Part Ⅱ： Tubular Mitochondrial AKT1 Is Activated During Ischemia Reperfusion Injury And Has A Critical Role in Predisposition To Chronic Kidney Disease.
Apr 04, 2023
Augmentation of renal tubule mitochondrial AKT1 protected against AKI and subsequent CKD
Since inhibition of AKT1 in renal epithelial cells exacerbates renal function and animal survival after IRI, we next investigated whether enhanced tubular mitochondrial AKT1 signaling could protect the kidney from IRI. To this end, we generated a two-gene mouse line with the tubular cell-specific expression of tam-induced mitochondrial-targeted constitutively active AKT1 (mcaAKT) (KMCAKT mice) by the same strategy we developed for KMDAKT mice. Western blot results showed that in the mitochondria of TAM-KMCAKT mice, constitutively active AKT1 was expressed specifically in the kidney, whereas it was absent and kidney-specific in control mice. As before, to verify that mutant AKT1 is localized to mitochondria in tubules, immunofluorescence staining showed co-localization of mutant AKT1 and mitochondria. Mutant AKT1 was not detected outside the tubules. to assess mcaAKT activity, we compared AKT1 enzyme activity in mitochondria isolated from TAM and corn oil-treated mice. As expected, renal mitochondrial AKT1 activity was significantly increased in TAM-KMCAKT mice.
To investigate whether enhancement of tubular mitochondrial AKT1 could improve the outcome of IRI in TAM-KMCAKT and corn oil- KMCAKT mice using unilateral IRI while contralateral Nx induced AKI. serum BUN was elevated in corn oil-KMCAKT mice on days 7 and 45 after IRI compared to TAM-KMCAKT mice (p=0.03). Cr was significantly lower in TAM-KMCAKT mice on days 2, 7, and 45 after IRI. These results suggest that enhancement of mitochondrial AKT in the renal tubules attenuated IRI-induced AKI and ameliorated the deterioration of renal function and subsequent development of CKD. Histology of these mice showed that renal injury was more severe in mice injected with corn oil. Compared to TAM KMCAKT kidneys, the tubular injury had higher Jablonski scores, loss of tubular brush border, tubular lysis, and more intra-tubular luminal debris (p=0.038). Masson trichrome staining showed reduced fibrotic area (%) in TAM-KMCAKT mice after IRI (p<0.001). there was less tubular injury and lower KIM-1 expression in TAM-KMCAKT kidneys. TUNEL assay showed increased apoptotic cells in corn oil KMCAKTkidneys after IRI. In TAM-KMCAKT kidneys, tubular apoptosis was reduced by 52% and glomerular apoptosis by 46% (p=0.0064 and p=0.0021). activation of Caspase 3 and 9 was correspondingly inhibited by activation of mitochondrial AKT1 in tubules. The severity of glomerulosclerosis assessed by PAS staining was reduced by 38% (p=0.001) in TAM-KMCAKT mice compared to corn oil KMCAKT mice 45 days after IRI. Thus, enhancement of renal tubular mitochondrial AKT1 protects the kidney from IRI-induced tubular apoptosis, prevents debris accumulation in the tubules, and attenuates glomerular damage after tubular injury.
Kaplan-Meier survival analysis was used to assess whether the aforementioned renal histological and functional alterations could infer survival. Indeed, the survival rate of TAMKMCAKT mice after IRI was much higher than that of corn oil KMCAKT mice (76.9% vs. 20.8%, p<0.001). This suggests that manipulation of tubular mitochondrial AKT1 signaling could be considered a potential target for developing new therapeutic approaches to improve outcomes.
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Mitochondrial AKT1 modulated mitochondria respiration and ATP production in renal tubules
To investigate the direct role of mitochondrial AKT1 in renal tubular epithelial cells, we isolated primary renal tubular epithelial (RTE) cells from KMDAKT mice. Analysis of cell preparations with an antibody to the proximal tubular epithelial cell marker aquaporin 1 (aquaporin1) showed that 97% of RTE cells expressed this marker (by direct cell counting under the microscope). 72 hours after 4-OH TAM or vector treatment, RTE cells were first stained with mitotic tracer red and then immunostained with anti-his-tag antibody (green). There was no His-Tag signal in vector-treated cells, consistent with the expected absence of mdnAKT expression. In contrast, His-Tag has been detected in 4-OH TAM-treated cells and co-localized with Mitotracker Red.
