The protective roles of GLP-1R signaling in diabetic nephropathy: possible mechanism and therapeutic potential

Transcription

1 & 213 Interntionl Society of Nephrology sic reserch The protective roles of GLP-1R signling in dietic nephropthy: possile mechnism nd therpeutic potentil Hiroki Fujit 1, Tsuks Morii 1, Hiromi Fujishim 1, Tkehiro Sto 1, Ttsunori Shimizu 1, Mihoko Hoso 1, Ktsushi Tsukiym 1, Tkum Nrit 1, Tkmune Tkhshi 2, Dniel J. Drucker 3,4, Yutk Seino 5 nd Yuichiro Ymd 1 1 Division of Endocrinology, Metolism nd Geritric Medicine, Akit University Grdute School of Medicine, Akit, Jpn; 2 Division of Nephrology nd Hypertension, Vnderilt University Medicl Center, Nshville, Tennessee, USA; 3 Deprtment of Medicine, University of Toronto, Toronto, Ontrio, Cnd; 4 The Lunenfeld-Tnenum Reserch Institute, Mt Sini Hospitl, Toronto, Ontrio, Cnd nd 5 Knsi Electric Power Hospitl, Osk, Jpn Glucgon-like peptide-1 (GLP-1) is gut incretin hormone tht hs n ntioxidtive protective effect on vrious tissues. Here, we determined whether GLP-1 hs role in the pthogenesis of dietic nephropthy using nephropthyresistnt nd nephropthy-prone KK/T-Akit mice. By in situ hyridiztion, we found the GLP-1 receptor (GLP-1R) expressed in glomerulr cpillry nd vsculr wlls, ut not in tuuli, in the mouse kidney. Next, we generted Glp1r knockout mice. These mice exhiited higher urinry lumin levels nd more dvnced mesngil expnsion thn wild-type mice, despite comprle levels of hyperglycemi. Incresed glomerulr superoxide, upregulted renl NAD(P)H oxidse, nd reduced renl camp nd protein kinse A (PKA) ctivity were noted in the Glp1r knockout mice. Tretment with the GLP-1R gonist lirglutide suppressed the progression of nephropthy in KK/T-Akit mice, s demonstrted y reduced luminuri nd mesngil expnsion, decresed levels of glomerulr superoxide nd renl NAD(P)H oxidse, nd elevted renl camp nd PKA ctivity. These effects were olished y n denylte cyclse inhiitor SQ22536 nd selective PKA inhiitor H-89. Thus, GLP-1 hs crucil role in protection ginst incresed renl oxidtive stress under chronic hyperglycemi, y inhiition of NAD(P)H oxidse, mjor source of superoxide, nd y camp-pka pthwy ctivtion. Kidney Interntionl (214) 85, ; doi:1.138/ki ; pulished online 23 Octoer 213 KEYWORDS: dietic nephropthy; glucgon-like peptide-1; oxidtive stress Correspondence: Hiroki Fujit, Division of Endocrinology, Metolism nd Geritric Medicine, Akit University Grdute School of Medicine, Hondo, Akit , Jpn. E-mil: hirofuji@gipc.kit-u.c.jp Received 5 Ferury 213; revised 8 Septemer 213; ccepted 12 Septemer 213; pulished online 23 Octoer 213 Dietic nephropthy (DN) is serious compliction of dietes nd the leding cuse of end-stge renl disese in developed countries. Recent evidence indictes tht oxidtive stress hs centrl role in the development nd progression of DN. 1,2 The increse in systemic oxidtive stress ecomes prominent from the incipient stge of DN. 3 In the kidney, rective oxygen species (ROS) including superoxide nion (O 2 ) re excessively produced y chronic hyperglycemi, leding to incresed levels of renl oxidtive stress. NAD(P)H oxidse is the most importnt source of superoxide nion, 4 7 nd this enzyme is shown to e upregulted in the dietic kidney. 5,8 1 A recent in vitro study using humn HEK293 cells demonstrted tht NAD(P)H oxidse NOX1-dependent ROS production is reduced y the elevtion of camp nd susequent ctivtion of protein kinse A (PKA). 11 Furthermore, tretment with camp-elevting gents such s isoproterenol nd forskolin normlized the levels of NAD(P)H oxidse ctivity nd superoxide in ortic vsculr smooth muscle cells of spontneously hypertensive rts. 12 Thus, the camp-pka pthwy ppers to work s n importnt inhiitory fctor for NAD(P)H oxidse dependent production of ROS or superoxide. Glucgon-like peptide-1 (GLP-1) is gut incretin hormone tht stimultes insulin secretion from pncretic -cells in glucose-dependent mnner. 13 Activtion of the GLP-1 receptor (GLP-1R) stimultes denylte cyclse nd enhnces the production of camp, the primry effector of GLP-1-induced insulin secretion. 13,14 Furthermore, incresed levels of camp ctivte PKA or camp-regulted gunine nucleotide exchnge fctor II (Epc2), nd contriute to mediting vrious physiologicl ctions including insulin secretion. 14,15 The GLP-1R is expressed in pncretic -cells nd in multiple extrpncretic tissues including the gut, rin, hert, lung, nd kidney. 16,17 Given the evidence indicting tht camp nd PKA pthwys link to ntioxidtive effects, it is likely tht GLP-1 protects vrious tissues from oxidtive injury. However, the roles of GLP-1 in the kidney nd DN hve not een fully Kidney Interntionl (214) 85,

2 sic reserch H Fujit et l.: GLP-1R signling nd dietic nephropthy elucidted. First, the precise locliztion of GLP-1R in the kidney remins uncler. Second, it is unknown whether gin or loss of GLP-1R signling modultes renl function nd the progression of renl injury under conditions of chronic hyperglycemi. In the present study, we investigted the role of endogenous GLP-1R signling in DN. First, we exmined the locliztion of GLP-1R in the mouse kidney y in situ hyridiztion nd reverse trnscriptse polymerse chin rection (RT-PCR) nlysis. Next, we studied two Ins2 Akit dietic mouse models showing different susceptiility to the development nd progression of DN, DN-resistnt C57BL/6- Ins2 Akit (), nd DN-prone KK/T-Ins2 Akit (KK/T-Akit). 