diabetic nephropathy ( dn ) is the leading cause of end - stage renal disease .
mesangial cell proliferation , excessive extracellular matrix ( ecm ) proteins accumulation , basement membrane thickening , mesangial expansion , and other mesangial region lesions are involved in the renal pathological process , which ultimately lead to glomerular sclerosis .
although hyperglycemia , hyperlipemia , hypertension , blood flow , and inflammation are participated in the pathogenesis of dn , the exact cause of dn remains unclear [ 14 ] .
naringenin ( 4,5,7-trihydroxy flavanone , nar ) is a flavanone compound found in citrus fruits , which is rich in the seeds and peels of fruits .
recent researches showed that nar had many potentially pharmacological effects such as antioxidant , anti - inflammation , anticancer , and antifibrosis [ 58 ] .
meanwhile , it was reported that nar possessed antiproliferative activity in lung cancer , colorectal cancer , and leukemia cell and regulated extracellular matrix synthesis in hepatic satellite cell [ 912 ] .
furthermore , nar could decrease the blood glucose level in stz induced diabetic rats [ 13 , 14 ] , suppress macrophage infiltration into adipose tissue in an early phase of high - fat diet - induced obesity , and inhibit production of il-1 , il-6 , type iv collagen , fibronectin , tgf-1 , and monocyte chemoattractant protein-1 ( mcp-1 ) in the kidney of diabetic mice .
however , the mechanism of the proliferation effect of nar in dn is unknown up to now .
microrna let-7 and its family members have been reported to participate in many diseases including kidney diseases . let-7a
regulated glucose metabolism and insulin synthesis / secretion with type 2 dm by targeting lin28 pathway [ 17 , 18 ] .
let-7a and let-7d could affect glucose metabolism by downregulating il-13 in the skeletal muscle of type 2 dm patients . also , let-7 family members regulated collagen expression in glomerular mesangial cells under diabetic conditions .
furthermore , our previous experiments showed that let-7a was downexpressed in dn patients , and the let-7a-3 promoter hypermethylation and the rs1143770 polymorphism of let-7a-2 were participated in dn [ 21 , 22 ] .
moreover , let-7a had been reported to modulate ecm deposition in breast and pancreas cells [ 2325 ] .
these findings suggested that let-7a may be an important factor in the effects of nar in dn . in the present study
, we aimed to explore the protective effects of nar on kidney as well as its effects on let-7a / tgfbr1 signaling in diabetic nephropathy rats and mesangial cell under high glucose condition .
this study demonstrated that let-7a and its related pathway - tgf-1/smad signaling formed a negative feedback to inhibit the deposition of ecm by targeting tgfbr1 in dn .
moreover , nar might repress glomerular mesangial cells proliferation and accumulation of ecm by let-7a / tgfbr1 signaling in dn , and let-7a may be a novel potential target for nar protecting against diabetic nephropathy .
twenty - five male sprague - dawley rats weighting 120  20  g ( four weeks old ) were provided by the experimental animal center at chongqing medical university .
the animals were housed in the clean environment under conditions of controlled temperature of 2025c and humidity 65~69% .
the rats were randomly divided into a control group ( con , n = 5 ) and a dn group ( n = 20 ) . the control rats were fed with the normal diet , while the rats in diabetic group were fed with the high - sugar - high - fat diet .
five weeks later , diabetic nephropathy was induced by a single intraperitoneal injection of stz ( sigma , st .
mmol / l ) and high urinary total protein level ( total protein level in dn group was more than twice higher than the control group ) were measured to confirm early dn in rats .
then , the dn rats ( n = 18 ) were assigned into two groups : dn rats treated with saline ( dn group , n = 8) and dn rats treated with nar ( nar group , n = 10 ) .
the rats in nar group were treated with naringenin ( sangon , shanghai , china ) ( 50  mgkgday , gavage ) , while the rats in dn group were treated with the equal volume of normal saline . among the three groups
, blood glucose was measured each week , and 24  h urine was collected every two weeks .
renal tissues from each rat were disposed as follows : one was immediately frozen in liquid nitrogen , and the other was fixed with 4% paraformaldehyde .
all animal experiments were performed in accordance with the protocols approved by chongqing medical university animal care committee .
