared with control-treated AAN mice at day 42, AAN mice received Smad7 treatment from day 14 to day 42 after induction of AAN were protected from progressive renal histological and functional injury (Fig.  7). Further studies also detected that Smad7 treatment largely inhibited a marked upregulation of collagen I and α-SMA and suppressed renal inflammation including expression of MCP-1 and TNFα, and infiltration of macrophages and T cells within the diseased kidney ( Fig.  8 and Figs. S7 and S8).
Next, we examined the therapeutic mechanisms by which Smad7 treatment attenuates AA-induced renal inflammation and fibrosis. As shown in Fig. 9 and Fig.  S9, immunohistochemistry, real-time PCR, and western blot analysis revealed that treatment with Smad7 in the established chronic AAN over days 14-42 abolished AA-induced activation of TGF-β/Smad and NF-κB/p65 signaling.

DIscUssION
We have demonstrated that TGF-β signaling is participated in the development of chronic AAN [12]. The present study provided new evidence for a protective role of Smad7, a negative regulator of TGF-β/Smad signaling, in the pathogenesis of chronic AAN. Importantly, we also reported here that Smad7 was an effective therapeutic agent for chronic AAN.
In this study, we found that loss of renal Smad7 may be a key mechanism leading to progression of chronic AAN. This was supported by the finding that genetic deletion of Smad7 enhanced a rapidly progressive renal injury in mice with chronic AAN. In contrast, restored renal Smad7 locally on Smad7 KO mice was capable of preventing the development of chronic AAN. These results demonstrated a protective role for Smad7 in the pathogenesis of chronic AAN, which is consistent with www.impactjournals.com/oncotarget Results show that compared with the WT mice, Smad7 KO mice show a significant increase in renal fibrosis and inflammation. Data are expressed as mean ± SE for groups of 6 mice. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the saline control mice. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with Smad7 WT mice with chronic AAN mice. Magnification: x400.
The most important finding in this study is that Smad7 may be a therapeutic agent for chronic AAN. This was supported by the finding that ultrasound-mediated gene transfer of Smad7 into the diseased kidneys with established chronic AAN in Smad7 WT mice at day 14 was capable of attenuating AA-induced progressive renal injury at day 42. These findings were in consistent with previous studies in a number of kidney disease models in which overexpression of Smad7 attenuates renal inflammation and fibrosis in obstructive nephropathy [14], remnant kidney disease [15,16], autoimmune nephritis [17], diabetic nephropathy [20], and hypertensive nephropathy [21,22]. However, it should also be pointed out that the therapeutic effect of Smad7 on chronic AAN was based on one time point study at day 42 after Smad7 treatment from day 14 of chronic AA. A prolong therapeutic effect of Smad7 on chronic AAN may be warranted. In addition, outcomes from this study may not be applied directly to the chronic AAN conditions in humans due to the potential difference in inflammatory responses between mice and humans. Nevertheless, the finding from this study provided new evidence for the therapeutic potential of Smad7 in chronic AAN.
Oncotarget 11941 www.impactjournals.com/oncotarget Smad7 was found in the kidney with chronic AAN, which may account for TGF-β/Smad3-mediated renal fibrosis. The functional importance of Smad7 in chronic AAN was demonstrated by the findings that deletion of Smad7 aggravated but restoration of Smad7 locally in the kidneys of Smad7 KO mice prevented AA-induced, TGF-β/ Smad3-mediated progressive renal fibrosis. In addition, inhibition of TGF-β/Smad3-mediated renal fibrosis by overexpressing renal Smad7 in the established chronic AAN further supported the inhibitory role of Smad7 in progressive chronic AAN and may also well explain the therapeutic effect of Smad7 on chronic AAN. Thus, blockade of TGF-β/Smad3-mediated renal fibrosis could be a mechanism by which Smad7 protects against chronic AAN.
Another mechanism for the protective role of Smad7 in chronic AAN may be due to its inhibitory role in the NF-κB-dependent renal inflammation. It is now clear that renal inflammation is also involved in chronic AAN [23,24]. NF-κB is a critical signaling pathway of the inflammatory cascade. We have previously reported that Smad7 functions as an inhibitor of NF-κB signaling to block NF-κB-dependent renal inflammation in vitro and in vivo [13,[16][17][18][19][20][21][22]. Previous study in an experimental AAN model demonstrated that progression of chronic AAN is associated with infiltration of macrophages and T cells [25]. In line with this finding, the present study added new evidence that the development of renal inflammation in chronic AAN was associated with a marked activation of NF-κB signaling, which was negatively regulated by Smad7 because deletion of Smad7 enhanced but restored renal Smad7 in Smad7 KO mice or overexpression of Smad7 in the diseased WT mice inhibited NF-κBmediated renal inflammation including upregulation of TNFα, MCP-1, and infiltration of macrophages and T cells in chronic AAN.
In conclusion, Smad7 plays a protective role in chronic AAN. Inhibition of TGF-β/Smad3 and NF-κB signaling pathways may be mechanisms by which Smad7 attenuates AA-induced renal fibrosis and inflammation. In addition, the ability of overexpression of renal Smad7 to inhibit the established chronic AAN suggests that Smad7 may have therapeutic potential for chronic AAN.