18,19 We exmined the renl phenotypes of mice with GLP-1R deficiency, nd in complementry experiments, we tested whether GLP-1R gonist, lirglutide, meliortes nephropthic chnges in KK/ T-Akit mice tht develop progressive DN. 18 RESULTS Renl expression nd locliztion of the Glp1r in mice As recent studies hve highlighted the lck of sensitivity nd specificity of multiple GLP-1R ntiser, 2,21 we used in situ hyridiztion nlysis to ssess Glp1r expression in kidneys of 8-week-old mle C57BL/6-wild-type () mice. As shown in Figure 1, Glp1r mrna trnscripts were loclized long glomerulr cpillry wlls nd throughout vsculr wlls, ut not in tuules nd collecting ducts, in the kidney. In prticulr, the in situ hyridiztion nlysis reveled tht the Glp1r is predominntly expressed in renl lood vessels. To further verify these findings, we exmined Glp1r mrna expression in isolted enriched preprtions of glomeruli, tuuli, nd renl rteries. Consistent with the results of in situ hyridiztion nlysis, Glp1r mrna trnscripts were detected in RNA from glomeruli nd to greter extent in renl rteries, ut not in tuuli (Figure 1e nd f). Genertion of GLP-1R- nd insulin-deficient mice nd sic metolic mesurements We next confirmed the lck of Glp1r expression in newly generted lines of Akit mice lcking the Glp1r. Figure 2 shows the dt of RT-PCR nlysis in 3-week-old GLP-1Rdeficient C57BL/6 strin mice (Glp1r ) nd GLP-1Rpresent littermtes (Glp1r þ / þ ). A complete sence of renl glomerulr Glp1r mrna ws confirmed in Glp1r mice. The levels of glomerulr Glp1r mrna trnscripts were similr etween nondietic nd dietic mice. As shown in Figure 2, GLP-1R deficiency did not ffect islet topogrphy nd the numer of insulin þ cells in the mice. Tle 1 shows iochemicl nd physiologicl prmeters t 3 weeks of ge in the WT nd Akit Glp1r nd Glp1r þ / þ mice. GLP-1R deficiency did not ffect plsm levels of ctive GLP-1, glucose, nd insulin. Furthermore, there were no significnt differences in ody weight, systolic lood pressure, lood ure nitrogen, nd plsm lipids etween Glp1r þ / þ nd Glp1r- 18S- Glomerulus Tuules Renl rtery Kidney Glp1r mrna (% glomerulus) Figure 1 Anlysis of kidney Glp1r mrna expression. Representtive in situ hyridiztion imges of Glp1r mrna expression re shown in upper pnels. () Glomerulus, () lood vessel, (c) cortex, nd (d) ppill in 8-week-old mle mouse kidney. Arrows indicte Glp1r mrna expression. Brs ¼ 5 mm. (e nd f) Lower pnels show the results of reverse trnscriptse polymerse chin rection (PCR) nlysis in glomeruli, tuuli, nd renl rteries isolted from 8-week-old mle mouse kidneys (n ¼ 5 per group; Po.1 vs. glomerulus). Glp1r mice. Interestingly, Glp1r mice exhiited significntly higher levels of urinry lumin, glomerulr filtrtion rte (GFR), nd kidney weight reltive to Glp1r þ / þ mice. The mild chnges in the renl phenotype of Glp1r mice were detected y 15 weeks of ge (dt not shown), nd these mice developed overt renl dietic chnges y 3 weeks of ge. In contrst, we did not oserve similr chnges in renl prmeters in Glp1r þ / þ C57BL/6- WT mice. Renl glomerulr histopthology in Glp1r mice Figure 3 shows glomerulr histopthology t 3 weeks of ge in Glp1r þ / þ versus Glp1r nd C57BL/ 6-Akit mice. Interestingly, periodic cid Schiff (PAS) stining exmintion reveled incresed mesngil expnsion in Glp1r mice s compred with Glp1r þ / þ mice. Nondietic mice displyed norml glomerulr histology. Ovious tuulointerstitil injury ws not oserved in ny groups (dt not shown). Fironectin (FN) is n extrcellulr mtrix component nd is present long glomerulr sement memrnes. An increse in FN deposition is oserved during glomerulr injury, 22 nd hence FN is used s mrker of dietic Glomerulus Tuules Renl rtery 58 Kidney Interntionl (214) 85,

3 H Fujit et l.: GLP-1R signling nd dietic nephropthy sic reserch +/+ / +/+ / G/p1r +/+ G/p1r / G/p1r G/p1r- 18S- G/p1r +/+ G/p1r / Glomerulr G/p1r mrna (% G/p1r +/+ ) /+ / +/+ / G/p1r Insulin-positive stining re (% G/p1r +/+ ) /+ / +/+ / G/p1r Figure 2 Genertion of glucgon-like peptide-1 receptor (GLP-1R)- nd insulin-deficient mice. () Reverse trnscriptse polymerse chin rection (PCR) nlysis of glomerulr Glp1r mrna expression in 3-week-old mle mice. Loss of Glp1r mrna trnscripts ws confirmed in Glp1r mouse glomeruli (n ¼ 5 per group). () Pncres insulin immunohistochemistry in 3-week-old mle mice (n ¼ 5 per group; Po.1 vs. Glp1r þ / þ ). Arrows indicte pncretic islet. Brs ¼ 5 mm. WT, wild type. Tle 1 Biochemicl nd physiologicl prmeters in 3-week-old mle mice Glp1r þ / þ Glp1r Glp1r þ / þ Glp1r n BW (g) 3.2± ± ±.4 22.±.7 SBP (mm Hg) 99±1 99±1 116±4 w 115±3 w BG (mg/dl) 153±5 156±5 5±38 56±21 Plsm insulin (ng/ml).63±.5.54±.11.9±.2.6±.2 Plsm ctive GLP-1 (pg/ml) 11.7± ± ± ±2.6 BUN (mg/dl) 2.9± ± ± ±1.6 Cre (mg/dl).74±.2.7±.2.84±.7.83±.4 TC (mg/dl) 78.2± ± ± ±4.6 TG (mg/dl) 123±5 19±9 121±7 113±13 ACR (mg/mg cretinine) 9.7± ±.9 5.±3.5 z 86.2±15.6,y GFR (ml/min/g BW) 1.±.5 1.2± ±.8 2.1±.5,z LKW/BW (g/kg) 5.2±.1 6.5±.3 9.3± ±.6,z Arevitions: ACR, urinry lumin-to-cretinine rtio; BG, lood glucose; BUN, lood ure nitrogen; BW, ody weight; Cre, plsm cretinine; GFR, glomerulr filtrtion rte; GLP-1, glucgon-like peptide-1; LKW, left kidney weight; SBP, systolic lood pressure; TC, totl cholesterol; TG, triglyceride; WT, wild type. Vlues re mens±s.e.m. Po.1, w Po.1, z Po.5 vs. Glp1r þ / þ ; y Po.5, z Po.1 vs. Glp1r þ / þ. glomerulr injury. Compred with Glp1r þ / þ mice, Glp1r mice displyed significntly incresed FN ccumultion in glomerulr cpillry wlls (Figure 3 nd c). WT1 stining nlysis reveled significnt reduction of podocyte numer in Glp1r mice reltive to Glp1r þ / þ mice (Figure 3 nd d). Furthermore, through electron microscopic nlysis, we oserved irregulr thickening of the glomerulr sement memrne (GBM) in Glp1r mice (Figure 3). Morphometric nlysis reveled significnt increse in GBM thickness in Glp1r - mice (Figure 3e). Renl chnges in camp nd PKA ctivity levels in Glp1r mice Figure 4 shows renl camp nd PKA ctivity levels t 3 weeks of ge in Glp1r þ / þ versus Glp1r nd mice. Renl levels of oth camp nd PKA ctivity were mrkedly reduced in Glp1r C57BL/6- WT nd Glp1r mice. In contrst, levels of renl camp nd PKA ctivity were similr in Glp1r þ / þ mice. These findings indicte tht renl camp nd PKA ctivity re regulted y the presence or sence of the Glp1r, independent of levels of glycemi in mice. Renl chnges in oxidtive stress mrkers in Glp1r mice The degree of renl oxidtive stress ws ssessed using dihydroethidium (DHE) histochemistry nd thiorituric cid rective sustnce (TBARS) ssy t 3 weeks of ge in Glp1r þ / þ versus Glp1r nd C57BL/6- Akit mice. As shown in Figure 5 nd, the glomeruli in dietic mouse groups showed intense DHE fluorescence, indicting incresed glomerulr superoxide production. Notly, glomerulr DHE fluorescence ws stronger in kidneys from Glp1r mice. Renl levels of TBARS, sensitive mrker of oxidtive stress, were elevted in dietic mouse groups (Figure 5e) nd significntly greter in Glp1r C57BL/6- Akit mice, suggesting tht GLP-1R deficiency contriutes to Kidney Interntionl (214) 85,

