Grape seed proanthocyanidins ameliorates cadmium-induced renal injury and oxidative stress in experimental rats through the up-regulation of nuclear related factor 2 and antioxidant responsive elements
Authors: Vaihundam Manoharan
Bashir Nazima
Selvaraj Miltonprabu
Journal: Biochemistry and Cell Biology (2015)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: ERO; Nrf2; ROS; apoptose; apoptosis
Affiliations: Department of Zoology, Faculty of Science, Annamalai University, Annamalainagar 608002, Tamilnadu, India.

Abstract:  Cadmium (Cd) preferentially accumulates in the kidney, the major target for Cd-related toxicity. Cd-induced reactive oxygen species (ROS) have been considered crucial mediators for renal injury. The biologically significant ionic form of cadmium (Cd(+)) binds to many bio-molecules, and these interactions underlie the toxicity mechanisms of Cd. The present study was hypothesized to explore the protective effect of grape seed proanthocyanidins (GSP) on Cd-induced renal toxicity and to elucidate the potential mechanism. Male Wistar rats were treated with Cd as cadmium chloride (CdCl2, 5 mg·kg(-1) bw, orally) and orally pre-administered with GSP (100 mg·kg(-1) bw) 90 min before Cd intoxication for 4 weeks to evaluate renal damage of Cd and antioxidant potential of GSP. Serum renal function parameters (blood urea nitrogen and creatinine) levels in serum and urine, renal oxidative stress (lipid peroxidation, protein carbonylation, enzymatic, and non-enzymatic antioxidants), inflammatory (NF-κB p65, NO, TNF-α, IL-6), apoptotic (caspase-3, caspase-9, Bax, Bcl-2), membrane bound ATPases, and Nrf2 (HO-1, keap1, γ-GCS, and μ-GST) markers were evaluated in Cd-treated rats. Pretreatment with GSP revealed a significant improvement in renal oxidative stress markers in kidneys of Cd-treated rats. In addition, GSP treatment decreases the amount of iNOS, NF-κB, TNF-α, caspase-3, and Bax and increases the levels Bcl-2 protein expression. Similarly, mRNA and protein analyses substantiated that GSP treatment notably normalizes the renal expression of Nrf2/Keap1 and its downstream regulatory proteins in the Cd-treated rats. Histopathological and ultra-structural observations also demonstrated that GSP effectively protects the kidney from Cd-induced oxidative damage. These findings suggest that GSP ameliorates renal dysfunction and oxidative stress through the activation of Nrf2 pathway in Cd-intoxicated rats. 

Retracted: Yes

PubPeer comments:
 Concern about Figure 8. In panel D, the same area appears to be visible twice (shown with red boxes). Could the authors please explain what happened here? ![file] , 
 Concern about Figure 10. Several panels appear to show repetitive elements. ![file] , 
 Figure 5A, 6A. ![file] Unexpected resemblances between bands. , 
 #2 Additional glitches in Fig 10 (Control). ![file] , 
 why should I give you the raw data , 
 Dear Dr. Nazima, serious concerns have been raised about figures in this paper. If you are not willing to share raw data - or at least provide some explanation about these unexpected repetitive features in the photos, you might be perceived as wanting to hide something. Could you please try again? , 
 #5 I don t think anyone asked for raw data, only for an explanation of various anomalies. #2  Concern about Figure 10. Several panels appear to show repetitive elements. I have re-annotated the GSP panel, out of concern that the original comment doesn t really show the size of the repeated section. ![file] , 
 #7 You might look in the upper left corner as well. Roughly indicated in green. *** ![file] , 
 Dear Pub peer collogues, Greetings. After a series of comments about our papers, especially about the histology and histopathology and immunohistochemistry slides pictures and this time is about westernblot figures. I want to clarify one thing, in the figures that you have shown repeated elements were not created by us and it is naturally found in our histological slides. And there is no need to modify and correct the figures in control and drug positive (grape seed proanthocyanidins, GSP) Eve if you can found the changes alterations in the treated slides were created by the toxicant (Cadmium, Cd) and the recovery has to be tested with our selected antioxidant phytochelating (GSP) substance. The alterations that are found in the tissues are produced by the metals through the process of oxidative damage via liberating more reactive oxygen species not by us. Repetitions that you have portrayed in the figures were not created by us and there was no need to manipulate in this particular panel of figures. We are still having all the slides. If you need specifically, Send your address, i will send my slides used for taking the