24  h urinary protein was measured by the coomassie brilliant blue ( cbb ) method .
urinary creatinine ( ucr ) and serum creatinine ( scr ) were measured by clinical laboratory .
urinary creatinine clearance ( ccr ) ratio was calculated using the following equation : ccr ( mlminkg ) = [ urinary cr ( mmol / l )  urinary volume ( ml)/serum cr ( mmol / l ) ]  [ 1/1,440  ( min ) ] .
right kidney weight ( kw ) and body weight ( bw ) were assessed to calculate kidney index :
the glomerular areas ( m ) with 50 fields of view were analyzed by image - pro plus 6.0 .
0.1  g nar powder was dissolved in 1  ml dmso and then filtrated using 0.22  m micropore filter , preserved at 4c for subsequent use .
the solution were diluted in dmem to different working concentration of 100  mol / l , 200  mol / l , 400  mol / l , 600  mol / l , and 1000  mol / l .
mouse glomerular mesangial cell ( mmc ) line ( academy of sciences , shanghai , china ) was cultured in dmem with 20% fbs in a humidified atmosphere containing 5% co2 at 37c .
mmcs were maintained in 5  mmol / l glucose plus 20  mmol / l mannitol as the control group ( lg ) .
cells were maintained in 25  mmol / l glucose as the high glucose group ( hg ) , and cells were treated with 25  mmol / l glucose plus nar as the nar treatment group ( nar ) .
293 t cells were preserved in our laboratory and grown in dmem with 10% fbs as previously described .
mmcs were seeded at a density of 1.0  10 cells / well in 96-well plates with nar treatment .
after treated with nar for 0  h , 12  h , 24  h , and 36  h , 20  l mtt reagent was added into each corresponding cultured well .
the absorbance in 490  nm was measured by the microplate reader ( bio - rad , usa ) .
mmcs were treated with nar for 24  h , then were digested with trypsin and were made into cell suspension , were washed with precooling pbs twice , were fixed with 75% precooling ethanol overnight at 4c , collected the cells by centrifuge , were washed with 1  ml pbs , were added 100  l rnasea incubated at 37c for 30  min , were added 400  l pi incubated at 4c for 30  min at dark , 1  10 cells for cell cycle analysis with bd flow cytometry by standard procedure .
mmcs were seeded at a density of 0.75  10  cells / ml in serum - free dmem , with the addition of a transfection agent and let-7a mimics oligonucleotides ( let7a - m ) , negative control mimics ( nc - m ) , let-7a inhibitor oligonucleotides ( let7a - i ) , and negative control inhibitor ( nc - i ) ( jima , shanghai , china ) .
six hours after transfection , the medium was changed and the cells were incubated with fresh serum - containing medium for another 24~48  h. in addition , mmcs were transfected with let-7a mimics plus nar as the nar + let7a - m group ; cells were transfected with let-7a inhibitor plus nar as the nar + let7a - i group .
all transfections were performed with the aid of a lipofectamine 2000 transfection agent ( invitrogen , usa ) following the manufacturer 's instructions .
total rna was extracted by trizol reagent ( invitrogen ) according to the manufacturer 's instructions .
small rnas ( < 300  nt ) were isolated when 2 to 5  g total rna sample was size - fractionated using a ym-100 microcon centrifugal filter ( millipore ) .
the primescript rt reagent kit ( takara , dalian , china ) and sybr premix ex taq tm ii ( takara , dalian , china ) were used for mirna and mrna quantification .
relative expressions were calculated using 2 method and normalized to the expression of u6 or gapdh .
primers for real - time pcr were as follows : col-4 , ( f ) : 5-caaaccacagccaatccttca-3 , ( r ) : 5-aagaagggaaaacccactgtagagt-3 ; fn , f : 5-catggctttaggcgaacca-3 , r : 5-catctacattcggcaggtatgg-3 ; tgf-1 , f : 5-agggctaccatgccaacttc-3 , r : 5-ccacgtagtagacgatgggc-3 ; tgfbr1 , f : 5-tggcggaatccacgaaga-3 , r : 5-acggatggatcagaaggtacaag-3 ; smad2 , f : 5-acaacaggcctttacagcttc-3 , r : 5-ctctgtggctcaattcctgc-3 ; smad7 , f : 5-ccatcaaggcttttgactatgaaa-3 , r : 5-ccatggctgctgcatgaac-3. all real - time rt pcr was performed in triplicate , and the data were presented as means  sd . total protein from cells and renal tissues
were extracted , subjected to 10% sds - polyacrylamide gels ( beyotime , shanghai , china ) and transferred to polyvinylidene difluoride membrane .