4 sic reserch H Fujit et l.: GLP-1R signling nd dietic nephropthy PAS Fironectin Podocyte (WT1) GBM G/p1r +/+ G/p1r / G/p1r +/+ G/p1r / c d e Mesngil expnsion scores Fironectin stining intensity (% G/p1r +/+ ) G/p1r +/+ / +/+ / Figure 3 Renl histopthology in glucgon-like peptide-1 receptor (GLP-1R)-deficient mice. () Representtive glomerulus imges of periodic cid Schiff (PAS) stining, fironectin immunohistochemistry, WT1 stining, nd electron microscopic nlysis in 3-week-old mle mouse kidneys. Brs ¼ 5 mm for PAS, fironectin, nd WT1 imges or 1 mm for glomerulr sement memrne (GBM) imges. () Glomerulr mesngil expnsion scores. (c) Semiquntified fluorescence intensity of glomerulr fironectin. (d) The numer of WT1-positive podocytes in the glomeruli. (e) GBM thickness (n ¼ 5 per group; Po.1 vs. Glp1r þ / þ ; w Po.1 vs. Glp1r þ / þ ). Podocyte numer (/glomerulus) GBM (nm) 3 Renl camp (pmol/mg protein) Renl PKA ctivity (ng/mg protein) /+ / +/+ / Glp1r incresing renl oxidtive stress in the setting of dietes (Figure 5e). Recent experimentl studies hve reported tht NAD(P)H oxidse NOX4 component is mjor source of renl superoxide in the dietic stte, 9 nd tht chronic Figure 4 Renl levels of camp nd protein kinse A (PKA) ctivity in glucgon-like peptide-1 receptor (GLP-1R)-deficient mice. Renl levels of () camp nd () PKA ctivity were determined in 3-week-old mle mice (n ¼ 5 per group; Po.1, w Po.1 vs. Glp1r þ / þ ). WT, wild type. hyperglycemi enhnces renl ctivity of NAD(P)H oxidse. 1,23 Consistent with these findings, we oserved tht glomerulr NOX4 expression nd renl NAD(P)H oxidse ctivity were incresed in dietic mice reltive to nondietic mice (Figure 5, c nd f). Reduction of glomerulr NO ccelertes the progression of luminuri nd mesngil expnsion in dietic mice Therefore, we exmined glomerulr NO levels y evlution of the fluorescent intensity of the diminofluorescein-2 dicette rection. As shown in Figure 5 nd d, dietic mice showed decresed glomerulr NO levels. Semiquntittive nlysis of glomerulr NO fluorescence intensity reveled significntly lower glomerulr NO levels in dietic Glp1r mice (Figure 5d). In contrst, there ws no significnt difference in glomerulr NO levels etween the two nondietic mouse groups (Figure 5 nd d). Renl glomerulr expression of firogenic cytokines in Glp1r mice Thromospondin-1 (TSP-1) is homeotrimetric glycoprotein identified s n endogenous ctivtor of trnsforming growth fctor-1 (TGF-1). 27 TGF-1 nd connective tissue growth fctor (CTGF) re well-known firogenic cytokines implicted in the development of renl hypertrophy nd mesngil expnsion in dietic nephropthy. 28,29 Hence, we 582 Kidney Interntionl (214) 85,

5 H Fujit et l.: GLP-1R signling nd dietic nephropthy sic reserch Superoxide (DHE) NOX4 NO G/p1r +/+ G/p1r / G/p1r +/+ G/p1r / e f # G/p1r +/+ / +/+ / G/p1r +/+ / +/+ / Renl TBARS (nmol/mg protein) Renl NAD(P)H oxidse ctivity (RLU/1 μg protein) DHE fluorescence intensity (% G/p1r +/+ ) c NOX4 fluorescence intensity (% G/p1r +/+ ) d NO fluorescence intensity (% G/p1r +/+ ) G/p1r +/+ / +/+ / Figure 5 Renl oxidtive stress, NAD(P)H oxidse, nd nitric oxide (NO) levels in glucgon-like peptide-1 receptor (GLP-1R)-deficient mice. () Representtive imges of glomerulr dihydroethidium (DHE) stining, NOX4 immunohistochemistry, nd in situ NO detection y diminofluorescein-2 dicette (DAF-2DA) perfusion method in 3-week-old mle mouse kidneys. Arrows indicte glomerulus. Brs ¼ 5 mm. Semiquntified fluorescence intensity of glomerulr () DHE stining, (c) NOX4, nd (d) NO were determined s descried in Mterils nd Methods (n ¼ 5 per group; Po.1 vs. Glp1r þ / þ ; w Po.5, z Po.1 vs. Glp1r þ / þ ). Renl levels of (e) thiorituric cid rective sustnce (TBARS) nd (f) NAD(P)H oxidse ctivity in 3-week-old mle mice re shown in lower pnels (n ¼ 5 per group; Po.1, # Po.5 vs. Glp1r þ / þ ; w Po.5, z Po.1 vs. Glp1r þ / þ ). RLU, reltive chemiluminescence (light) unit; WT, wild type. exmined renl glomerulr expression of TSP-1, TGF-1, nd CTGF in 3-week-old mice. Strikingly, Glp1r mice exhiited incresed TSP-1 expression in glomerulr cpillry wlls (Figure 6 nd ) nd significntly higher glomerulr mrna levels of TGF-1 nd CTGF (Figure 6c nd d). In contrst, these glomerulr chnges were not oserved in nondietic Glp1r þ / þ C57BL/ 6-WT mice tht displyed norml glomerulr histology. Effects of GLP-1R gonist lirglutide on renl function, histologicl chnges, nd oxidtive stress in DN-prone KK/T- Akit mice We next ssessed whether the ctivtion of GLP-1R using the GLP-1R gonist lirglutide suppresses the progression of renl injury in KK/T-Akit mice, mouse model of progressive DN. 18 Mice were treted with lirglutide lone or in comintion with n denylte cyclse inhiitor SQ22536 or selective PKA inhiitor H-89. Tle 2 shows physiologicl nd iochemicl dt fter 4-week tretment period in 8-week-old KK/T-Akit mice. Although ll groups of mice were similr with respect to ody weight, systolic lood pressure, lood glucose, plsm insulin, lood ure nitrogen, plsm cretinine, nd serum lipids, mice treted with lirglutide lone exhiited lower levels of urinry