Succinylation of Park7 activates a protective metabolic response to acute kidney injury

Abstract

Acute kidney injury (AKI) is extremely prevalent among hospitalizations and presents a significant risk for the development of chronic kidney disease and increased mortality. Ischemia caused by shock, trauma, and transplant are common causes of AKI. To attenuate ischemic AKI therapeutically, we need a better understanding of the physiological and cellular mechanisms underlying damage. Instances of ischemia are most damaging in proximal tubule epithelial cells (PTECs) where hypoxic signaling cascades, and perhaps more rapidly, posttranslational modifications (PTMs), act in concert to change cellular metabolism. Here, we focus on the effects of the understudied PTM, lysine succinylation. We have previously shown a protective effect of protein hypersuccinylation on PTECs after depletion of the desuccinylase sirtuin5. General trends in the results suggested that hypersuccinylation led to upregulation of peroxisomal activity and was protective against kidney injury. Included in the list of changes was the Parkinson's-related deglycase Park7. There is little known about any links between peroxisome activity and Park7. In this study, we show in vitro and in vivo that Park7 has a crucial role in protection from AKI and upregulated peroxisome activity. These data in combination with published results of Park7's protective role in cardiovascular damage and chronic kidney disease lead us to hypothesize that succinylation of Park7 may ameliorate oxidative damage resulting from AKI and prevent disease progression. This novel mechanism provides a potential therapeutic mechanism that can be targeted.NEW & NOTEWORTHY Succinylation is an understudied posttranslational modification that has been shown to increase peroxisomal activity. Furthermore, increased peroxisomal activity has been shown to reduce oxidative stress and protect proximal tubules after acute kidney injury. Analysis of mass spectrometry succinylomic and proteomic data reveals a novel role for Parkinson's related Park7 in mediating Nrf2 antioxidant response after kidney injury. This novel protection pathway provides new insights for kidney injury prevention and development of novel therapeutics.

Keywords: Nrf2; Park7; acute kidney injury; sirtuin5; succinylation.

Graphical abstract

Figure 1.

Bioinformatics of kidneys before and after injury highlight candidates for protective enzyme activity. A: succinylome analysis of wild-type and sirtuin 5 (Sirt5) knockout kidneys via targeted mass spectrometry showing the overlap of proteins that are succinylated after AKI in order to find the protein activity responsible for the protection conferred to Sirt5 knockouts (“protected”). Of the 65 succinylated proteins only in Sirt5 knockout, 73% of the proteins are solely mitochondrially localized, underlining the role of succinylated metabolic proteins as the mechanism for protection (A′). Interestingly, Park7 is one of the cytoplasmically localized proteins without a known fatty acid oxidation role. B: according to the Kidney Precision Medicine Project (atlas.kpmp.org), Park7 is expressed most highly in the proximal tubule epithelium, and the levels are significantly decreased in both acute kidney injury and chronic kidney disease. These data highlight the rationale for further study of Park7 in acute kidney injury, and its relationship with Sirt5-mediated protection. The fold change was calculated by dividing average expression of the gene in the segment of interest by its average expression in all other segments being compared. C: there are four lysine residues that are succinylated in the kidney, K32, K41, K93, and K148. They are shown spatially in reference to the “active” C106 site in the mouse Park7 (Q99LX0), space-filling model with charged colors (blue basic, red acidic, cysteines shown in yellow) obtained from the Biozentrum database (swissmodel.expasy.org). TAL, thick ascending limb; Glom, glomerulus; PT, proximal tubule; INT, interstitium; DCT, distal convoluted tubule; CD, collecting duct; HRT, healthy reference tissue; AKI, acute kidney injury; CKD, chronic kidney disease.

Figure 2.