membranes were blocked with 5% nonfat dry milk dissolved in tbst and then incubated with primary antibody overnight at 4c .
antibodies and dilutions included the following : rabbit monoclonal to col4 antibody ( 1  :  1,000 , proteintech ) , rabbit polyclonal to fn antibody ( 1  :  1,000 , proteintech ) , rabbit monoclonal to smad2 antibody ( 1  :  1,500 , proteintech ) , rabbit polyclonal to p - smad2 antibody ( 1  :  500 , bioworld ) , rabbit polyclonal to tgf-1 antibody ( 1  :  800 , proteintech ) , rabbit polyclonal to tgfbr1 antibody ( 1  :  700 , bioworld ) , and rabbit polyclonal to gapdh antibody ( 1  :  2,000 , proteintech ) .
the membranes were incubated with the goat anti - rabbit igg ( 1  :  5,000 , abcam ) for 1.5  h. signals were detected with chemidocxrs system ( bio - rad , usa ) .
kidney tissue paraffin sections ( 4  m ) were subjected to immunofluorescence staining.antibodies and dilutions were as follows : rabbit monoclonal to col4 antibody ( 1  :  50 , proteintech ) , rabbit polyclonal to fn antibody ( 1  :  100 , proteintech ) , rabbit polyclonal to tgf-1 antibody ( 1  :  50 , proteintech ) , and rabbit polyclonal to tgfbr1 antibody ( 1  :  50 , bioworld ) .
the tissue sections were then incubated with fitc or tritc for 1  h. images were acquired using the fluorescence microscope ( olympus japan ) .
tgfbr1 ( nm_009370.2 ) 3-utr - luciferase constructs were made using the pmir - rb - report vector , and the primers for tgfbr1 3 utr - wt were as follows : tgfbr1-wt - f : 5-gcgctcgaggggtgtttaggaggctggt-3 ; tgfbr1-wt - r : 5-aatgcggccgccatacaacttttccttcgg-3. the pcr products were excised with noti and xhoi .
the potential binding sequences of let-7a on the tgfbr1 3utr were mutated by the quikchange site - directed mutagenesis kit ( stratagene ) .
for transfection , 293 t cells were seeded into 96-well plates at a density of 1.5  10 per well ; then cells were transfected with tgfbr1 - 3utr - wt ( 200  ng / ml ) , tgfbr1 - 3utr - mut ( 200  ng / ml ) , and let-7a mimics ( 50  nmol / l ) using lipofectamine 2000 . after 48  h
data for multiple variable comparisons were analyzed by one - way analysis of variance ( anova ) .
results showed blood glucose level went up steadily with time in dn rats , while blood glucose level was decreased at 6 weeks in nar group ( figure 1(a ) ) .
the 24  h urinary protein went up steadily with time in dn rats , while after nar treatment , it was decreased ( figure 1(b ) ) . and creatinine clearance ratio ( ccr )
is generally considered as the feature of renal function , reflecting renal filtration function . kidney index ( ki ) and glomerular area ( ga ) are the morphological markers of glomerular hypertrophy .
we evaluated the effects of nar on 24  h urinary protein , ccr , ki , and ga .
