PKCŒ¥ promotes high glucose induced renal tubular oxidative damage via regulating activation and translocation of p66Shc
Authors: Chengyuan Tang
Lin Sun
Xiaoxuan Xu
Shikun Yang
Yuyan Yang
Jiefu Zhu
Fuyou Liu
Mingming Ma
Li Xiao
Panai Song
Journal: Oxidative Medicine and Cellular Longevity (2014)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Diabetic kidney disease; p66Shc; PKCŒ¥; ROS production; mitochondrial injury
Affiliations: Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410000, China.

Abstract:  Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease (ESRD). Renal tubular injury by overproduction of ROS in mitochondria plays a critical role in the pathogenesis of DKD. Evidences have shown that p66Shc was involved in renal tubular injury via mitochondrial-dependent ROS production pathway, but little is known about the upstream signaling of p66Shc that leads to tubular oxidative damage under high glucose conditions. In this study, an increased PKCŒ¥ and p66Shc activation and ROS production in renal tissues of patients with diabetic nephropathy were seen and further analysis revealed a positive correlation between the tubulointerstitial damage and p-PKCŒ¥, p-p66Shc, and ROS production. In vitro, we investigated the phosphorylation and activation of p66Shc and PKCŒ¥ during treatment of HK-2 cells with high glucose (HG). Results showed that the activation of p66Shc and PKCŒ¥ was increased in a dose- and time-dependent manner, and this effect was suppressed by Rottlerin, a pharmacologic inhibitor of PKCŒ¥. Moreover, PKCŒ¥ siRNA partially blocked HG-induced p66Shc phosphorylation, translocation, and ROS production in HK-2 cells. Taken together, these data suggest that activation of PKCŒ¥ promotes tubular cell injury through regulating p66Shc phosphorylation and mitochondrial translocation in HG ambient. 

Retracted: Yes

PubPeer comments:
 This paper has a 2013 correction for an error in Figure 1. However, there is a potential other concern about Actin blot lanes that appear to be visible in figures 3C, 5A, 5B, and 6B, while not always in the same order or displaying the same experiments. Shown with arrows in two shades of green. Note that the reuse between Figure 3C and 5A is acceptable because these show the same experiments in the same order. This was reported to the journal in October 2015.  , 
 The concern you have was already raised in 2017 and the editors invited us to respond, which we did extensively. We have also provided uncropped scans for all experiments for proof. The answer has remained the same. **Western blot procedures for analysis of 2-Deprenyl-rheediaxanthone B (XB)** Our routine western blot protocols were adapted to the limited amount of compound we had available for this project. When isolating natural compounds from plants, material is limited from the beginning. In addition, substance goes into structure elucidation and then the basic cytotoxicity and apoptosis assays. By the time we were able to address mechanistic questions at the protein level we had sufficient material to do 2 independent experiments for protein isolation and western blot with the original cell line SW480 (20u00b5M XB; 20 and 24 hours of exposure) and keep some in reserve for revisions. With that reserve compound, we later did the experiments using a second cell line (Caco2; 10u00b5M and 20u00b5M XB; 20 hours of exposure). All together, we had 6 lysates for each cell line: control and 2 XB-groups for each SW480 experiment and 2 controls + 2 each of 10u00b5M and 20u00b5M XB for Caco 2. Protein detection and identification was done using peroxidase coupled second antibodies, chemoluminiscence detection and exposure of X-ray films. Compound limitation is the reason, why individual blots were repeatedly re-probed and several panels ended up with identical loading controls. To avoid too much stripping we optimized antibody combinations according to antibody type and behavior as well as antigen molecular weight. When a blot had acquired too many bands, it was stripped, re-numbered and probed again. It is a consequence of this procedure that some of the original blot scans show unrelated bands caused either by unspecific reaction or by antibodies used previously. In that case, the correct bands were identified by molecular weight and the unrelated bands were cropped away. When scanning the original X-ray films, we optimized brightness and contrast (like everybody does). However, we always did that uniformly for the complete panel and we never applied any other alterations. Most of all we never altered individual lanes or the background. If we made mistakes when putting together the panels for the publication, they were honest mistakes caused by the fact that the loading controls were all fairly uniform and similar. They never altered the result in any way. I sincerely hope that this will become obvious, when you examine the original uncropped scans. **As to your specific concerns:** The panels in figures 3 and 5 actually come from the same blot and have the same loading controls. This also includes the caspase 2 panel in figure 5B. Why we presented caspase 2 with only 1 XB lane, I cannot really explain, because the uncropped scan shows 3 perfect lanes with the increased pro-caspase and activated caspase bands for both SW480 experiments. The mistake we made, when we chose the panels, was to crop the caspase different from the u00df-actin, which definitely creates the wrong expression. However, again it did not alter the result. With regard to figure 6: from the uncropped scans, I cannot really decide whether we put the correct (K12) or incorrect loading control (K 6). They do look very similar and they are both clear uniform loading controls. However, I found one mistake that had not been obvious: the cdc25C panel should have gotten a loading control of its own (K7), which again is a uniform loading control. In summary, I want to underline that there was no manipulation of the data that could have altered the results and conclusions. Unfortunately, the uncropped scan files are too big for uploading. I can provide the by e-mail to anybody who wants to see them (brigitte.marian@meduniwien.ac.at) ,