lumin-to-cretinine rtio, GFR, nd kidney weight. These results suggest tht lirglutide ttenutes the development of dietic renl chnges such s overt luminuri, glomerulr hyperfiltrtion, nd renl hypertrophy without ffecting the severity of dietes nd dietesrelted fctors. Furthermore, SQ22536 nd H-89 olished the renl protective effects of lirglutide. We next exmined chnges in renl histopthology, oxidtive stress, NO, camp, nd PKA ctivity fter 4-week tretment with lirglutide, with or without SQ22536 or H-89, in 8-week-old KK/T-Akit mice. As shown in Figure 7 (PAS stining) nd 7, moderte mesngil expnsion ws oserved in vehicle-treted KK/T-Akit mice; however, mesngil expnsion ws reduced y lirglutide dministrtion. FN ccumultion in glomerulr cpillry wlls ws significntly diminished in the KK/T-Akit mice treted with lirglutide lone (Figure 7 nd c). Furthermore, the KK/T-Akit mice treted with lirglutide lone exhiited higher podocyte numer (Figure 7 nd d) nd lower GBM thickness (Figure 7 nd e) thn vehicle-treted KK/T-Akit mice. Notly, the meliortion of glomerulr histopthologicl dmge y lirglutide ws eliminted in KK/T-Akit Kidney Interntionl (214) 85,

6 sic reserch H Fujit et l.: GLP-1R signling nd dietic nephropthy GIp1r +/+ GIp1r / GIp1r +/+ GIp1r / TSP1 c d TSP1 stining intensity (% GIp1r +/+ ) Glomerulr TGF-β1 mrna (% GIp1r +/+ ) /+ / +/+ / GIp1r 1 5 +/+ / +/+ / GIp1r Glomerulr CTGF mrna (% GIp1r +/+ ) /+ / +/+ / GIp1r Figure 6 Glomerulr expression of thromospondin-1 (TSP-1) nd firogenic cytokines in glucgon-like peptide-1 receptor (GLP-1R)-deficient mice. () Representtive imges of glomerulr TSP-1 immunohistochemistry in 3-week-old mle mouse kidneys. Brs ¼ 5 mm. () Semiquntified fluorescence intensity of glomerulr TSP-1 (n ¼ 5 per group; Po.1 vs. Glp1r þ / þ ; w Po.1 vs. Glp1r þ / þ ). Glomerulr expression levels of (c) trnsforming growth fctor-1 (TGF-1) nd (d) connective tissue growth fctor (CTGF) were determined y quntittive reverse trnscriptse polymerse chin rection (PCR) in 3-week-old mle mice (n ¼ 5 per group; Po.1 vs. Glp1r þ / þ ; w Po.1, z Po.5 vs. Glp1r þ / þ ). WT, wild type. Tle 2 Physiologicl nd iochemicl prmeters fter 4-week tretment with lirglutide either lone or in comintion with SQ22536 or H-89 in mle KK/T-Akit mice Prmeter þ SQ22536 þ H-89 lone n BW (g) 21.5± ± ±.4 2.1±.7 SBP (mm Hg) 116±3 112±7 114±4 115±6 BG (mg/dl) 54±41 481±31 57±39 492±22 Plsm insulin (ng/ml).6±.4.8±.2.7±.2.6±.5 BUN (mg/dl) 38.± ±2.6 4.± ±1.5 Cre (mg/dl).94±.8.94±.8.95±.7.73±.4 TC (mg/dl) 136±14 128±1 122±8 135±4 TG (mg/dl) 229±21 238±2 272±3 268±2 ACR (mg/mg cretinine) 63±56 618±1 76±84 21±26 GFR (ml/min/g BW) 16.4±.8 ND ND 1.8±1.1 LKW/BW (g/kg) 14.9± ± ±.3 1.6±.5 w Arevitions: ACR, urinry lumin-to-cretinine rtio; BG, lood glucose; BUN, lood ure nitrogen; BW, ody weight; Cre, plsm cretinine; GFR, glomerulr filtrtion rte; LKW, left kidney weight; SBP, systolic lood pressure; TC, totl cholesterol; TG, triglyceride. Vlues re mens ±s.e.m. Po.1, w Po.5 vs. vehicle. mice treted with lirglutide in comintion with either SQ22536 or H-89. Figure 8 shows renl camp nd PKA ctivity levels in 8-week-old KK/T-Akit mice. KK/T-Akit mice treted with lirglutide lone for 4 weeks displyed higher renl levels of camp nd PKA ctivity thn vehicle-treted KK/T-Akit mice. In contrst, renl camp did not increse fter tretment with SQ22536, nd ws only modestly incresed in mice treted with H-89, wheres PKA ctivity levels were not incresed in kidneys of KK/T-Akit mice treted with lirglutide in comintion with SQ22536 or H-89. Figure 9 shows the degree of renl oxidtive stress nd glomerulr NO levels in 8-week-old KK/T-Akit mice. The tretment with lirglutide lone for 4 weeks significntly reduced glomerulr levels of superoxide nd NOX4 expression nd incresed glomerulr NO levels in KK/T-Akit mice (Figure 9 d). Furthermore, we oserved significnt reduction of renl TBARS nd NAD(P)H oxidse ctivity levels in the KK/T-Akit mice treted with lirglutide lone s compred with vehicle-treted KK/T-Akit mice (Figure 9e nd f). In contrst, the renoprotective effects of lirglutide were olished y codministrtion of either SQ22536 or H-89. DISCUSSION The present study, using in situ hyridiztion nd RT-PCR nlyses, demonstrtes tht Glp1r mrna trnscripts re loclized in glomerulr cpillry wlls nd throughout vsculr wlls, ut not in tuules nd collecting ducts, in the mouse kidney. More recently, Pnjwni et l. 2 hve demonstrted tht commercilly ville widely used GLP-1R ntiser re neither sensitive nor specific, nd detect comprle immunorective nds in tissue extrcts of oth Glp1r þ / þ nd Glp1r mice. 2 Similr concerns surrounding the use of commercilly ville GLP-1R ntiser hve een rised y other groups. 21 Hence, to void the pitflls inherent in using ntiser with suoptiml sensitivity nd specificity, we used in situ hyridiztion nd RT-PCR nlysis to ssess the expression nd locliztion of Glp1r expression within the kidney. To explore whether the presence or sence of GLP-1R signling hs crucil role in the development nd progression of DN, we disrupted the Glp1r gene in the DN-resistnt mouse model nd investigted its renl phenotype. The mice exhiit less oxidtive nd dietic renl dmge despite hving reltively high renl ctivity of NAD(P)H oxidse, mjor source of superoxide, ecuse superoxide dismutse ntioxidnt 584 Kidney Interntionl (214) 85,