Acute kidney injury causes localization of Park7 to the nucleus. A: we validated the KPMP data (Fig. 1B) with in vivo staining of a commercially available Park7 antibody. Quantification of immunofluorescence with markers for proximal tubule localization (LTL) or collecting duct localization (DBA) confirmed that Park7 was most highly expressed in the proximal tubules. Both mRNA and protein levels of Park7 were decreased after ischemia-reperfusion injury, but not significantly in our hands. The immunofluorescence after injury showed that in contralateral kidneys, Park7 was localized in the cytoplasm and significantly concentrated in the nucleus after ischemia-reperfusion, quantified in A′. B and C: histological sections of contralateral kidney tissue from adults (B) or post-IRI (C) were stained for Park7 and counterstained with DAPI (N = 6). Images taken at ×20 and ×40 were acquired with a Leica DMi8 system (N = 3 mice, ∼20 ROIs per slide). IRI, ischemia-reperfusion injury; KPMP, Kidney Precision Medicine Project; PTEC, proximal tubule epithelial cell.

Figure 3.

Park7 is protective after AKI and upstream of sirtuin 5 (Sirt5)-mediated protection. A: HK-2 cells were treated with 5 nM Silencer Select Park7-specific siRNA (P7siR) or negative control siRNA (Neg siR) for 48 h. B: MitoSOX SuperOxide indicator was added to the media of HK-2 cells (pretreated with siRNAs as A) for 30 min before imaging and quantifying the signal intensity (n = 2 experimental runs, approximately 100 cells per well). Park7 siRNA and control cells were fixed after in vitro injury models. Combined glucose and oxygen deprivation (CGOD) or 20 μM cisplatin for 24 h was used. TUNEL staining of these cells was quantified and shown in C (control siRNA) and C′ (Park7 siRNA) (n = 2 experiments, two slides per condition). Similarly, immunofluorescence and quantification of the damage marker NGAL were performed on control and Park7 siRNA cells (D and D′). These changes show a trend toward increased damage at baseline or after CGOD when Park7 levels were decreased. However, statistically significant damage was seen in the quantification of peroxisomal activity [D^″, E(control siRNA) and E′ (Park7 siRNA)] (N = 4 coverslips containing approximately 300 cells). Given the link between peroxisomal activity and Sirt5 established previously, we assayed Sirt5 protein levels after Park7 siRNA and saw a significant decrease (F). The opposite was seen in Sirt5 knockout mice (G). Immunofluorescence of Park7 antibody from histological sections of wild-type and Sirt5 global knockout kidneys (both contralateral and ischemia-reperfusion) showed that Sirt5 knockout had higher levels of Park7 protein. AKI, acute kidney injury.

Figure 4.

Increased succinylation correlates with more Park7 activity and Nrf2-mediated protection from AKI. A: quantification of pan-succinyl lysine levels in contralateral and ischemia-reperfused kidneys plus or minus 10% DC-8 supplementation was quantified and shown to be significantly higher in those kidneys pretreated with DC-8. B: the protein levels of Park7, while decreased after IRI, remained not statistically changed in DC-8 treatment, indicating that increased succinylation is protective in part by maintaining Park7 levels after injury. N = 4 experimental runs, at least three samples per condition. C: examining the individual succinylation sites of Park7 with DC8 treatment, we saw a trend toward upregulated succinylation with DC8 after injury; however, the most significant change was in the K93 site. We suggest that the succinylation of this residue changes the charge of the residue, preventing oxidation of cysteine 106 and subsequent apoptosis. D: creating a mask of nuclear or cytoplasmic regions in vivo kidneys plus or minus 10% DC8 showing that increased nuclear Park7 correlated with increased nuclear Nrf2 translocation. This relocalization was dependent on Park7 activity as Park7 siRNA disrupted the Nrf2 localization, seen as a decrease in colocalization of Nrf2 and DAPI. n = 5 samples, three experimental runs. E: a pictographic model of DC8-induced protection from AKI due in part to succinylation of K93 on Park7 levels, increasing peroxisomal FAO, decreasing oxidative stress through Nrf2 signaling into the nucleus. FAO, fatty acid oxidation.