Man J. Livingston

Autophagy in Acute Kidney Injury

Acute kidney injury is a major kidney disease associated with poor clinical outcomes. The pathogenesis of acute kidney injury is multifactorial and is characterized by tubular cell injury and death. Recent studies have shown autophagy induction in proximal tubular cells during acute kidney injury. The regulatory mechanisms of tubular cell autophagy are poorly understood; however, some recent findings have set up a foundation for further investigation. Although autophagy may promote cell death under certain experimental conditions, pharmacologic and autophagy-related gene knockout studies have established a renoprotective role for autophagy in acute kidney injury. The mechanisms by which autophagy protects cells from injury and how, possibly, its pro-survival role switches to pro-death under certain conditions are discussed. Further research is expected to help us understand the regulatory network of tubular cell autophagy, define its precise roles in the specific context of acute kidney injury, and identify autophagy-targeting strategies for the prevention and treatment of acute kidney injury.

Reciprocal regulation of cilia and autophagy via the MTOR and proteasome pathways

Primary cilium is an organelle that plays significant roles in a number of cellular functions ranging from cell mechanosensation, proliferation, and differentiation to apoptosis. Autophagy is an evolutionarily conserved cellular function in biology and indispensable for cellular homeostasis. Both cilia and autophagy have been linked to different types of genetic and acquired human diseases. Their interaction has been suggested very recently, but the underlying mechanisms are still not fully understood. We examined autophagy in cells with suppressed cilia and measured cilium length in autophagy-activated or -suppressed cells. It was found that autophagy was repressed in cells with short cilia. Further investigation showed that MTOR activation was enhanced in cilia-suppressed cells and the MTOR inhibitor rapamycin could largely reverse autophagy suppression. In human kidney proximal tubular cells (HK2), autophagy induction was associated with cilium elongation. Conversely, autophagy inhibition by 3-methyladenine (3-MA) and chloroquine (CQ) as well as bafilomycin A1 (Baf) led to short cilia. Cilia were also shorter in cultured atg5-knockout (KO) cells and in atg7-KO kidney proximal tubular cells in mice. MG132, an inhibitor of the proteasome, could significantly restore cilium length in atg5-KO cells, being concomitant with the proteasome activity. Together, the results suggest that cilia and autophagy regulate reciprocally through the MTOR signaling pathway and ubiquitin-proteasome system.

Protein Kinase Cδ Suppresses Autophagy to Induce Kidney Cell Apoptosis in Cisplatin Nephrotoxicity

Nephrotoxicity is a major adverse effect in cisplatin chemotherapy, and renoprotective approaches are unavailable. Recent work unveiled a critical role of protein kinase Cδ (PKCδ) in cisplatin nephrotoxicity and further demonstrated that inhibition of PKCδ not only protects kidneys but enhances the chemotherapeutic effect of cisplatin in tumors; however, the underlying mechanisms remain elusive. Here, we show that cisplatin induced rapid activation of autophagy in cultured kidney tubular cells and in the kidneys of injected mice. Cisplatin also induced the phosphorylation of mammalian target of rapamycin (mTOR), p70S6 kinase downstream of mTOR, and serine/threonine-protein kinase ULK1, a component of the autophagy initiating complex. In vitro, pharmacologic inhibition of mTOR, directly or through inhibition of AKT, enhanced autophagy after cisplatin treatment. Notably, in both cells and kidneys, blockade of PKCδ suppressed the cisplatin-induced phosphorylation of AKT, mTOR, p70S6 kinase, and ULK1 resulting in upregulation of autophagy. Furthermore, constitutively active and inactive forms of PKCδ respectively enhanced and suppressed cisplatin-induced apoptosis in cultured cells. In mechanistic studies, we showed coimmunoprecipitation of PKCδ and AKT from lysates of cisplatin-treated cells and direct phosphorylation of AKT at serine-473 by PKCδin vitro Finally, administration of the PKCδ inhibitor rottlerin with cisplatin protected against cisplatin nephrotoxicity in wild-type mice, but not in renal autophagy-deficient mice. Together, these results reveal a pathway consisting of PKCδ, AKT, mTOR, and ULK1 that inhibits autophagy in cisplatin nephrotoxicity. PKCδ mediates cisplatin nephrotoxicity at least in part by suppressing autophagy, and accordingly, PKCδ inhibition protects kidneys by upregulating autophagy.

Autophagy is activated to protect against endotoxic acute kidney injury.

Endotoxemia in sepsis, characterized by systemic inflammation, is a major cause of acute kidney injury (AKI) in hospitalized patients, especially in intensive care unit; however the underlying pathogenesis is poorly understood. Autophagy is a conserved, cellular catabolic pathway that plays crucial roles in cellular homeostasis including the maintenance of cellular function and viability. The regulation and role of autophagy in septic or endotoxic AKI remains unclear. Here we show that autophagy was induced in kidney tubular cells in mice by the endotoxin lipopolysaccharide (LPS). Pharmacological inhibition of autophagy with chloroquine enhanced LPS-induced AKI. Moreover, specific ablation of autophagy gene 7 (Atg7) from kidney proximal tubules worsened LPS-induced AKI. Together, the results demonstrate convincing evidence of autophagy activation in endotoxic kidney injury and support a renoprotective role of autophagy in kidney tubules.