To analyze how mitochondrial AKT1 signaling regulates RTE cell bioenergetics, we used a hippocampal extracellular analyzer to assess mitochondrial function. OCR indicates the measured value of oxidative respiration. The overall basal respiration of KMDAKT RTE cells treated with 4-OH TAM was significantly higher than control (p<0.001). the alternate respiration profile of KMDAKT RTE cells cultured with 4-OH TAM was also higher than the control (p=0.00076). the ATP-dependent respiration profile of KMDAKT RTE cells cultured with 4-OH TAM was significantly higher than the control (p<0.001). higher proton leakage in 4-OH TAM-treated KMDAKT RTE cells indicated uncoupled respiration (p<0.001). reduced ATP production in KMDAKT-derived RTE cells after 4-OH TAM induction. tam-induced cellular lipid peroxidation was significantly higher, indicating elevated ROS. The alterations in oxidative phosphorylation were not caused by changes in mitochondrial content in these cells. However, in TAM-KMDAKT kidneys, we observed higher levels of Drp1, a marker of mitochondrial fission, suggesting that mitochondrial dynamics are regulated. These results suggest that inhibition of mitochondrial AKT1 signaling leads to respiratory uncoupled mitochondrial dysfunction, which, together with reduced ATP production, exacerbates apoptosis in KMDAKT kidneys under IRI.
We also investigated how the activation of mitochondrial AKT1 regulates oxidative phosphorylation in RTE cells isolated from KMCAKT. The results showed the opposite effect of mitochondrial AKT1 compared to KMDAKT RTE cells. Basal respiration, alternate respiration, ATP-dependent respiration, proton leakage, and glycolytic potential were lower in cells cultured with 4OH-TAM. These findings suggest that mitochondrial AKT1 activation increases the efficiency of oxidative phosphorylation and reduces respiratory uncoupling. drp1 analysis showed lower drp1 staining in TAM-KMCAKT kidneys, further confirming higher drp1 in TAM-KMDAKT kidneys and the involvement of mitochondrial AKT1 in the regulation of mitochondrial dynamics.
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Discussion
The present study shows that IRI induces acute activation and translocation of AKT1 in the mitochondria of proximal tubular epithelial cells. Activation of mitochondrial AKT1 in the proximal tubule in response to IRI appears to represent a self-protective mechanism during ischemia-reperfusion. This is because inhibition of tubular mitochondrial AKT1 exacerbates AKI and promotes the subsequent development of CKD after IRI. Conversely, enhanced tubular mitochondrial AKT1 signaling protected the kidney from IRI and attenuated the development of CKD. These data identify a novel role for the tubular mitochondrial AKT1 signaling pathway during the evolution of renal failure after renal ischemia-reperfusion.
The Role of Mitochondria in Renal Tubule AKI
Most of the renal mitochondria are located in the proximal tubules. Mitochondria serve as the major organelle metabolizing nutrient ATP production. Mitochondrial AKT1 regulates cellular oxidative phosphorylation, ROS production, and cell survival As shown in this study, impaired mitochondrial AKT1 signaling produces uncoupled respiration and reduces ATP production. Although renal tubular apoptosis is a common finding in various AKI models, signaling pathways upstream from the mitochondria remain incompletely understood Renal IRI leads to the induction of apoptotic genes and activation of Caspases and endonucleases, which contribute to the induction of apoptotic renal tubular cell injury and death is thought to be a key factor in the development of AKI. In addition, tubular repair and regeneration are thought to be the main events in AKI recovery. Although the sublethal injury may be reversible, tubular cell death leads to the inevitable loss of tubular function In vitro and in vivo models, activation of endowed apoptotic pathways in AKI including the Bcl-2 protein family and mitochondrial apoptotic mechanisms have been characterized. The absence of mitochondrial transmembrane electrochemical gradients is thought to be a key control point for triggering apoptosis Mitochondrial proton leakage, regulated by mitochondrial AKT1, combined with reduced ATP synthesis in this energy-demanding organ may contribute to the overall balance of signaling in favor of apoptosis.