24  h urinary protein ( 8.49  1.87 versus 40.41  4.68  mg/24  h , p < 0.001 ) .
ki ( 2.87  0.17 versus 4.83  0.30 , p < 0.001 ) and ga ( 146.32  23.74 versus 194.29  40.60  m ,
p < 0.05 ) were significantly increased in dn group compared with those in con group , whereas ccr ( 3.31  0.74 versus 1.39  0.30  ml / min , p < 0.01 ) was decreased in dn group .
after treatment with nar , ccr was obviously increased ( 1.39  0.30 versus 2.08  0.55  ml / min , p < 0.05 ) ( figure 1(c ) ) ; ki was significantly decreased ( 4.83  0.30 versus 4.33  0.34 , p < 0.05 ) ( figure 1(d ) ) .
ga was reduced ( 194.29  40.60 versus 165.15  41.15  m , p < 0.05 ) in the nar group compared with that in dn group ( figures 1(e ) and 1(f ) ) .
to evaluate the effect of nar on mesangial cells proliferation , mtt and cell cycle analysis were used .
mtt results showed that nar obviously inhibited cell proliferation at the concentration of 400 , 600 , and 1000  mol / l for 24  h , 36  h , and 200  mol / l for 36  h , whereas cell proliferation rates were unchanged at the concentration of 100  mol / l ( figure 2(a ) ) .
moreover , the cell cycle analysis showed cells were treated with 400 , 600 , and 1000  mol / l nar for 24  h , respectively .
the results showed that g2 phase of nar group was significantly increased when compared with that in hg group , while s phase was shortened in nar group at the concentration of 600 and 1000  mol / l .
therefore , these data indicated that nar delayed mesangial cell growth at g2 phase ( figure 2(b ) ) .
a key feature of dn was the accumulation of excessive extracellular matrix proteins , predominant collagens ( col4 ) , and fibronectin ( fn ) . to determine whether nar affected col4 and fn expression in vivo and in vitro , col4 and fn mrnas and proteins of kidney tissues were examined by real - time rt pcr ( figure 2(c ) ) , western blot ( figures 2(d ) , 2(e ) , and 2(f ) ) , and immunofluorescence ( figures 2(g ) and 2(h ) ) .
the results showed that the mrna and protein of col4 and fn were remarkably increased in the dn or hg group compared with the con or lg group and decreased when nar was treated in mice and cells .
taken together , our results suggested that nar could improve the excessive deposition of ecm .
our previous study demonstrated that several mirnas were differentially expressed in blood samples of dn patients by mirna array and real - time pcr , including let-7a , let-7d , let-7f , mir-363 , and mir-4429 ( data not shown ) .
furthermore , our result displayed that only let-7a was reversed when treated with nar among the five mirnas in mesangial cells with high glucose ( figure 3(a ) ) . to determine whether nar affected the expression of let-7a in vivo ,
the results showed that let-7a was significantly decreased in the dn group compared with the con group .
after the treatment of nar , the expression of let-7a was significantly enhanced in the nar group compared with the dn group ( figure 3(b ) ) .
these results demonstrated that nar upregulated let-7a expression both in vivo and in vitro in dn .
we further observed whether nar affected fibrosis markers col4 and fn expressions by regulating let-7a in cells .
mmcs were transfected with let-7a mimics ( let7a - m ) , let-7a inhibitor ( let7a - i ) , nar , or nar with let-7a intervention .
the results showed that let-7a was highly expressed in let-7a mimics group and decreased in inhibitor group .
the expression of let-7a was significantly elevated in nar + let-7a group , whereas let-7a declined in nar + let-7a inhibitor group ( figure 3(c ) ) .
moreover , the expressions of col4 and fn were decreased in let-7a or nar group compared with those in the untreated hg group .
interestingly , the expressions of col4 and fn were lower after transferring let-7a mimic and nar into mesangial cell compared with nar or let-7a group ( figures 3(d ) and 3(e ) ) .
so our results suggested that nar could improve the excessive deposition of ecm by upregulating let-7a expression . to evaluate possible functions of let-7a , we screened potential target genes of let-7a .
firstly , according to bioinformatics software targetscan ( http://www.targetscan.org/ ) , we found that dn related gene - tgfbr1 was a potential target of let-7a . by analyzing homology
, we found that the putative mmu - let-7a target site in tgfbr1 3-utr was highly conserved in sixteen genomes ( figure 4(a ) ) .
transcripts carrying the tgfbr1 3-utr - wt exhibited a significant reduction in luciferase activity in the presence of let-7a .
in contrast , the mimics negative control had no significant effect on the luciferase activity ( figure 4(b ) ) .
meanwhile , western blot results showed that the expression level of tgfbr1 protein was reduced significantly in the let-7a mimics group compared with the hg group ( figures 4(c ) and 4(d ) ) .
