Jun-ya Kaimori

Autophagy Protects the Proximal Tubule from Degeneration and Acute Ischemic Injury

Autophagy is a bulk protein degradation system that likely plays an important role in normal proximal tubule function and recovery from acute ischemic kidney injury. Using conditional Atg5 gene deletion to eliminate autophagy in the proximal tubule, we determined whether autophagy prevents accumulation of damaged proteins and organelles with aging and ischemic renal injury. Autophagy-deficient cells accumulated deformed mitochondria and cytoplasmic inclusions, leading to cellular hypertrophy and eventual degeneration not observed in wildtype controls. In autophagy-deficient mice, I/R injury increased proximal tubule cell apoptosis with accumulation of p62 and ubiquitin positive cytoplasmic inclusions. Compared with control animals, autophagy-deficient mice exhibited significantly greater elevations in serum urea nitrogen and creatinine. These data suggest that autophagy maintains proximal tubule cell homeostasis and protects against ischemic injury. Enhancing autophagy may provide a novel therapeutic approach to minimize acute kidney injury and slow CKD progression.

Autophagy guards against cisplatin-induced acute kidney injury.

Autophagy is a highly conserved bulk protein degradation pathway involved in cellular homeostasis. Although emerging evidence indicates involvement of autophagy in various conditions, efforts to clarify the role of autophagy in renal tubules are beginning to be elucidated. In the present study, we examined the hypothesis that autophagy guards against acute kidney injury (AKI) by modulating several deteriorative pathways that lead to tubular cell death using a cisplatin-induced model of AKI. Cisplatin treatment of GFP-LC3 (green fluorescent protein-microtubule-associated protein 1 light chain 3) transgenic mice induced autophagy in kidney proximal tubules in a time-dependent manner. Proximal tubule-specific autophagy-deficient mice exhibited more severe cisplatin-induced AKI than did control mice, as assessed via kidney function and morphologic findings. In addition, cisplatin induced more severe DNA damage and p53 activation, concomitant with an increase in apoptotic cell number, and a massive accumulation of protein aggregates in autophagy-deficient proximal tubules. Cisplatin treatment significantly increased reactive oxygen species-producing damaged mitochondria in immortalized autophagy-deficient proximal tubular cells when compared with autophagy-retrieved control cells. In conclusion, autophagy guards kidney proximal tubules against AKI, possibly by alleviating DNA damage and reactive oxygen species production and by eliminating toxic protein aggregates. Enhancing autophagy may provide a novel therapeutic option to minimize AKI.

Activation of the Signal Transducer and Activator of Transcription Signaling Pathway in Renal Proximal Tubular Cells by Albumin

Renal proximal tubular cells activated by reabsorption of protein are thought to play significant roles in the progression of kidney diseases. It was hypothesized that the signal transducer and activator of transcription (STAT) proteins may be activated by proteinuria in proximal tubular cells. To test this hypothesis, murine proximal tubular cells were treated with albumin (30 mg/ml medium) for various lengths of time. The results showed that albumin could activate Stat1 and Stat5 within 15 min in proximal tubular cells. The activation of STATs was mediated mostly by Jak2 and required no protein synthesis. In addition, activation of Stat1 occurred even after neutralization of IFN-gamma. The activation of STATs was inhibited by N-acetyl-L-cysteine, a precursor of glutathione and a reactive oxygen species (ROS) scavenger, and fluorescence-activated cell sorter analysis showed upregulation of intracellular ROS after albumin overloading, suggesting that albumin per se could generate ROS in proximal tubular cells. The activation of STATs occurred by way of the ROS generating system, and especially through the membrane-bound NADPH oxidase system. Reduced activities of glutathione peroxidase and catalase could also be responsible for the accumulation of intracellular ROS. Hence, not only the ROS generating system, but also the ROS scavenging system may contribute to the induction of ROS by albumin. These findings support the hypothesis that proximal tubular cells are activated and generate ROS by reabsorption of abundant urinary proteins filtered through the glomerular capillaries, and as a consequence, various IFN-gamma-inducible proteins are synthesized through IFN-gamma-independent activation of STAT signaling.