7 H Fujit et l.: GLP-1R signling nd dietic nephropthy sic reserch PAS Fironectin Podocyte (WT1) GBM + SQ H-89 lone c d e Mesngil expnsion scores Fironectin stining intensity (% vehicle) Podocyte numer (/glomerulus) GBM (nm) SQ22536 H Figure 7 Renl histopthology in lirglutide-treted KK/T-Akit mice. Tretment with lirglutide (2 mg/kg per dy) either lone or in comintion with SQ22536 or H-89 strted t 8 weeks of ge nd ended t 12 weeks of ge in mle KK/T-Akit mice. () Representtive glomerulus photomicrogrphs of periodic cid Schiff (PAS) stining, fironectin immunohistochemistry, WT1 stining, nd electron microscopic nlysis in the kidneys removed fter 4-week lirglutide tretment. Brs ¼ 5 mm for PAS, fironectin, nd WT1 imges or 1 mm for glomerulr sement memrne (GBM) imges. () Glomerulr mesngil expnsion scores. (c) Semiquntified fluorescence intensity of glomerulr fironectin. (d) The numer of WT1-positive podocytes in glomeruli. (e) GBM thickness (n ¼ 5 per group; Po.1 vs. vehicle). Renl camp (pmol/mg protein) Renl PKA ctivity (ng/mg protein) SQ22536 H-89 + # + Figure 8 Renl levels of camp nd protein kinse A (PKA) ctivity in lirglutide-treted KK/T-Akit mice. Renl levels of () camp nd () PKA ctivity were determined in 4-week lirglutidetreted mice (n ¼ 5 per group; Po.1 vs. vehicle; w Po.1 vs. SQ22536; # Po.1 vs. H-89). defense system works well in their kidneys. 18 Interestingly, the present dt indicte tht loss of the GLP-1R induces the upregultion of glomerulr NOX4 expression nd further elevtion of renl NAD(P)H oxidse ctivity, resulting in elevtion of glomerulr superoxide nd renl oxidtive stress levels in the mice. These renl ltertions secondry to GLP-1R deficiency contriute to the development of overt DN in the DN-resistnt mice, s evidenced y pronounced mesngil expnsion, podocyte reduction, nd GBM thickening. Therefore, it is conceivle tht loss of GLP-1 ction resulting from GLP-1R deficiency directly reduces the renl ntioxidnt defense cpcity ginst incresed oxidtive stress under hyperglycemic conditions. Furthermore, consistent with the lck of significnt islet phenotype rising in other insulin-resistnt dietic murine models lcking the Glp1r such s the o/o Glp1r mouse, 3 we found tht GLP-1R deficiency does not ffect insulin nd GLP-1 secretion, glucose tolernce, islet topogrphy, or other metolic fctors in the mice. To further elucidte the mechnism underlying renl ltertions oserved in the Glp1r mice, we investigted renl camp-pka, n importnt second Kidney Interntionl (214) 85,

8 sic reserch H Fujit et l.: GLP-1R signling nd dietic nephropthy Superoxide (DHE) e NOX4 No Renl TBARS (nmol/mg protein) + SQ SQ22536 H f Renl NAD(P)H oxidse ctivity (RLU per 1 μg protein) + H SQ22536 H-89 + lone + DHE fluorescence intensity (% vehicle) c NOX4 fluorescence intensity (% vehicle) d N fluorescence intensity (% vehicle) SQ22536 H Figure 9 Renl oxidtive stress, NAD(P)H oxidse, nd nitric oxide (NO) levels in lirglutide-treted KK/T-Akit mice. () Representtive photomicrogrphs of glomerulr dihydroethidium (DHE) stining, NOX4 immunohistochemistry, nd in situ NO detection y diminofluorescein-2 dicette (DAF-2DA) perfusion method in 4-week lirglutide-treted mice. Arrows indicte glomerulus. Brs ¼ 5 mm. Semiquntified fluorescence intensity of glomerulr () DHE stining, (c) NOX4, nd (d) NO were determined s descried in Mterils nd Methods (n ¼ 5 per group; Po.1 vs. vehicle). Renl levels of (e) thiorituric cid rective sustnce (TBARS) nd (f) NAD(P)H oxidse ctivity in 4-week lirglutide-treted mice re shown in lower pnels (n ¼ 5 per group; w Po.5, Po.1 vs. vehicle). RLU, reltive chemiluminescence (light) unit. messenger system downstrem of GLP-1R ctivtion. Our results indicte tht GLP-1R deficiency cuses mrked sl reduction of intrrenl camp formtion nd PKA ctivity in the mice. Given the evidence tht camp nd PKA represent importnt inhiitory fctors for NAD(P)H oxidse, 11,12,31,32 it is conceivle tht Glp1r C57BL/6- Akit mice gretly incresed their renl NAD(P)H oxidse ctivity owing to reduction of camp levels in the setting of chronic hyperglycemi, resulting in renl superoxide overproduction eyond their superoxide dismutse superoxide scvenging cpcity. It is well known tht oxidtive stress upregultes firogenic cytokines such s TGF-1 nd CTGF, leding to mesngil cell prolifertion nd extrcellulr mtrix production. 29,33 Our study indicted tht Glp1r mice hve higher renl expression levels of TGF-1 nd CTGF in ddition to TSP-1, n endogenous ctivtor of TGF1. Hence, incresed ctivity of these firogenic cytokines my contriute to the pronounced mesngil expnsion oserved in the Glp1r mice. Another interesting finding oserved in the kidneys of Glp1r mice is glomerulr NO reduction. Excessive superoxide nion could reduce glomerulr NO levels y scvenging NO from glomerulr endothelil cells. 34,35 NO is n importnt regultor for chrge selectivity in the endothelil cell lyer, nd therefore glomerulr NO reduction could enhnce the permselectivity of plsm lumin through glomerulr cpillry wll. Arcos et l. 36 showed tht chronic NO inhiition impirs glomerulr chrge selectivity rrier nd cuses luminuri in rts. Furthermore, experimentl studies of dietic rts nd mice indicted tht renl NO reduction is ssocited with the development of renl histologic lesions, 37 nd tht NO deficiency y endothelil NO synthse knockout cuses glomerulr endothelil injury nd overt luminuri. 25 Tken together, it is plusile tht glomerulr NO reduction triggered y excessive oxidtive stress contriuted to incresed luminuri in Glp1r mice. Recently, we generted mouse model of progressive DN: KK/T-Akit. In contrst to mice, the KK/T- Akit mice show high susceptiility to DN, s evidenced y the development of severe luminuri nd prominent mesngil expnsion. 18 We reported tht the dietic renl chnges in the KK/T-Akit mice re ttriutle to excessive renl oxidtive stress. 18 In this regrd, the KK/T-Akit mouse represents useful model to ssess whether ctivtion 586 Kidney Interntionl (214) 85,