tgf-1 is recognized as a major mediator of renal fibrosis because it is able to stimulate the accumulation of ecm protein and tgf-1/smad signaling activation involving in this process . to determine that whether nar affected the tgf-1/smad signaling in vivo , we examined tgf-1 , tgfbr1 , smad2 , and smad7 mrna and protein expressions in kidney tissue of dn rats .
the results of real - time rt pcr showed that tgf-1 , tgfbr1 , and smad2 mrnas were remarkably increased in the dn group compared with the con group , while smad7 mrna was declined .
nar reduced the expressions of tgf-1 , tgfbr1 , and smad2 and increased smad7 mrnas ( figure 5(a ) ) .
meanwhile , the western blot results showed that tgf-1/smad signaling was highly activated in dn group as revealed by a marked upregulation of tgf-1 , tgfbr1 , and p - smad2/smad2 and downexpression of smad7 , whereas nar directly reduced tgf-1 , tgfbr1 , and p - smad2/smad2 and increased smad7 protein expression ( figures 5(b ) and 5(c ) ) .
we further observed the effects of nar on tgf-1 , tgfbr1 , smad2/p - smad2 , and smad7 protein in mmcs .
we found that the expressions of tgf-1 , tgfbr1 , and smad2/p - smad2 significantly decreased and smad7 increased in nar group compared with those in hg group ( figures 5(b ) and 5(d ) ) .
the results of immunofluorescence for tgf-1 and tgfbr1 were consistent with the results of western blot ( figure 5(e ) ) .
together , our results suggested that tgf-1/smad signaling was activated in dn rats and mmcs in high glucose condition and nar could inhibit tgf-1/smad signaling activation in vivo and vitro .
furthermore , we observed whether nar affected tgf-1/smad signaling by regulating let-7a in mmcs or not .
we found that overexpression of let-7a and nar treatment decreased the ratio of p - smad2/smad2 and tgfbr1 proteins compared with mmcs without treatment .
additionally , nar alleviated the change in the ratio of p - smad2/smad2 and tgfbr1 proteins cooperation with let7a - m more significantly than nar group and let7a - m group , whereas the ratio of p - smad2/smad2 and tgfbr1 protein was increased in the nar + let-7a inhibitor group compared with nar group ( figures 5(f ) and 5(g ) ) .
our results suggested that tgf-1/smads signaling was activated in dn rats and mmcs in high glucose condition and nar inhibited tgf-1/smads signaling activation through upregulating let-7a and suppressing tgfbr1 expression .
glomerular mesangial cell acts actively in glomeruli , which can secrete ecm and produce cytokine , phagocyte , and clear macromolecules , and it has systolic function like smooth muscle cell .
studies have confirmed that mesangial cell was the important glomerular resident cells in the synthesis of ecm proteins [ 30 , 31 ] .
deposition of excessive ecm molecules , predominant collagens , and fibronectin in the kidney will lead to kidney hyperplasia and glomerular area enlargement , which adversely affected the structure and function of the kidney .
it played an important role in diabetes and its complications . researches revealed that nar could affect dyslipidemia , apob overproduction , and hyperinsulinemia in ldl - receptor null mice with diet - induced insulin resistance .
the antidiabetic effect of nar might be insulin - like effect in type 2 diabetic rats . additionally , it was reported that nar supplement increased its deposit in the liver and kidney of diabetic mice , and plasma levels of glucose and blood urea nitrogen were both decreased in nar group , while insulin level and creatinine clearance were increased in nar group compared with the diabetic control group . in this study ,
our results showed nar could not only decrease the blood glucose , 24  h urinary protein , kidney index , and glomerular area but also increase ccr in dn rats .
also , we examined the therapeutic effects of nar in dn rats and mesangial cells under diabetic condition .
our data showed that nar inhibited mesangial cells proliferation by delaying cell cycle at g2 phase .
moreover , col4 and fn were highly expressed in dn rats and mmcs under high glucose condition , while nar directly reduced the expressions of col4 and fn mrna and proteins .
therefore , these findings displayed that nar might participant in the development and progress of dn and suggested that nar could play an important role in dn .