Febuxostat suppressed renal ischemia-reperfusion injury via reduced oxidative stress.

Febuxostat is a novel selective inhibitor of xanthine oxidase (XO), approved for treating hyperuricemia. XO inhibits the generation of uric acid (UA) as well as the resulting generation of superoxide. During renal ischemia-reperfusion (I/R) injury, the burst of reactive oxygen species (ROS) can trigger the inflammation and the tubular cell injury. As XO is a critical source of ROS, inhibition of XO could be a therapeutic target for I/R injury. Therefore, we performed this study to test the therapeutic effect of febuxostat on renal I/R injury. Sprague-Dawley rats, received vehicle or febuxostat, were subjected to right nephrectomy and left renal I/R injury. Febuxostat significantly suppressed XO activity, and thereby reduced oxidative stress, assessed by nitrotyrosine, thiobarbituric acid-reactive substances (TBARS) and urine 8-isoprostane. Furthermore, febuxostat reduced the induction of endoplasmic reticulum (ER) stress, assessed by GRP-78, ATF4, and CHOP. Vehicle-treated I/R injured rats exhibited elevated serum creatinine and UN, which were significantly suppressed in febuxostat-treated I/R-injured rats. Histological analysis revealed that fubuxostat-treated rats showed less tubular injury and interstitial fibrosis with reduction in ED1-positive macrophage infiltration, TUNEL positive apoptotic tubular cells, and interstitial smooth muscle α actin (SMαA) expression, compared to vehicle-treated rats. In conclusion; novel XO inhibitor, febuxostat, can protect kidney from renal I/R injury, and may contribute to preserve kidney function.

Autophagy protects kidney proximal tubule epithelial cells from mitochondrial metabolic stress

Chronic metabolic stress is related to diseases, whereas autophagy supplies nutrients by recycling the degradative products. Cyclosporin A (CsA), a frequently used immunosuppressant, induces metabolic stress via effects on mitochondrial respiration, and thereby, its chronic usage is often limited. Here we show that autophagy plays a protective role against CsA-induced metabolic stress in kidney proximal tubule epithelial cells. Autophagy deficiency leads to decreased mitochondrial membrane potential, which coincides with metabolic abnormalities as characterized by decreased levels of amino acids, increased tricarboxylic acid (TCA) ratio (the levels of intermediates of the latter part of the TCA cycle, over levels of intermediates in the earlier part), and decreased products of oxidative phosphorylation (ATP). In addition to the altered profile of amino acids, CsA decreased the hyperpolarization of mitochondria with the disturbance of mitochondrial energy metabolism in autophagy-competent cells, i.e., increased TCA ratio and worsening of the NAD+/NADH ratio, coupled with decreased energy status, which suggests that adaptation to CsA employs autophagy to supply electron donors from amino acids via intermediates of the latter part of the TCA cycle. The TCA ratio of autophagy-deficient cells was further worsened with decreased levels of amino acids in response to CsA, and, as a result, the deficiency of autophagy failed to adapt to the CsA-induced metabolic stress. Deterioration of the TCA ratio further worsened energy status. The CsA-induced metabolic stress also activated regulatory genes of metabolism and apoptotic signals, whose expressions were accelerated in autophagy-deficient cells. These data provide new perspectives on autophagy in conditions of chronic metabolic stress in disease.

