Harumi Kitamura

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.

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.

Impact of Age and Overt Proteinuria on Outcomes of Stage 3 to 5 Chronic Kidney Disease in a Referred Cohort

BACKGROUND AND OBJECTIVES Population-based studies have reported outcomes and risk factors for patients with chronic kidney disease (CKD), defined primarily by decreased estimated GFR (eGFR). They are characterized by old age, low proteinuria level, and stage 3 CKD. However, many patients referred to nephrologists are younger and have overt proteinuria and advanced CKD. This study evaluated the association between outcomes and those factors among referred CKD patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We retrospectively reviewed 461 referred patients with stage 3 to 5 CKD from January 2003 to December 2007. Key outcomes were death and ESRD. Patients were followed from the time of first serum creatinine measurement to December 2009. RESULTS The median age of subjects was 67.0 years, and median follow-up was 3.2 years. Overt proteinuria was present in 57.0% of subjects. For stage 3, 4, and 5 CKD, cumulative mortality and probability of ESRD at 3 years was 9.5 and 6.5%, 11.2 and 27.8%, and 16.5 and 79.1%, respectively. Using proportional-hazards regression models, age was a determinant for death, whereas overt proteinuria was strongly associated with ESRD. Among stage 3 CKD patients older than 65 years without overt proteinuria, the incidence of death before renal replacement therapy (RRT) was 2.8/100 patient-years and none had ESRD. In patients with advanced CKD and overt proteinuria, the incidence of ESRD was substantially higher than that of death before RRT. CONCLUSIONS Stratification by age, proteinuria level, and CKD stage could predict the competing outcomes of death before RRT and ESRD among CKD patients.

Nonerythropoietic derivative of erythropoietin protects against tubulointerstitial injury in a unilateral ureteral obstruction model

BACKGROUND Erythropoietin (EPO), a member of the cytokine type I superfamily, acts to increase circulating erythrocytes primarily by preventing apoptosis of erythroid progenitors, is known to protect tissues and can raise haemoglobin (Hb) concentrations. Recently, a second receptor for EPO comprising the EPO receptor and beta-common receptor has been reported to mediate EPO-induced tissue protection. EPO modified by carbamylation (CEPO) only signals through this second receptor. Accordingly, we hypothesized that treatment with CEPO, which would not increase Hb concentrations, would protect against tubular damage and thereby inhibit tubulointerstitial injuries. METHODS We evaluated therapeutic effects of CEPO using a rat unilateral ureteral obstruction model. RESULTS CEPO decreased tubular apoptosis and alpha-smooth muscle actin (alphaSMA) expression in the absence of polycythaemia, while the untreated obstructed kidneys exhibited increased tubular apoptosis with expanded (alphaSMA) expression. While EPO treatment similarly inhibited tubular apoptosis and alphaSMA expression, EPO treatment increased Hb concentrations and induced a wedge-shaped infarction. CONCLUSION We established a therapeutic approach using CEPO to protect against tubulointerstitial injury. The therapeutic value of this approach warrants further attention and preclinical studies.

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.

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.

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.

Histological reaction of sintered nanohydroxyapatite-coated cuff and its fibroblast-like cell hybrid for an indwelling catheter.

Rapid tissue-ingrowth of a sintered hydroxyapatite(HAp)-coated and cell-hybrid subcutaneous cuff equipped with an indwelling catheter was developed. The rod-like HAp nanoparticles were coated by covalent bonding on the surface of the silk fibroin (SF) fibers for about 100 microm of the length. The fibers were transplanted three-dimensionally on a cuff substrate made of silicone elastomer with an adhesive. The fibroblast-like cells, explanted and proliferated from skin tissue containing the epidermis, dermis, and subcutaneous tissue of Japanese white rabbits, were incubated on the three-dimensional cuff for three days. Three types of cuff--polyester, HAp-coated, and cell-hybrid cuffs--were percutaneously implanted into the backs of the same animals for 3 and 7 days. The subcutaneous tissues around the cuffs were stained with hematoxylin-eosin. Immunohistochemical staining to identify macrophages and alpha-smooth muscle actin (alpha-SMA) was also done and examined by light microscopy. The alpha-SMA-positive area was very limited in the polyester cuff group even after 7 days, although many macrophages infiltrated into the fibers. In the cell-hybrid cuff group, on the other hand, an alpha-SMA-positive area was formed extensively after 3 and 7 days, causing severe inflammation. In the HAp-coated cuff group, an alpha-SMA-positive area was formed among the fibers with little inflammation. The extent order of the alpha-SMA-positive area was cell-hybrid cuff >> HAp-coated cuff >> polyester cuff, while the degree of inflammatory cells order was cell-hybrid cuff >> polyester cuff >> HAp-coated cuff.

Towards developing new strategies to reduce the adverse side-effects of nonsteroidal anti-inflammatory drugs

The antipyretic and analgesic actions of nonsteroidal anti-inflammatory drugs (NSAIDs) are caused by the inhibition of prostaglandin E2 (PGE2), thromboxane A2 and prostacyclin (PGI2) production. Accumulating evidence suggests that the inhibition of PGE2 production can cause adverse side-effects of NSAIDs on fluid and blood pressure regulation, such as hypertension and edema formation. Since both cyclooxygenase (COX)-1 and COX-2 isoforms contribute to the production of PGE2, selective COX-2 inhibitors are not free of these adverse side-effects although they may be less severe. Four subtypes of PGE2 receptors have been identified. The antipyretic action of blunted PGE2 production is mediated predominantly by a reduced input to the prostaglandin E receptor 3 (EP3) pathway, whereas the analgesic action is mediated predominantly by a reduced input to the EP1 pathway and perhaps by contributions from the other EP receptors. Accordingly, some of the adverse side-effects might be moderated by combined use of NSAIDs with selective EP2 or EP4 agonists that do not block the antipyretic or analgesic actions of NSAIDs that are mediated by reduced activation of EP1 or EP3 receptors. Moreover, EP2 receptor-deficient mice had salt-sensitive hypertension and EP4 receptor blockade moderated salt and water excretion and both EP2 and EP4 agonists had renoprotective effects. This suggests that strategies to maintain activation of EP2 and EP4 receptors during NSAID administration may not only reduce adverse effects but might confer additional benefits. In conclusion, enhancing EP2 and EP4 receptor activity by administration of selective agonists during the administration of NSAIDs has the potential to permit treating fever, inflammation and pain but with marginal adverse effects on fluid or blood pressure regulation.