however , the exact mechanism of nar in dn is still unclear . to explore the underlying mechanism of nar in dn , several dn
results showed let-7a was the only mirna which reversed the expression level when treated with nar in mesangial cells cultured with high glucose . furthermore
, numerous researches showed that let-7a was closely linked to modulate cell proliferation in various diseases [ 3638 ] .
also , our previous studies also showed that the promoter hypermethylation and single nucleotide polymorphism of let-7a played important roles in dn [ 21 , 22 ] .
therefore , these studies suggested that let-7a might be related to the therapeutic effects of nar in dn . in our study , real - time rt pcr results exhibited that let-7a expression was significantly decreased in both blood and kidney tissues of dn rats and mesangial cells under hyperglycemic condition . and
additionally , nar alleviated the changes of col4 and fn proteins cooperated with let7a mimics more significantly than nar group , whereas the inhibitory effect of nar cooperated with let-7a inhibitor was weakened .
so these results suggested that nar alleviated the deposition of ecm proteins by upregulating let-7a expression . in the famous dn related pathway - tgf-1/smad signaling pathway
subsequently , phosphorylated smad2 and smad3 bound to the common smad4 and formed the smad complex to regulate the downstream gene transcription . in this process , smad7 could block the activation of tgf-1/smad signaling as an inhibitory factor .
therefore , tgfbr1 , tgf-1 , and smads were key factors in the tgf-1/smad signaling pathway .
research showed nar decreased cell invasion and metastasis by inhibition of tgf-1/smads signaling pathway in pancreatic cancer , and nar also reduced tgf-1-induced hepatic satellite cell extracellular matrix deposition by inhibiting tgf-1/smads signals [ 12 , 40 ] . in this study , our data displayed that there was a significant reduction of tgfbr1 protein in the presence of let-7a mimics by dual - luciferase reporter assay , and overexpression of let-7a could decrease the expression of tgfbr1 protein by western blot .
our results showed that tgf-1/smads signaling was highly activated in dn rats and mmcs under high glucose condition , whereas nar directly suppressed activation of tgf-1/smad signaling .
more importantly , in vitro experiments , overexpression of let-7a decreased the expression of p - smad2 and tgfbr1 proteins compared with hg group and it suggested that let-7a may negatively regulate tgf-1/smads signaling by targeting tgfbr1 .
meanwhile , nar cooperated with let7a mimics ; its effects were more remarkable than nar or let-7a mimics ' individual effect in the reductions of p - smad2 and tgfbr1 proteins , while the reductions of p - smad2 and tgfbr1 proteins in the nar cooperated with let-7a inhibitor group were weakened .
taken together , our data exhibited that nar regulated the alteration of tgf-1/smads signaling through upregulating let-7a expression and suppressing tgfbr1 . in summary ,
our study suggested that nar ameliorated kidney injury and inhibited mesangial cells proliferation and accumulation of ecm in dn .
/ tgfbr1 , and let-7a might be a novel therapeutic target for nar protect against dn .
 diabetic nephropathy ( dn ) is one of the most common complications of diabetes mellitus ( dm ) . 
 however , the exact mechanism is not clearly understood . in this study 
 , our results showed that 24  h urinary protein , kidney index , and glomerular area were decreased , while creatinine clearance ratio was increased in dn rats when the rats were treated with nar 50 mg / d for 6 weeks . 
 mesangial cell ( mmcs ) proliferation was inhibited in the nar group by 3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide ( mtt ) , and the cell cycle analysis showed that cells stayed in g2 phase in nar group . 
 and nar treatment attenuated the deposition of ecm in dn rats and mmcs . 
 moreover , our data showed that let-7a was downexpressed in both dn rats and mmcs under high glucose condition . 
 surprisingly , nar affected the expressions of col4 and fn through upregulating let-7a in mmcs . 
 in addition , we found that let-7a negatively regulated the expression of transforming growth factor-1 receptor 1 ( tgfbr1 ) , and tgfbr1 was required for the let-7a - mediated downregulation of tgf-1/smad signaling . 
 interestingly , nar inhibited tgf-1/smads signaling activation by upregulating let-7a . therefore , our findings indicated that nar ameliorated kidney injury by regulating let-7a 
 / tgfbr1 signaling . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion