PartⅠ： Tubular Mitochondrial AKT1 Is Activated During Ischemia Reperfusion Injury And Has A Critical Role in Predisposition To Chronic Kidney Disease.
Apr 03, 2023
Abstract
Renal tubular dysfunction can lead to acute kidney injury and transformation into chronic kidney disease. Although tubular mitochondria are associated with the pathophysiology of renal failure, the mechanism is unclear. Here, we demonstrate that ischemia-reperfusion injury induces acute translocation and activation of mitochondrial protein kinase B (also known as AKT1) in the renal tubules. We hypothesized that mitochondrial AKT1 signaling could prevent the development of acute kidney injury and subsequent chronic kidney disease. To test this prediction, we used the Cre-Lox strategy to generate two new renal tubule-specific transgenic mouse strains that induced expression of either mitochondria-targeted dominant-negative AKT1 or constitutively active AKT1. in mitochondria-targeted dominant AKT1-negative mice, inhibition of mitochondrial AKT1 exacerbated azotemia, tubular injury, renal fibrosis, glomerulosclerosis, and survival after ischemia-reperfusion injury negatively. In contrast, in mitochondria-targeted constitutively active AKT1 mice, enhanced tubular mitochondrial AKT1 signaling attenuated renal injury, protected renal function, and significantly improved survival after ischemia-reperfusion injury (76.9% vs. 20.8%, respectively). When mitochondrial AKT1 was inhibited, an increase in uncoupled mitochondrial respiration and oxidative stress was found in renal tubules, supporting the role of mitochondrial dysfunction in the pathophysiology of renal failure. Thus, our study suggests that the tubular mitochondrial AKT1 signaling pathway may be a novel target for the development of new strategies to better prevent and treat kidney injury.
Translational Statement
Acute kidney injury (AKI) and the subsequent development of chronic kidney disease (CKD) are major health problems. We developed a transgenic mouse model and determined the role of AKT activation of proximal renal tubular mitochondria in AKI and CKD. This rescue mechanism of renal protection may be a novel target for developing new strategies to better prevent and treat AKI and CKD. Future studies should confirm the involvement of the mitochondrial AKT signaling pathway in human kidney injury and explore drug targets of the mitochondrial AKT1 pathway.
Keywords
Mitochondrial Akt1; Ischemia Refusion Injury; Acute Kidney Injury; Chronic Kidney Disease; Cistanche extract.
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Introduction
Acute kidney injury (AKI) and the subsequent development of chronic kidney disease (CKD) are major health problems. Currently, specific medical treatments for these disease states are limited, in part due to a lack of understanding of the molecular mechanisms underlying kidney injury. The kidney is one of the most energy-demanding organs in the body, and mitochondria, which are necessary to facilitate active transport, maintain proper electrochemical gradients, and regulate fluid homeostasis, are the primary source of cellular ATP production through oxidative phosphorylation. disturbances in ATP production and mitochondrial dysfunction can lead to increased reactive oxygen species (ROS) production, which in turn impairs renal cell function and induces cell death. Although mitochondrial damage and dysfunction have been reported in patients with kidney disease and animal models of acute and chronic kidney injury, knowledge of the pathophysiological role of mitochondria in kidney injury remains incomplete.
The enhanced mitochondrial function may prevent ischemia-reperfusion injury (IRI). During the ischemic phase of IRI, oxygen depletion inhibits electron transfer in the mitochondrial respiratory chain and reduces ATP production. The subsequent influx of sodium ions and efflux of protons help restore normal pH and may promote cell survival by restoring intracellular volume During reperfusion, restoration of blood flow is associated with increased reactive oxygen species (ROS), Ca2+ inward flow to mitochondria, loss of mitochondrial transmembrane electrochemical gradients, and activation of apoptotic pathways These intracellular changes may be detrimental to cells that survive after initial ischemic injury.
AKI is more common in hospitalized patients and can accelerate the onset of new CKD, exacerbate existing CKD, and lead to renal death The mechanisms underlying the transition from AKI to CKD are unclear. Recent animal studies suggest that renal tubular dysfunction plays an important role in the progression of AKI to CKD, mirroring the transition from AKI to CKD in humans. Understanding the signaling pathways that regulate kidney injury will allow us to identify new therapeutic targets to mitigate kidney injury and prevent the progression of AKI to CKD.
The link between cytoplasmic signaling pathways and mitochondrial function during renal injury is largely unknown. Mitochondrial function is closely linked to cytoplasmic signaling, other organelles, and nuclear pathways Our laboratory has identified the activation and translocation of AKT1 to mitochondria as a key step in protecting cardiomyocytes from IRI injury by regulating oxidative phosphorylation complexes, increasing the efficiency of oxidative phosphorylation and reducing ROS. However, whether IRI can trigger the translocation of activated AKT1 from the cytoplasm to the mitochondria in the renal tubules is unknown. If mitochondrial AKT1 can be activated in renal tubules in response to renal injury, it would be important to assess the pathophysiological role of mitochondrial AKT1 in the development of acute kidney injury (AKI) and chronic kidney disease (CKD). Here, we used two inducible renal tubule-specific transgenic mouse models that modulate mitochondrial AKT1 signaling in vivo and demonstrated the protective role of renal tubular mitochondrial AKT1 activation during IRI against AKI and subsequent CKD development.
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Results
Activation of Mitochondrial AKT1 in Renal Tubules upon IRI
We first investigated whether IRI activates AKT1 in renal mitochondria. To this end, we established a renal IRI model by ligating the left renal artery for 30 min followed by contralateral nephrectomy (Nx). The ligation of the left renal artery was reversed to end the ischemic phase, reperfusion was performed, and the left kidney was harvested at the indicated time interval.
The renal cortex enriched in renal tubules was isolated and mitochondria were prepared by subfractionation. Upon reperfusion, the abundance of mitochondrial pAKT1 and AKT1 was increased, suggesting the translocation of activated AKT1 into the mitochondria (Figure 1A). This is similar to what we observed in the myocardium compared to sham-operated controls or resected kidney controls, where mitochondrial AKT1 was significantly elevated 30-60 minutes after reperfusion (Figure 1A and B) (n= 11-15). Mitochondrial pAKT1 was also increased. These results suggest that IRI-induced activation and translocation of AKT1 to renal mitochondria. iHC staining confirmed phosphorylation of AKT1 in renal tubules after IRI (Figure 1C). Background staining for pAKT1 was minimal in the renal tubules of sham-operated controls, whereas pAKT1 was dramatically elevated in the renal tubules after IRI (Figures 1C and 1D). No pAKT1 was seen in any renal region except the tubules. to confirm the subcellular localization of pAKT1 in the tubules, pAKT1 was stained in combination with mitochondrial markers. significant co-localization of pAKT1 and mitochondria was observed in renal tubular cells at IRI (Figure 1E). Together, these results suggest IRI translocation and activation of AKT1 to mitochondria.
Figure 1. Ischemia reperfusion-induced acute translocation and activation of AKT1 in renal mitochondria.
Generation of transgenic mice with inducible renal tubule-specific expression of a dominant negative AKT in mitochondria
To test the hypothesis that activation of renal tubular mitochondrial AKT1 during IRI protects the kidney from AKI and subsequent CKD development, we generated a transgenic mouse model that enables tamoxifen (TAM)-induced expression of tubular-specific mitochondrial targeting of dominant-negative AKT1. Dominant negative AKT1 (mdnAKT) and mitochondrial targeting sequences were added to the N terminus. Conditional expression of tamoxifen of AKT1 transgenes was mediated using pCALNL (see Materials and Methods). To exclude confounding effects associated with the random integration of transgene arrays within the genome, a single copy of the transgene was introduced by homologous recombination in ES cells with targeted transgenes at the Gt(ROSA) 26or locus on a chromosome. Two-gene mice (KMDAKT) were obtained by crossing tam-induced of the mdnAKT mice with hemizygous KSP-CreERT2 mice, where tam-induced Cre activity was driven by the renal tubular cell-specific calmodulin 16 promoters. tam injection induced Cre activity, isolated mitochondria, and SDS-PAGE separated mitochondrial proteins for immunoblotting. The results showed that TAM successfully induced the expression of mutant AKT1. The mutant protein was undetectable in KMDAKT mice, KSPCreERT2 mice, and wild-type mice (corn oil injection).
To verify kidney-specific expression, mitochondrial proteins were extracted from different tissues after TAM injection. his-tagged mutant AKT1 was expressed only in the kidney. Kidney sections were stained with anti-his-tag antibodies and mitochondrial Mitotracker for immunofluorescence imaging. The results showed that all mutants AKT1 were stained with His-tag antibody and localized to mitochondria. No AKT1 mutations were detected in the glomeruli. The enzymatic activity of mitochondrial AKT1 was analyzed using recombinant GSK3α to confirm a major negative role in the mitochondria of tam-treated KMDAKT tubules. Since renal tubule mitochondrial AKT1 is activated by IRI, we compared the enzymatic activity of AKT1 in mitochondria isolated from tam-treated mice and corn oil-treated mice after IRI. The results showed that the renal mitochondrial AKT1 activity was significantly inhibited in the TAM-treated KMDAKT mice. Therefore, this transgenic mouse line is a good model for studying the role of AKT1 in tubular mitochondria.