photograph. And this is the supporting evidence along with the other evidences even without these figures; we could prove the positive effect of our selected phytochelator drugs with our biochemical results. Regarding immunohistological studies, the tissues were fixed and dispatched to the out sourcing lab for preparation of the slides using immunohistochemical staining and a pathologist would take the photograph from different groups and send their results with comments along the slides. Based on the intensity of staining, we semi quantitatively measure and portrayed in the graph table. Here also, I could mention or assure that, there is no need for manipulation, because it is a supporting data. Even with this our other biochemical findings could support our hypothesis. Regarding the western bloting figures, even by looking in to the band intensity of gst and bax, it showed a mild difference, because gst (26kda) bax (24kda) values and the difference in band intensity has also been calculated and portrayed along with the graphical representation calculated by the softwares. B-actin (42kda) is a control housekeeping gene expressed protein and it could be used as a control element in all the apoptotic, inflammatory and antioxidant proteins expressions, so it could not be altered in all the bloting panels. And we are routinely working on the effects of phytoantioxidants against metal toxicities through the upregulation of Nrf2 and its related protein expressions. We are having the raw data and band intensities of the reported proteins. So here in this portion also, there is no need for us to manipulate. I would give some of the published papers with these criteria, you can check it down with your so called figure plagiarism software. And i have reviewed more than 300 papers peer reviewed paper from around every publishing house and i have also evaluated the originality and correctness of every article to be published in the journals. By just going through the manuscript i could identify the originality of the articles even we are not having such sophisticated softwares and microscopes. And for me there is no need for manipulating the figure for my articles. The photos have to be received by me, based on the figures and reports, i have written and compiled my findings along with the published supporting evidences. And every journal system is having the iThenticate Urkund softwares and other similar softwares for finding plagiarism and duplication manipulation of materials figures in the submitted manuscript. After these peer review process only our papers are accepted by the reputed journals. Do you want to disparage disapprove all the systems, softwares and reviewers were inefficient and useless? This is ridiculous. If you really asking for unintentional doubts, in the subject of pathology i am ready to respond. But your criticisms are so intensional and only targeting the Annamalai authoru2019s alones. Even without any proper sophisticated instruments, with limited funding opportunities, we have done lot of very good works based on the out sourcing process only for histopathology, immunohistochemistry and western bloting. We did not have the necessity to modify or manipulate our figures only. Based on the results we got we had written and submit our reports to the publications and they have also done a very long screening and reviewing process with a wide array of experts in their own areas of expertise. And above all these things you have reported to be done before the publication of an article. As well, it is the supportive evidence only along with the other series of evidences to prove the efficacy of this particular phytochelator (GSP) against Cadmium induced oxidative damage in the kiney of rats. Even without this part, we could prove the effect of this drug. Thank you with warm regards on behalf of all authors Dr.S.Miltonprabu , 
 Dear Dr. Miltonprabu, Thank you for your long reply. * You write that there was no need to manipulate, but that does not take away our concerns about the duplicated features that both Hoya and I see. * You write that these duplications were not caught during peer review, but peer review is not a clean bill of health. Later flaws or concerns are always allowed to be discussed after publication in a process called Post Publication Peer Review, which is what we are doing now. * You write that the images were actually not made by you, but outsourced. That appeared to not have been disclosed in the paper. More importantly, that does not remove your responsibility for these images. You and your coauthors accepted full responsibility for all the data presented in your paper. Given the outsourcing of the images in which we see duplications, could you please disclose which of the figures in your paper were generated in your lab, and which ones were outsourced? Also, we would very much appreciate it if you could post the raw photos here. You can just draw a png or jpg from your computer into PubPeer, as long as the files are 1Mb. If larger, you can host them on a site like Imgur and embed the link here. , 
 #3 Figures 5 and 6 again:  Fig.5. Effect of GSP on western blotting renal apoptotic markers Bax and Bcl2 in **kidney** of control and Cd-treated rats. Values are expressed as mean u00b1 SD for groups of six rats in each. Fig.6. Effect of GSP on renal western blot protein Nrf2, HO-1, u03b3-GCS, u00b5-GST and Keap-1 in **kidney** of control and experimental rats. Values are expressed as mean u00b1 SD for groups of six rats in each. ![file] Please compare with Fig 9 A from [Nazimabashir et al. (2015)] :  Fig. 9. Effect of GSP on renal western blot protein Nrf2, HO-1, and Keap-1 in **heart** of control and experimental rats (9A). Lane 1 u00bc Control, Lane 2 u00bc Control u00b1 GSP (100 mg kg bw), Lane 3 u00bc Cd u00b1 control (5 mg kg bw) Lane 4 u00bc GSP (100 mg kg bw) u00b1 Cd (5 mg kg bw). ![file] Could the authors check whether the kidneys or the hearts of the experimental animals were the sources of the proteins? , 
 *[Left]* Fig 4(A).  Effect of GSP on western blotting of Cytochrome C, Caspase 9 and Caspase 3 on control and experimental rats in **kidney**. Values are expressed as mean u00b1 SD for groups of six rats in each.  *[Right]* Fig. 8.  Effect of GSP on... cytochrome C (8B)... in the **heart tissue** of control and Cd treated rats by western blot. Lane 1. Control.; Lane 2. Control u00b1 GSP (100 mg kg bw), Lane 3. Cd u00b1 control (5 mg kg bw) Lane 4. GSP (100 mg kg bw) u00b1 Cd (5 mg kg bw). ... Values are expressed as mean u00b1 SD for groups of six rats in each . From [Nazimabashir et al. (2015)] : ![file] Cytochrome-C and beta-actin bands appear to be the same. Could the authors check whether the kidneys or the hearts of the experimental animals were the sources of the proteins? , 
 Fig 11:  Electron micrographs of kidney in control and experimental rats. (11 A-B); In control group of rats, Part of distal convoluted tubule: spherical apical Nucleus (N), Nuclear envelope , Nuclear pores, Mitochondria, Golgi bodies , few short Microvilli (Mv), mesangial cell (Mc), with large nucleus surrounded by little amount of electron-dense cytoplasm, is seen between the capillary loops, podocyte (PC) of the inner capsular membrane having an irregular nucleus (N) and numerous secondary processes on the basement membrane (BM) of the glomerular capillaries are also seen. (11 C-D); GSP -treated rats there are partial fusion of the secondary foot processes of the podocytes (PC) on the basement membrane (BM) of the glomerular capillary (Ca) and dilation and congestion between the glomerular capillary, Microvilli, apical vesicles, tight Junctions, oval Nuclei (N), are more organized with dense Mitochondria (M) and Golgi bodies. (11 E-F) many mesangial cells (Mc) containing vacuoles are observed in the glomerulus of Cd rats, absence of microvilli, increased tight junctions, severe cellular blabbing with irregular Nuclear outlines (N) of different sizes, pleomorphic Mitochondria (M) with swelling profiles. (11 G-H) dilation capillary loops is seen within the glomerulus in combined GSP and Cd-treated rats group, Microvilli (Mv), tight Junctions (J), euchromatic Nucleus (N),numerous well oriented dense Mitochondria (M), RER, Golgi bodies, ribosomes. In addition, the glomerular capillary is lined by discontinuous endothelial cells and contains blood cells within its wide lumen. ![file] Close inspection reveals small repeated sections within each panel. 11B: ![file] 11C: ![file] , 
 Hoya s analysis of panels in Figure 11 made me see even more possible duplications: Figure 11A: ![file] Figure 11B: ![file] Figure 11C: ![file] Figure 11D: ![file] Other panels in this figure might show similar glitches. , 
 #14 ![file] , 
 #14 11G: ![file] , 
 Retracted [20 March 2020] .  The Editors-in-Chief of Biochemistry and Cell Biology were alerted to cases of image manipulation in the article entitled u201cGrape seed proanthocyanidins ameliorates cadmium-induced renal injury and oxidative stress in experimental rats through the up-regulation of nuclear related factor 2 and antioxidant responsive elements,u201d which we published in 2015 (see https: www.nrcresearchpress.com doi pdf 10.1139 bcb-2014-0114). Thus, the Editors-in-Chief have retracted the paper from *Biochemistry and Cell Biology*. This decision is in compliance with the publishing policy of *Biochemistry and Cell Biology* and its publisher, Canadian Science Publishing. ,