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The Crosstalk between Renal Tubules and Glomerulus
Anatomically, tubular interstitial injury can lead to the narrowing of the glomerular-tubular junction and eventual formation of tubular glomeruli, i.e., dysfunction of glomerular tubular epithelial cells with no obvious connection to the tubules, compression, and obstruction of adjacent tubules by the interstitial matrix, and transformation of mural epithelial cells to fibroblast-like cells are potential mechanisms of tubular glomeruli formation. Incomplete repair of the RTE may occur in the proximal RTE, especially in the S1 region, which is sensitive to ischemic injury in renal injury, leading to tubular atrophy and interstitial fibrosis.
Tubular injury may affect glomerular filtration function through tubular-glomerular feedback. Tubular feedback is a physiological crosstalk mechanism between the tubules and the glomerulus. Little is known about the sequence of events following AKI and the relationship between glomerular filtration rate and tubular changes. In a clinical study, extensive formation of tubular glomeruli in patients with severe renal artery stenosis suggested that chronic ischemia may lead to structural damage and disintegration of renal tubules. On the other hand, there is ample evidence that renal tubular dysfunction can lead to loss of tubular cell polarity, loss of gap junctions, tubular cell death, subsequent tubular obstruction, and retrograde glomerular injury.
Our findings provide new insights into how mitochondria are involved in defense against IRI-induced kidney injury. inhibition of mitochondrial AKT1 during IRI leads to activation of caspases and tubular cell death, which may trigger retrograde glomerular apoptosis, glomerulosclerosis, and renal fibrosis. Activation of mitochondrial AKT1 may play an important role in the transition from AKI to CKD, as inhibition of mitochondrial AKT1 during IRI significantly affects BUN/Cr 45 days after initial IRI.
The transition from AKI to CKD
The exact mechanism of the transition from AKI to CKD is unclear. Histologically, both AKI and CKD are associated with renal tubular injury For many years, it has been known that renal tubular interstitial pathology is a feature of many types of CKD. Disruption of intracellular signaling in the renal tubules is thought to play a role in the initiation and progression of AKI to CKD.
In CKD, the transforming growth factor (TGF)-β signaling pathway is activated and promotes glomerulosclerosis and tubulointerstitial fibrosis by inducing the production of profibrotic extracellular matrix proteins However, the role of TGF-β signaling in AKI is inconsistent across experimental models Our data do not show increased TGF-β expression. Hypoxia activates hypoxia-inducible factor (HIF) to regulate gene transcription in AKI, and overall expression of HIF in various experimental systems exerts a renoprotective effect but other studies have failed to confirm the renoprotective role of HIF-1.
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Current evidence suggests that mitochondria may be key regulators in the transition from AKI to CKD. Ablation of pro-apoptotic BAK and BAX ameliorates ischemic and cisplatin-induced AKI. inhibition of mitochondrial fragmentation has been shown to attenuate the severity of ischemic AKI in diabetic mice in a model of ischemic and nephrotoxic AKI associated with high activation of mitochondrial apoptosis CKD-related inhibition of mitochondrial function and biogenesis sensitizes renal cells and tissues to AKI and prevents recovery of AKI. In CKD, renal mitochondrial dysfunction also occurs during the onset and progression of CKD. In CKD, high glucose and albumin overload induce apoptotic mitochondrial signaling in renal cells. In diabetes-induced experimental CKD, mitochondrial fragmentation is the result of pathological alterations in mitochondrial fusion and fission.
The identification of renal tubular interstitial histopathology in the pathogenesis of CKD shifts the Glomerulus Central paradigm of renal injury to a new focus on the pathophysiological role of the proximal tubule in AKI and its pathogenesis toward CKD,s. Our findings suggest that mitochondrial AKT1 may indirectly prevent the development of Glomerulosclerosis by maintaining the structural and functional integrity of the renal tubules. The data provided in this study help to fill an important gap in knowledge regarding the role of proximal renal tubular mitochondria in AKI and its subsequent transition to CKD by activating and translocating AKT1 into the mitochondria. Future studies should confirm the role of mitochondrial AKT1 in human kidney injury and explore potential targets in the mitochondrial AKT1 signaling pathway that could be used to improve the outcome of renal failure.
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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
7. Department of Diabetes, Endocrinology, and Metabolism, City of Hope National Medical Center, Duarte, California