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Inhibition of renal tubule mitochondrial AKT aggravated IRI in vivo
IRI was induced in unilateral renal ischemia. In the last series of experiments, phosphorylated AKT1 was barely detectable in the mitochondria of the proximal renal tubules. No significant differences in renal histology, blood urea nitrogen (BUN), and serum creatinine (Cr) were observed in corn oil-injected and tam-injected KMDAKT mice, suggesting that mitochondrial AKT1 signaling has no significant role in renal structure and function at baseline before the induction of IRI. To investigate whether mitochondrial AKT1 plays a protective role during IRI, unilateral IRI was used to induce AKI in corn oil mice or TAM-KMDAKT mice. there were no differences in BUN or Cr levels obtained immediately after IRI induction, but in TAM-KMDAKT mice, both BUN and Cr levels were significantly elevated after 45 days. These results suggest that blocked activation of mitochondrial AKT1 in the renal tubules during IRI exacerbated the late development of renal dysfunction. histological analysis of H&E-stained renal sections showed that renal injury was exacerbated in TAM-KMDAKT mice. Jablonski's score of renal tubular injury was higher with loss of tubular brush border, tubular lysis, and more debris in the tubular lumen (p=0.018). To exclude the effect of TAM or Cre recombinase, we also analyzed TAM-injected KSP/CreERT2 mice, and Jablonski scores were similar in corn oil KMdakt and TAM-KSP/CreERT2 mice at day 7 after IRI (Figure 3D) (p=0.7613). These data suggest that our findings were not confounded by the effects of TAM or Cre recombinase. In TAM-KMDAKT mice after IRI, Masson trichrome staining showed higher fibrotic area (%) (p<0.001), accompanied by disordered tubular structures with intra-luminal casts and debris. col1a expression was increased, consistent with Masson trichrome staining. tGFβ expression was not increased, suggesting that mitochondrial AKT1 did not regulate the inflammatory response. After IRI, the expression of tubular injury marker- KIM -1 was increased in the TAM-KMDAKT group, confirming the increased renal injury in the TAM-KMDAKT group (p=0.002).
TUNEL staining was used to evaluate renal cell apoptosis. The number of apoptotic cells was significantly increased in the tubules (p=0.0054) and glomeruli (p<0.001) of TAM-KMDAKT mice compared to the control group. It is suggested that inhibition of tubular mitochondrial AKT1 during IRI exacerbates tubular cell death and glomerular injury. Consistent with this, activation of both caspase 9 (promoter) and caspase 3 (executor) was significantly increased in TAM-KMDAKT renal tubular epithelial cells. To further explore chronic glomerular changes, periodic acid-Schiff (PAS) staining analysis was performed on kidney sections to quantify glomerulosclerosis. Although no difference in glomerulosclerosis was seen at 7 days post-IRI (p=0.2634), more glomerulosclerosis was found in TAM-KMDAKT mice at 45 days post-IRI (p<0.001). This finding suggests that the renal tubular mitochondrial AKT signaling pathway may regulate glomerular scar formation during CKD development. Kaplan-Meier survival analysis showed the effect of inhibition of tubular mitochondrial AKT on IRI outcome. Survival was reduced after IRI in TAM-KMDAKT mice compared to controls (p=0.0013).
The survival rate of TAM- KMDAKT mice were significantly lower than that of controls within 7 days after IRI, but the BUN/Cr levels of TAM- and corn oil- KMDAKT mice were similar during this period. The reported BUN/Cr changes in TAM-treated mice may have been underestimated due to the unavailability of BUN/Cr data from dead mice. In addition, the higher mortality in TAM-KMDAKT mice may be caused by distal organ dysfunction (lung, heart, brain) rather than just BUN/Cr changes.
The role of tubular mitochondrial AKT1 was also assessed in female KMDAKT mice. serum BUN/Cr and renal histology were relatively higher in TAM-KMDAKT female mice 45 days after IRI. No significant sex effect was observed.
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Hugo Y-H Lin1,2,3,4, Yummy Chen1, Yu-Han Chen1, Albert P. Ta1,2, Hsiao-Chen Lee1,5, Grant R. MacGregor6, Nosratola D. Vaziri1,2, Ping H. Wang1,2,7
1. UC Irvine Diabetes Center and Department of Medicine, University of California, Irvine, California
2. Department of Physiology and Biophysics, the University of California, Irvine, California
3. Department of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
4. Department of Internal Medicine, Kaohsiung Municipal Ta-Tung Hospital, Taiwan
5. Department of Plastic Surgery, Kaohsiung Medical University, Kaohsiung, Taiwan
6. Department of Developmental and Cell Biology, University of California, Irvine, California
7. Department of Diabetes, Endocrinology, and Metabolism, City of Hope National Medical Center, Duarte, California