9 H Fujit et l.: GLP-1R signling nd dietic nephropthy sic reserch of GLP-1R signling suppresses the progression of DN vi meliortion of renl oxidtive stress. The present dt clerly show tht the GLP-1R gonist lirglutide meliortes oxidtive stress y elevting camp nd PKA ctivity levels, downregulting NOX4, nd reducing NAD(P)H oxidse ctivity in the kidneys of KK/T-Akit mice. Furthermore, lirglutide suppresses the progression of dietic renl pthology, s evidenced y renl ltertions such s reduced luminuri, ttenuted mesngil expnsion, enhnced glomerulr NO levels, nd improved glomerulr hyperfiltrtion nd renl hypertrophy, in KK/T-Akit mice. It is noteworthy tht renl improvement ws induced without mjor chnges in insulin secretion, glucose tolernce, nd other metolic fctors. Collectively, these findings support the concept tht GLP-1R signling directly exerts ntioxidtive nd protective effects on the hyperglycemic kidney. Furthermore, the eneficil ctions of lirglutide were inhiited y the denylte cyclse inhiitor SQ22536 nd the selective PKA inhiitor H89, consistent with criticl role for camp nd PKA-dependent pthwys downstrem of GLP-1R ctivtion in renl protection. However, we cnnot rule out the possiility tht other signling pthwys my lso e involved, given the possiility for off-trget inhiition when these nd other inhiitors re used t high concentrtions. Consistent with the gin-of-function studies reported here, GLP-1R gonists such s exendin-4 nd lirglutide ttenuted dietic renl injury, including mesngil expnsion nd luminuri through the protection of glomerulr endothelil cells, 38 reduction of renl oxidtive stress, 39 nd suppression of renl inflmmtory cytokines 4 in high-dose streptozotocin (STZ)-induced dietic rts nd mice. However, there re severl importnt differences in renl outcomes etween our Akit mouse models nd the STZ dietic nimls. Notly, luminuri in the STZ rts is more severe thn tht in KK/T-Akit dietic mice, exceeding 2. mg/24 h. 39,4 Furthermore, STZ is known to induce nonspecific toxicity in multiple orgns including kidney, liver, nd rteries, especilly when it is used t high doses. 41,42 Therefore, it is likely tht these deleterious effects of STZ cused greter kidney dmge nd more severe luminuri in the STZ rts. More importntly, it hs een shown tht STZ stimultes superoxide production 43 nd induces roust oxidtive stress in the kidneys s compred with spontneous dietic mice. 44 In this context, our KK/ T-Akit mouse model tht develops overt DN without chemicls such s STZ or lloxn provides vlule informtion regrding the role of GLP-1R signling in DN rising in the sence of chemiclly induced renl injury. Finlly, our dt support model for GLP-1R-dependent intrrenl signling pthwy, s summrized in Figure 1. The present study provides the first evidence tht loss of GLP-1R signling upregultes renl NAD(P)H oxidse nd increses renl oxidtive stress, with reduced levels of renl camp-pka ctivity in the setting of chronic hyperglycemi ccentuting the progression of DN. Furthermore, the Renl glomerulus nd lood vessels Dietes Oxidtive renl injury GLP1 GLP-1 receptor GLP-1 receptor signling camp NAD(P)H oxidse O 2 H 2 O 2 PKA Ctlse GSH peroxidse H 2 O SOD Figure 1 Proposed glucgon-like peptide-1 receptor dependent signl-trnsduction pthwy in the kidney. Glucgon-like peptide-1 receptors re expressed in the glomerulus nd lood vessels in the kidney. After glucgon-like peptide (GLP-1) inds to its receptor on glomerulr cpillry nd vsculr wlls, glucgon-like peptide-1r signls upregulte the production of mjor second messenger camp nd consequently ctivte protein kinse A (PKA). The upregulted camp nd PKA re expected to contriute to the meliortion of oxidtive renl injury vi inhiition of mjor source of superoxide nion (O 2 ), NAD(P)H oxidse, ctivted under chronic dietic condition. GSH, glutthione; H 2 O 2, hydrogen peroxide; SOD, superoxide dismutse. present study illustrtes tht GLP-1R gonists suppress the progression of DN through ntioxidtive ctions. These dt support the concept of future clinicl studies investigting whether renl outcomes will e similrly modified in sujects with dietic kidney disese. MATERIALS AND METHODS Experimentl nimls nd lirglutide tretment mice re ville from SLC (Hmmtsu, Shizuok, Jpn). The genertion of Glp1r C57BL/6 mice hs een descried previously. 45 Glp1r mice were generted using nd Glp1r C57BL/6 mice. Mle nd mice with or without GLP- 1R deficiency were used for the study. Biochemicl nd physiologicl prmeters, renl histopthology, nd renl oxidtive stress mrkers were exmined t 3 weeks of ge in these mice. KK/T-Akit mice were generted s descried previously. 18 Eight-week-old mle KK/ T-Akit mice were sucutneously injected with the GLP-1R gonist lirglutide (2 mg/kg per dy; Novo Nordisk, Novo Alle, Bgsverd, Denmrk) either lone or in comintion with n denylte cyclse inhiitor SQ22536 (2 mg/kg per dy; Sigm-Aldrich, St Louis, MO) or selective PKA inhiitor H-89 (2 mg/kg per dy; Sigm-Aldrich) dministered y retro-oritl injection under light isoflurne nesthesi for 4 weeks. A prllel group of control ge-mtched mle KK/T-Akit mice ws injected with n equivlent volume of the vehicle (sline). The mice were llowed unrestricted ccess to stndrd rodent chow nd wter. Animl experiments were crried out in ccordnce with the Animl Kidney Interntionl (214) 85,

10 sic reserch H Fujit et l.: GLP-1R signling nd dietic nephropthy Welfre Guidelines of Akit University. All procedures were pproved y the Committee on Animl Experimenttion of Akit University. Blood nd urine prmeters Blood glucose ws mesured on smples otined fter 6-h dytime fst using Glucocrd Dimeter (Arkry, Tokyo, Jpn). Blood ure nitrogen, plsm cretinine, plsm totl cholesterol, nd plsm triglycerides were enzymticlly mesured using n utonlyzer (Fuji Dry-Chem 55; Fuji Film, Tokyo, Jpn). Urinry lumin excretion ws ssessed y the determintion of lumin-tocretinine rtio on morning spot urine, s descried previously. 46 Plsm ctive GLP-1 nd insulin were determined using GLP-1 (ctive) ELISA kit (Shiygi, Gunm, Jpn) nd n insulin ELISA kit (Moring, Yokohm, Jpn), respectively. Physiologicl prmeters Systolic lood pressure ws mesured in conscious trined mice using noninvsive til cuff nd pulse trnsducer system (BP-98A; Softron, Tokyo, Jpn). GFR ws mesured y single-olus fluorescein isothiocynte-inulin injection nd clernce method, s descried previously. 47 In situ hyridiztion The sense nd ntisense rioproes were directed ginst mouse Glp1r (p 18 5; GenBnk NM_21332) nd generted y RT- PCR to mplify 393-p frgment. Detiled protocol for in situ hyridiztion is descried in Supplementry Mteril online. Isoltion of glomeruli nd quntittive RT-PCR Glomeruli nd tuuli were seprted from the renl cortex of 8- week-old mle mice y Dyned perfusion method s reported previously, with some modifictions. 48 To further investigte the locliztion of Glp1r mrna trnscripts in the kidney in ddition to in situ hyridiztion nlysis, we performed RT-PCR nlysis using totl RNA extrcted from glomeruli, tuuli, nd renl rteries. Furthermore, RT-PCR nlysis of glomerulr TGF-1 nd CTGF in ddition to GLP-1R ws performed t 3 weeks of ge in mle nd mice with or without GLP-1R deficiency, s descried previously. 19 Histologic nlysis The kidneys were perfused vi the left ventricle with phosphteuffered sline, followed y 4% prformldehyde in phosphteuffered sline, removed, nd fixed in 4% prformldehyde in phosphte-uffered sline overnight t 4 1C. Two-mm-thick prffin sections were stined with PAS. A semiquntittive score ws used to evlute the degree nd extent of glomerulr mesngil expnsion, s descried previously. 18 Podocyte numer ws evluted y WT1 immunohistochemistry nd clculted using the Weiel Gomez method, s reported previously. 49,5 Five mice per group were nlyzed, nd more thn 6 corticl glomeruli were ssessed in ech mouse. The thickness of GBM ws evluted y electron microscopic exmintion in five mice per group, s descried previously. 18 Immunofluorescence histochemistry, DHE histochemistry, nd in situ NO detection Histochemicl nlysis ws performed s descried previously. 5,18,19,51 Five mice per group were nlyzed, nd more thn 2 corticl glomeruli were ssessed in ech mouse. Detiled protocols for immunofluorescence histochemistry, DHE histochemistry, nd in situ NO detection re descried in Supplementry Mteril online. Mesurement of renl camp nd PKA ctivity levels Kidney lyste ws prepred using phosphte-uffered sline-perfused nd freshly removed renl corticl tissue, s descried previously. 18 Renl camp nd PKA ctivity levels were mesured in these kidney lyste smples using DetectX Direct camp Immunossy kit (Aror Assys, Ann Aror, MI) nd PKA kinse ctivity kit (Enzo Life Sciences, Frmingdle, NY), respectively. The levels were expressed s renl corticl camp nd PKA ctivity to protein rtio. Mesurement of renl TBARS nd NAD(P)H oxidse ctivity Renl TBARS levels were mesured in renl corticl tissue lyste smples using TBARS ssy kit (Cymn Chemicl, Ann Aror, MI). The levels were expressed s renl corticl TBARS to protein rtio. Renl NAD(P)H oxidse ctivity ws mesured in the sme smples using lucigenin-enhnced chemiluminescence ssy, s descried previously. 1 Enzymtic ctivity of NAD(P)H ws expressed in reltive chemiluminescence (light) units (RLU) per 1 mg of protein. Sttisticl nlysis All dt were presented s mens±s.e.m. Sttisticl nlysis of the dt ws performed using the GrphPd Prism softwre (GrphPd, Sn Diego, CA). Differences etween multiple groups were determined y one-wy nlysis of vrince followed y Bonferroni s multiple comprison test. DISCLOSURE DJD receives reserch support for crdiovsculr studies from, nd is consultnt to, Novo Nordisk, the mnufcturer of lirglutide. All the other uthors declred no competing interests. ACKNOWLEDGMENTS This work ws supported y Grnt-in-Aid for Scientific Reserch (no to HF) from the Ministry of Eduction, Science nd Culture of Jpn nd y the Cnd Reserch Chirs Progrm (to DJD). SUPPLEMENTARY MATERIAL The protective roles of GLP-1R signling in dietic nephropthy: possile mechnism nd therpeutic potentil Supplementry mteril is linked to the online version of the pper t REFERENCES 1. Brownlee M. The pthoiology of dietic complictions: unifying mechnism. Dietes 25; 54: Fores JM, Coughln MT, Cooper ME. Oxidtive stress s mjor culprit in kidney disese in dietes. Dietes 28; 57: Fujit H, Skmoto T, Komtsu K et l. Reduction of circulting superoxide dismutse ctivity in type 2 dietic ptients with microluminuri nd its modultion y telmisrtn therpy. Hypertens Res 211; 34: Guzik TJ, Muss S, Gstldi D et l. Mechnisms of incresed vsculr superoxide production in humn dietes mellitus: role of NAD(P)H oxidse nd endothelil nitric oxide synthse. Circultion 22; 15: Stoh M, Fujimoto S, Hrun Y et l. NAD(P)H oxidse nd uncoupled nitric oxide synthse re mjor sources of glomerulr superoxide in rts with experimentl dietic nephropthy. Am J Physiol Renl Physiol 25; 288: F1144 F Kidney Interntionl (214) 85,

11 H Fujit et l.: GLP-1R signling nd dietic nephropthy sic reserch 6. Soccio M, Tonito E, Evngelist V et l. Oxidtive stress nd crdiovsculr risk: the role of vsculr NAD(P)H oxidse nd its genetic vrints. Eur J Clin Invest 25; 35: Wrdle EN. Cellulr oxidtive processes in reltion to renl disese. Am J Nephrol 25; 25: Fujit H, Fujishim H, Morii T et l. Modultion of renl superoxide dismutse y telmisrtn therpy in C57BL/6-Ins2(Akit) dietic mice. Hypertens Res 212; 35: Gorin Y, Block K, Hernndez J et l. Nox4 NAD(P)H oxidse medites hypertrophy nd fironectin expression in the dietic kidney. J Biol Chem 25; 28: Kitd M, Koy D, Sugimoto T et l. Trnsloction of glomerulr p47phox nd p67phox y protein kinse C-et ctivtion is required for oxidtive stress in dietic nephropthy. Dietes 23; 52: Kim JS, Dieold BA, Bior BM et l. Regultion of Nox1 ctivity vi protein kinse A-medited phosphoryltion of NoxA1 nd inding. J Biol Chem 27; 282: Sh S, Li Y, Annd-Srivstv MB. Reduced levels of cyclic AMP contriute to the enhnced oxidtive stress in vsculr smooth muscle cells from spontneously hypertensive rts. Cn J Physiol Phrmcol 28; 86: Bggio LL, Drucker DJ. Biology of incretins: GLP-1 nd GIP. Gstroenterology 