1366 Diabetes Volume 63, April 2014 Li Xiao,1 Xuejing Zhu,1 Shikun Yang,1 Fuyou Liu,1 Zhiguang Zhou,2 Ming Zhan,3,4 Ping Xie,3,4 Dongshan Zhang,1 Jun Li,1 Panai Song,1 Yashpal S. Kanwar,3,4 and Lin Sun1 Rap1 Ameliorates Renal Tubular Injury in Diabetic Nephropathy Rap1b ameliorates high glucose (HG)-induced the endogenous PGC-1a promoter and the mitochondrial dysfunction in tubular cells. interaction between PGC-1a and catalase or However, its role and precise mechanism in diabetic mitochondrial superoxide dismutase, indicating nephropathy (DN) in vivo remain unclear. We that Rap1b ameliorates tubular injury and hypothesize that Rap1 plays a protective role in slows the progression of DN by modulation of tubular damage of DN by modulating primarily the mitochondrial dysfunction via C/EBP-b–PGC-1a mitochondria-derived oxidative stress. The role and signaling. precise mechanisms of Rap1b on mitochondrial Diabetes 2014;63:1366–1380 | DOI: 10.2337/db13-1412 dysfunction and of tubular cells in DN were examined in rats with streptozotocin (STZ)-induced diabetes that have Rap1b gene transfer using an ultrasound Although glomerular injury is believed to initiate kidney microbubble-mediated technique as well as in damage in diabetic nephropathy (DN), recently emerging COMPLICATIONS renal proximal epithelial tubular cell line (HK-2) evidence suggests that tubular injury also plays a key role exposed to HG ambiance. The results showed that in the causation of damage in DN (1). Most of these Rap1b expression decreased significantly in studies have examined the tubular damage in the ad- tubules of renal biopsies from patients with DN. vanced stages of DN, but the mechanism(s) initiating Overexpression of a constitutively active Rap1b tubular injury during this process have not been thor- G12V notably ameliorated renal tubular oughly explored. mitochondrial dysfunction, oxidative stress, and Renal proximal tubule is uniquely susceptible to a va- apoptosis in the kidneys of STZ-induced rats, which riety of metabolic and hemodynamic factors, which is was accompanied with increased expression of related to the events of apoptosis. Interestingly, in- transcription factor C/EBP-b and PGC-1a. creased apoptosis has been observed in the proximal and Furthermore, Rap1b G12V also decreased distal tubular epithelia in patients with diabetes (2), phosphorylation of Drp-1, a key mitochondrial as well as in proximal tubular epithelial cells under fission protein, while boosting the expression of high-glucose (HG) ambience (3). Thus, it is believed that genes related to mitochondrial biogenesis and the events leading to apoptosis in tubular epithelial and antioxidants in HK-2 cells induced by HG. These further progression to tubulointerstitial lesions are effects were imitated by transfection with C/EBP-b among the main features in DN (4). In addition, HG and or PGC-1a short interfering RNA. In addition, angiotensin II could additively aid in the generation of Rap1b could modulate C/EBP-b binding to reactive oxygen species (ROS), which may mediate renal 1Department of Nephrology, Second Xiangya Hospital, Central South University, Received 15 September 2013 and accepted 4 December 2013. Changsha, Hunan, China This article contains Supplementary Data online at http://diabetes 2 Diabetes Center, Institute of Metabolism and Endocrinology, Second Xiangya .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1412/-/DC1. Hospital, Central South University, Changsha, Hunan, China L.X. and X.Z. contributed equally to this work. 3Department of Pathology, Northwestern University, Chicago, IL 4Department of Medicine, Northwestern University, Chicago, IL © 2014 by the American Diabetes Association. See http://creativecommons .org/licenses/by-nc-nd/3.0/ for details. Corresponding author: Lin Sun, [email protected]. diabetes.diabetesjournals.org Xiao and Associates 1367 tubular cell apoptosis (5). In addition, following the by the Institutional Human Experimentation Ethics uptake of glucose metabolic intermediaries via various Committee, Second Xiangya Hospital, Central South glucose transporters, the mitochondrial electron trans- University. port system is overwhelmed in proximal tubular cells, Measurements of Blood Glucose, thus causing intracellular oxidative stress and cell g-Glutamyltranspeptidase, b-N-Acetyl-b-D- damage (6). Indicating that HG itself is an initiating Glucosaminidase, and Urine Albumin Excretion Levels factormaybedirectlyresponsibleforthecausationof Blood glucose was detected by a blood glucose monitor tubular damage and apoptosis in DN. Nonetheless, the (Boehringer Mannheim, Mannheim, Germany). The mechanism by which HG underpins the mitochondrial g g dysfunction and tubular or tubulointerstitial damage is -glutamyltranspeptidase ( -GT) concentrations were measured using a human g-GT GGT ELISA kit unknown. (Biocompare), and urine b-N-acetyl-b-D-glucosaminidase Rap1 is a small GTPase (7) that has been shown to (b-NAG) was measured by automated colorimetric regulate cell adhesion, migration, proliferation, and cell method (Pacific Biomarkers, Inc.). Urine albumin was survival (8). We previously demonstrated decreased ac- measured with a rat urine albumin ELISA kit (Bethyl tivation of Rap1b under HG ambience in vitro and found Laboratories), and urine creatinine levels were tested HG-induced mitochondrial dysfunction was rescued by using the QuantiChrom Creatinine Assay Kit (BioAssay overexpression of Rap1b in tubular cells (9). However, ’ whether Rap1 can dampen the progression of DN in vivo Systems) following the manufacturer sprotocol.Urine albumin excretion (UAE) was normalized with creati- by modulating mitochondrial-derived oxidative stress is nine excretion and expressed per milligram of unclear, and it needs to be investigated along with creatinine. the delineation of the signaling pathways that may be involved. Animal Experimental Design RESEARCH DESIGN AND METHODS A total of 60 adult male Sprague-Dawley rats at 8 weeks of age (body weight 210–230 g) were divided into four Antibodies, Plasmids, and Other Reagents groups of 15 animals each. The first group was injected – Polyclonal anti-Rap1b antibody, polyclonal anti with a normal saline only, which served as a control. The phospho-Drp1 (Ser637) and (Ser656) antibodies, second group of rats received a single dose of strepto- – – monoclonal anti PGC-1a,andanti C/EBP-b were zotocin (STZ; 65 mg/kg i.p.). The third group included from Cell Signaling Technology; human/mouse/rat rats with STZ-induced diabetes but injected with Rap1 cytochrome c monoclonal antibody was from BD V12G using ultrasound microbubble gene transfer Biosciences; procaspase-3 antibody and procaspase-9 technique (12). The fourth group included rats with fi antibody were from Thermo Fisher Scienti c; mono- STZ-induced diabetes but injected with empty vector – clonal anti cleaved caspase-3 (Asp175), rabbit poly- control (STZ + empty vector). All animals were killed at clonal IgG antibodies including anti-mitofusin2 (Mfn2), 8 weeks following STZ administration. The animal ex- – anti-catalase, anti manganese superoxide dismutase perimental protocols as described above were approved – (Mn-SOD), anti nuclear respiratory factor-1 (NRF-1), by the Institutional Animal Experimentation Ethics – anti glutathione peroxidase (GSH-Px), and anti-mtTFA Committee. were from Santa Cruz Biotechnology; and plasmids containing pcDNA/Rap1b G12V and pcDNA/Rap1b Ultrasound-Mediated Gene Transfer of Inducible S17N mutant were generated in our laboratory as pre- Rap1 V12G Gene-Bearing Microbubble Into the Rat viously described (10). Extracellular signal–related ki- Kidneys nase 1/2 (ERK1/2) short interfering RNA (siRNA), PGC- To control Rap1 V12G transgene expression within the 1a siRNA, DFC, MitoRed, and MitoSOX were purchased kidney, a doxycycline-induced Rap1 V12G–expressing from Invitrogen. plasmid was constructed as previously described (10,14). Briefly, pTRE-Rap1b G12V was generated by subcloning Morphological Analysis of Kidney a rat full-length Rap1b G12V cDNA into pTRE (Clontech, Human kidney biopsy tissues were obtained from DN Palo Alto, CA), a tetracycline-inducible vector. An im- (N = 12) of 10–15 years’ duration, and an equal number proved pTet-on vector (Clontech), pEFpurop-Tet-on, was of nondiabetic patients (N = 12) were recruited for the constructed as described previously (15,16). To achieve study. The renal sections were stained with periodic acid doxycycline-inducible Rap1 G12V transgene expression Schiff (PAS) and periodic acid–silver methenamine in the kidney, the ultrasound microbubble-mediated (PASM). Tubulointerstitial lesion index was determined system was applied as previously described (14,15). using a semiquantitative scoring system (11). Tubular Briefly, 24 h following STZ injection, the left rat kidney damage was also scored (12), and the mitochondrial was transfected with a mixture of pTR-Flag-Rap1b and alterations in renal tubules were gauged by electron mi- pEFpurop-Tet-on with Optison (Mallinckrodt, St. croscopy (EM) as previously described (13). The human Louis, MO) in 1:1 volume:volume ratio. The mixture experimental protocols as described above were approved contained 25 mg of each of the plasmids in 0.5 mL 1368 Rap1b in Diabetic Nephropathy Diabetes Volume 63, April 2014 saline, and it was introduced via the left renal artery Mitochondrial Enzyme Activities following a temporary cessation of the renal blood Mn-SOD and CuZn-SOD activity was determined using supply for 3–5 min with a microclamp. Control animals