Animal models
A chronic AAN model was induced in genetically identical littermate Smad7 KO and wild-type (WT) mice (CD-1 background mice, male, aged 6-8weeks, 26-31g) by intraperitoneal injection of AA (Sigma, St. Louis, MO) at a dose of 5mg/kg every other day for 6 weeks, as described previously [12]. Smad7 KO mice were generated by functionally deleting exon I in the Smad7 gene as previously described [26]. Groups of 6 mice were killed at 42 days after the initial AA injection for histology, immunohistochemistry, real-time PCR, and western blot analysis. In addition, groups of 6 normal Smad7 KO and WT mice received saline injection were used as normal age-matched control.
To further investigate the role of renal Smad7 in the development of chronic AAN, a rescued study was performed in Smad7 KO mice (male, aged 6-8 weeks, 26-31g) by restoring Smad7 locally into the kidneys of Smad7 KO mice using the ultrasound-microbubble gene transfer technique (see below), which was performed immediately after intraperitoneal injection of AA. Groups of 6 Smad7 KO mice were used. Control mice with chronic AAN had the same procedure but received empty vector treatment. In addition, a group of mice with chronic AAN without ultrasound treatment and a group of normal Smad7 KO mice treated with saline were used as controls.
To test the therapeutic effect of Smad7 in the established chronic AAN, groups of 6 mice with chronic AAN induced in Smad7 WT mice were randomized into three groups at day 14 after the initial AA injection. Group 1 animals received AA without any treatment (AAN); Group 2 mice received AA but treated with empty vector control (VC) from day 14 onwards after the initial injection of AA; Group 3 mice received AA and treated with Smad7 (Smad7) from day 14 onwards after the initial injection of AA. In addition, a groups of AAN mice (n=6) at day 14 before treatment were euthanized as disease control before Smad7 treatment and a group of 6 normal age-matched mice treated with saline were used as normal control. Smad7 gene transfer was performed in groups of 6 Smad7 WT mice with established AAN at day 14 as described below. The experimental procedures were approved by the Institutional Animal Experimentation Ethics Committee (Permit No. 12-352).

A non-invasive ultrasound-microbubble-mediated smad7 gene transfer into kidneys
A non-invasive ultrasound-microbubble-mediated Smad7 gene transfer was performed following the previous protocol [17,21]. Briefly, pTRE 2 -Flag-M2-Smad7 and Tet-on plasmids (100ug/mouse, respectively) were mixed with Sonovue (Bracco Diagnostics, Princeton, NJ,USA) in a 1:1 ratio (volume:volume). The mixture (400ul) was injected into Smad7 KO (for the rescued study) or Smad7 WT (for the treatment study) mice via tail vein, followed by placing the ultrasound probe on the skin of the mouse back opposite to the bilateral kidneys with a plus-wave output (2 W/cm 2 ) for a total of 5 min with 30 seconds intervals. After ultrasound treatment, 200ug/ ml of doxycycline (Sigma, St, Louis, MO) were injected intraperitoneally, followed by the addition of doxycycline Oncotarget 11942 www.impactjournals.com/oncotarget in the daily drinking water (200ug/ml) for the entire study period. Control ultrasound treatment group had the same protocol but received the pTRE 2 -Tet-on empty vectors without Smad7. According to the previous studies that ultrasound-microbubble-mediated Smad7 transgene expression peaked at days 3-7 and declined 2 weeks later [ 17,21], Smad7 gene transfer was repeated every 2 weeks until animals were sacrificed.
The experimental procedures were approved by the Institutional Animal Experimentation Ethics Committee (Permit No. 12-352).

renal function and proteinuria
Twenty-four hour urine samples were collected in all animals in the metabolic cages at time 0 (before induction of AAN) and at weeks 2,4,6 after induction of AAN for proteinuria assay. Urine protein levels were measured using the Quick start Bradford Dye Reagent (BioRAD). Serum creatinine was used to evaluate renal function. Levels of serum creatinine were detected by the Enzymatic creatinine LiquiColor Reagent (Stanbio Laboratory, Boerne, TX), according to the manufacturer's instructions.

real-time Pcr
RNA was collected from renal tissues and purified by an RNeasy kit according to the manufacturer's instructions (Qiagen, Valencia, CA), and real-time PCR was performed with Sybergreen on an Opticon real-time PCR machine (MJ Research, Waltham, MA) as previously described. [28][29][30] Primers used for detection of mRNA expression of collagen I, α-SMA, TGF-β1, MCP-1, TNFα, and GAPDH were described previously [28][29][30]. House keep gene GAPDH was used as an internal standard. The ratio for the mRNA was examined against GAPDH and was expressed as mean±SE.

statistical analysis
Data obtained from this study are expressed as means±SE. Statistical analyses were performed using oneway analysis of variance followed by a Newman-Keuls posttest (Prism 5.0 GraphPad Software, San Diego, CA).