27; 132: Holz GG. Epc: new camp-inding protein in support of glucgon-like peptide-1 receptor-medited signl trnsduction in the pncretic etcell. Dietes 24; 53: Leech CA, Chepurny OG, Holz GG. Epc2-dependent rp1 ctivtion nd the control of islet insulin secretion y glucgon-like peptide-1. Vitm Horm 21; 84: Hirt K, Kume S, Arki S et l. Exendin-4 hs n nti-hypertensive effect in slt-sensitive mice model. Biochem Biophys Res Commun 29; 38: Bullock BP, Heller RS, Hener JF. Tissue distriution of messenger rionucleic cid encoding the rt glucgon-like peptide-1 receptor. Endocrinology 1996; 137: Fujit H, Fujishim H, Chid S et l. Reduction of renl superoxide dismutse in progressive dietic nephropthy. J Am Soc Nephrol 29; 2: Fujit H, Fujishim H, Tkhshi K et l. SOD1, ut not SOD3, deficiency ccelertes dietic renl injury in C57BL/6-Ins2(Akit) dietic mice. Metolism 212; 61: Pnjwni N, Mulvihill EE, Longuet C et l. GLP-1 receptor ctivtion indirectly reduces heptic lipid ccumultion ut does not ttenute development of therosclerosis in dietic mle ApoE( ) mice. Endocrinology 213; 154: Pyke C, Knudsen LB. The glucgon-like peptide-1 receptor or not? Endocrinology 213; 154: Vn Vliet A, Belde HJ, Vleming LJ et l. Distriution of fironectin isoforms in humn renl disese. J Pthol 21; 193: Thlls-Bonke V, Thorpe SR, Coughln MT et l. Inhiition of NADPH oxidse prevents dvnced glyction end product-medited dmge in dietic nephropthy through protein kinse C-lph-dependent pthwy. Dietes 28; 57: Zho HJ, Wng S, Cheng H et l. Endothelil nitric oxide synthse deficiency produces ccelerted nephropthy in dietic mice. J Am Soc Nephrol 26; 17: Knetsun Y, Tkhshi K, Ngt M et l. Deficiency of endothelil nitricoxide synthse confers susceptiility to dietic nephropthy in nephropthy-resistnt inred mice. Am J Pthol 27; 17: Wng CH, Li F, Hiller S et l. A modest decrese in endothelil NOS in mice comprle to tht ssocited with humn NOS3 vrints excertes dietic nephropthy. Proc Ntl Acd Sci USA 211; 18: Dniel C, Schu K, Amnn K et l. Thromospondin-1 is n endogenous ctivtor of TGF-et in experimentl dietic nephropthy in vivo. Dietes 27; 56: Reeves WB, Andreoli TE. Trnsforming growth fctor et contriutes to progressive dietic nephropthy. Proc Ntl Acd Sci USA 2; 97: Elmrky AA, Sullivn JC. Reltionship etween oxidtive stress nd inflmmtory cytokines in dietic nephropthy. Crdiovsc Ther 21; 3: Scrocchi LA, Hill ME, Sleh J et l. Elimintion of glucgon-like peptide 1R signling does not modify weight gin nd islet dpttion in mice with comined disruption of leptin nd GLP-1 ction. Dietes 2; 49: Bengis-Grer C, Gruener N. Protein kinse A downregultes the phosphoryltion of p47 phox in humn neutrophils: possile pthwy for inhiition of the respirtory urst. Cell Signl 1996; 8: Svith G, Slimth BP. Cross-tlk etween protein kinse C nd protein kinse A down-regultes the respirtory urst in polymorphonucler leukocytes. Cell Signl 1993; 5: Ohshiro Y, M RC, Ysud Y et l. Reduction of dietes-induced oxidtive stress, firotic cytokine expression, nd renl dysfunction in protein kinse Cet-null mice. Dietes 26; 55: Evns JL, Goldfine ID, Mddux BA et l. Oxidtive stress nd stress-ctivted signling pthwys: unifying hypothesis of type 2 dietes. Endocr Rev 22; 23: Forstermnn U, Munzel T. Endothelil nitric oxide synthse in vsculr disese: from mrvel to mence. Circultion 26; 113: Arcos MI, Fujihr CK, Sesso A et l. Mechnisms of luminuri in the chronic nitric oxide inhiition model. Am J Physiol Renl Physiol 2; 279: F16 F Prhkr S, Strnes J, Shi S et l. Dietic nephropthy is ssocited with oxidtive stress nd decresed renl nitric oxide production. JAmSoc Nephrol 27; 18: Mim A, Hirok-Ymomoto J, Li Q et l. Protective effects of GLP-1 on glomerulr endothelium nd its inhiition y PKCet ctivtion in dietes. Dietes 212; 61: Hendrto H, Inoguchi T, Med Y et l. GLP-1 nlog lirglutide protects ginst oxidtive stress nd luminuri in streptozotocin-induced dietic rts vi protein kinse A-medited inhiition of renl NAD(P)H oxidses. Metolism 212; 61: Koder R, Shikt K, Ktok HU et l. Glucgon-like peptide-1 receptor gonist meliortes renl injury through its nti-inflmmtory ction without lowering lood glucose level in rt model of type 1 dietes. Dietologi 211; 54: Breyer MD, Bottinger E, Brosius FC 3rd et l. Mouse models of dietic nephropthy. J Am Soc Nephrol 25; 16: Ind A, Knmori H, Ari H et l. A model for dietic nephropthy: dvntges of the inducile camp erly repressor trnsgenic mouse over the streptozotocin-induced dietic mouse. J Cell Physiol 28; 215: Szkudelski T. The mechnism of lloxn nd streptozotocin ction in B cells of the rt pncres. Physiol Res 21; 5: Luec B, Hermon M, Hoeger H et l. Aromtic hydroxyltion in niml models of dietes mellitus. FASEB J 1998; 12: Hnsoti T, Bggio LL, Delmeire D et l. Doule incretin receptor knockout (DIRKO) mice revel n essentil role for the enteroinsulr xis in trnsducing the glucoregultory ctions of DPP-IV inhiitors. Dietes 24; 53: Qi Z, Fujit H, Jin J et l. Chrcteriztion of susceptiility of inred mouse strins to dietic nephropthy. Dietes 25; 54: Qi Z, Whitt I, Meht A et l. Seril determintion of glomerulr filtrtion rte in conscious mice using FITC-inulin clernce. Am J Physiol Renl Physiol 24; 286: F59 F Tkemoto M, Asker N, Gerhrdt H et l. A new method for lrge scle isoltion of kidney glomeruli from mice. Am J Pthol 22; 161: Tne K, Lnsp MA, Kitgw W et l. Nicorndil s novel therpy for dvnced dietic nephropthy in the enos-deficient mouse. Am J Physiol Renl Physiol 212; 32: F1151 F Nichols SB, Bsgen JM, Sinh S. Using stereologic techniques for podocyte counting in the mouse: shifting the prdigm. Am J Nephrol 211; 33(Suppl 1): Fujit H, Kkei M, Fujishim H et l. Effect of selective cyclooxygense-2 (COX-2) inhiitor tretment on glucose-stimulted insulin secretion in C57BL/6 mice. Biochem Biophys Res Commun 27; 363: Kidney Interntionl (214) 85,