Vitamin D Deficiency Predicts Decline in Kidney Allograft Function: A Prospective Cohort Study

CONTEXT Vitamin D, often deficient in kidney transplant (KTx) recipients, has potential immunomodulatory effects. OBJECTIVE This study aimed to evaluate whether vitamin D status affects the rate of decline in kidney allograft function. DESIGN, SETTING, AND PATIENTS The study included a prospective cohort of 264 ambulatory KTx recipients at a single Japanese center. MAIN OUTCOME MEASURES We measured the baseline 25-hydroxyvitamin D (25D) concentration and examined its association with annual decline in estimated glomerular filtration rate (eGFR). Secondary outcome was rescue treatment with iv methylprednisolone (IV-MP) as an index of rejection episodes. RESULTS The mean serum 25D concentration was 17.1 (SD 6.5) ng/mL, and 68.4% patients had vitamin D inadequacy or deficiency. Time after KTx was a significant effect modifier for the association of serum 25D concentration with annual eGFR change and need for IV-MP (P for interaction < .1). We divided patients according to the median time after KTx (10 y) and found that low vitamin D was significantly associated with a rapid eGFR decline at less than 10 years after KTx but not at 10 or more years after KTx. The same was true for rescue treatment with IV-MP. Overall, propensity score matching showed independent associations of low vitamin D with both outcomes. Stratified matching confirmed pronounced associations at less than 10 years after KTx. CONCLUSIONS Vitamin D deficiency predicts a rapid decline in eGFR and need for IV-MP at less than 10 years after KTx. Future studies are warranted to evaluate the clinical efficacy of vitamin D supplementation.

Hydrogen-rich University of Wisconsin solution attenuates renal cold ischemia-reperfusion injury.

Background Renal ischemia-reperfusion (I/R) injury is unavoidable in kidney transplantation and frequently influences both short- and long-term allograft survival rates. One of the major events in I/R injury is the generation of cytotoxic oxygen radicals. Recently, hydrogen gas has been reported to display antioxidant properties and protective effects against organ dysfunction induced by various I/R injuries. We investigated whether hydrogen-rich University of Wisconsin (HRUW) solution attenuates renal cold I/R injury. Methods We prepared HRUW solution by a novel method involving immersion of centrifuge tubes containing UW solution into hydrogen-saturated water. Hydrogen readily permeates through the centrifuge tubes, and thus, the hydrogen concentration of the UW solution gradually increases in a time-dependent manner. Syngeneic rat kidney transplantation was performed, and the animals were divided into three groups: recipients with nonpreserved grafts (control group), recipients with grafts preserved in UW solution for 24 to 48 hr (UW group), and recipients with grafts preserved in HRUW solution for 24 to 48 hr (HRUW group). Results In the early phases, HRUW solution decreased oxidative stress, tubular apoptosis, and interstitial macrophage infiltration in the kidney grafts. Consequently, HRUW solution improved renal function and prolonged recipient survival rate compared with simple cold storage using UW solution. Histopathologically, HRUW treatment alleviated tubular injury and suppressed development of interstitial fibrosis. Conclusions HRUW solution improved graft function and prolonged graft survival compared with simple cold storage using UW solution by protecting tubular epithelial cells from inflammation and apoptosis. Our new method of organ preservation is a groundbreaking, safe, and simple strategy that may be applied in the clinical setting.

Time-dependent dysregulation of autophagy: Implications in aging and mitochondrial homeostasis in the kidney proximal tubule.

ABSTRACT Autophagy plays an essential role in cellular homeostasis through the quality control of proteins and organelles. Although a time-dependent decline in autophagic activity is believed to be involved in the aging process, the issue remains controversial. We previously demonstrated that autophagy maintains proximal tubular cell homeostasis and protects against kidney injury. Here, we extend that study and examine how autophagy is involved in kidney aging. Unexpectedly, the basal autophagic activity was higher in the aged kidney than that in young kidney; short-term cessation of autophagy in tamoxifen-inducible proximal tubule-specific autophagy-deficient mice increased the accumulation of SQSTM1/p62- and ubiquitin-positive aggregates in the aged kidney. By contrast, autophagic flux in response to metabolic stress was blunted with aging, as demonstrated by the observation that transgenic mice expressing a green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3B fusion construct, showed a drastic increase of GFP-positive puncta in response to starvation in young mice compared to a slight increase observed in aged mice. Finally, proximal tubule-specific autophagy-deficient mice at 24 mo of age exhibited a significant deterioration in kidney function and fibrosis concomitant with mitochondrial dysfunction as well as mitochondrial DNA abnormalities and nuclear DNA damage, all of which are hallmark characteristics of cellular senescence. These results suggest that age-dependent high basal autophagy plays a crucial role in counteracting kidney aging through mitochondrial quality control. Furthermore, a reduced capacity for upregulation of autophagic flux in response to metabolic stress may be associated with age-related kidney diseases.

Transplantation of allogenic fetal membrane-derived mesenchymal stem cells protects against ischemia/reperfusion-induced acute kidney injury.

Mesenchymal stem cells (MSCs) are an attractive therapeutic cell source for treating renal diseases. MSC administration has been shown to improve renal function, although the underlying mechanisms are not completely understood. We recently showed that allogenic fetal membrane-derived MSCs (FM-MSCs), which are available noninvasively in large amounts, had a renoprotective effect in an experimental glomerulonephritis model. Here we investigated whether allogenic FM-MSC administration could protect kidneys from ischemia/ reperfusion (I/R) injury. Lewis rats were subjected to right nephrectomy and left renal I/R injury by clamping the left renal artery as an acute kidney injury (AKI) model. After declamping, FM-MSCs (5 × 105 cells) obtained from major histocompatibility complex (MHC)-mismatched ACI rats were intravenously administered. I/R-injured rats exhibited increased serum creatinine and BUN, whereas FM-MSC administration significantly ameliorated renal function. Histological analysis revealed that FM-MSC administration significantly suppressed tubular apoptosis and infiltration of macrophages and T-cells. Administration of FM-MSCs mainly homed into the lung, but increased serum IL-10 levels. Of interest is that renoprotective effects of FM-MSCs were abolished by using anti-IL-10 neutralization antibody, suggesting that IL-10 would be one of the candidate factors to protect rat kidney from I/R injury in this model. We concluded that allogenic FM-MSC transplantation is a potent therapeutic strategy for the treatment of AKI.

Autophagic Clearance of Mitochondria in the Kidney Copes with Metabolic Acidosis

Metabolic acidosis, a common complication of CKD, causes mitochondrial stress by undefined mechanisms. Selective autophagy of impaired mitochondria, called mitophagy, contributes toward maintaining cellular homeostasis in various settings. We hypothesized that mitophagy is involved in proximal tubular cell adaptations to chronic metabolic acidosis. In transgenic mice expressing green fluorescent protein-tagged microtubule-associated protein 1 light chain 3 (GFP-LC3), NH4Cl loading increased the number of GFP puncta exclusively in the proximal tubule. In vitro, culture in acidic medium produced similar results in proximal tubular cell lines stably expressing GFP-LC3 and facilitated the degradation of SQSTM1/p62 in wild-type cells, indicating enhanced autophagic flux. Upon acid loading, proximal tubule-specific autophagy-deficient (Atg5-deficient) mice displayed significantly reduced ammonium production and severe metabolic acidosis compared with wild-type mice. In vitro and in vivo, acid loading caused Atg5-deficient proximal tubular cells to exhibit reduced mitochondrial respiratory chain activity, reduced mitochondrial membrane potential, and fragmented morphology with marked swelling in mitochondria. GFP-LC3-tagged autophagosomes colocalized with ubiquitinated mitochondria in proximal tubular cells cultured in acidic medium, suggesting that metabolic acidosis induces mitophagy. Furthermore, restoration of Atg5-intact nuclei in Atg5-deficient proximal tubular cells increased mitochondrial membrane potential and ammoniagenesis. In conclusion, metabolic acidosis induces autophagy in proximal tubular cells, which is indispensable for maintaining proper mitochondrial functions including ammoniagenesis, and thus for adapted urinary acid excretion. Our results provide a rationale for the beneficial effect of alkali supplementation in CKD, a condition in which autophagy may be reduced, and suggest a new therapeutic option for acidosis by modulating autophagy.