Emerging Functions of Autophagy in Kidney Transplantation

In response to ischemic, toxic or immunological insults, the more frequent injuries encountered by the kidney, cells must adapt to maintain vital metabolic functions and avoid cell death. Among the adaptive responses activated, autophagy emerges as an important integrator of various extracellular and intracellular triggers (often related to nutrients availability or immunological stimuli), which, as a consequence, may regulate cell viability, and also immune functions, both innate or adaptive. The aim of this review is to make the synthesis of the recent literature on the implications of autophagy in the kidney transplantation field and to discuss the future directions for research.

Histone deacetylase inhibitors protect against cisplatin-induced acute kidney injury by activating autophagy in proximal tubular cells

Histone deacetylase inhibitors (HDACi) have therapeutic effects in models of various renal diseases including acute kidney injury (AKI); however, the underlying mechanism remains unclear. Here we demonstrate that two widely tested HDACi (suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA)) protect the kidneys in cisplatin-induced AKI by enhancing autophagy. In cultured renal proximal tubular cells, SAHA and TSA enhanced autophagy during cisplatin treatment. We further verified the protective effect of TSA against cisplatin-induced apoptosis in these cells. Notably, inhibition of autophagy by chloroquine or by autophagy gene 7 (Atg7) ablation diminished the protective effect of TSA. In mice, TSA increased autophagy in renal proximal tubules and protected against cisplatin-induced AKI. The in vivo effect of TSA was also abolished by chloroquine and by Atg7 knockout specifically from renal proximal tubules. Mechanistically, TSA stimulated AMPK and inactivated mTOR during cisplatin treatment of proximal tubule cells and kidneys in mice. Together, these results suggest that HDACi may protect kidneys by activating autophagy in proximal tubular cells.

Lithium in Kidney Diseases: Big Roles for the Smallest Metal

Lithium, the lightest metal, with an atomic number of 3, was formally introduced into medicine in 1949, when John Cade discovered the therapeutic effects of lithium carbonate on bipolar disorder. Since then, lithium has become one of the most effective and widely prescribed drugs for mood stabilization in the treatment of psychiatric disorders. 1 Recent studies in both experimental and clinical settings have further revealed the potential of lithium as a neurotrophic and neuroprotective agent for the treatment of acute brain injury (e.g., stroke or ischemia), as well as chronic neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. Multiple signaling pathways and molecular and cellular targets have been identified to account for lithium’s neurologic effects.2 In addition, lithium has been shown to modulate several aspects of hematopoiesis, such as enhancing the production and release of G-CSF, improving the quantity and quality of neutrophil, increasing monocyte differentiation and activity, and assisting in hematopoietic stem cell mobilization in bone marrow transplantation. 3 The clearance of lithium depends exclusively on renal excretion, and, as people age, the ability of lithium clearance decreases. Interestingly, after glomerular filtration, 80% of the filteredlithiumisreabsorbedviarenaltubules.Becauseof its unique interactions with kidney cells, lithium has been an important and versatile tool for understanding renal physiology and pathophysiology. 4 In medicine, despite its usefulness in psychiatric and neurologic disorders, lithium is knowntohaveadverse effects inkidneys, which, notsurprisingly, depend on the dose and duration of use, patient age and health status, and the presence of concurrent medications. 5 The notable adverse effects of lithium include acute lithium toxicity, CKD, and nephrogenic diabetes insipidus (NDI), which are especially common in persons with longterm use and in elderly patients.5 In stark contrast, if used briefly and at low doses, lithium can be renoprotective. In this regard, recent studies have shown the protective effects of lithium in experimental models of AKI induced by renal ischemia-reperfusion, 6,7 nephrotoxin, 8 and endotoxin. 9 In this issue of JASN ,B aoet al. report that a single dose of lithium given after AKI promotes the recovery and repair of kidneys, whereas de Groot and colleagues show that lithium induces G2 cell cycle arrest in the principal cells of the collecting ducts, which may contribute to the development of NDI. 10,11

Autophagy is activated to protect against podocyte injury in adriamycin-induced nephropathy

Podocytes are highly differentiated epithelial cells wrapping glomerular capillaries to form the filtration barrier in kidneys. As such, podocyte injury or dysfunction is a critical pathogenic event in glomerular disease. Autophagy plays an important role in the maintenance of the homeostasis and function of podocytes. However, it is less clear whether and how autophagy contributes to podocyte injury in glomerular disease. Here, we have examined the role of autophagy in adriamycin-induced nephropathy, a classic model of glomerular disease. We show that autophagy was induced by adriamycin in cultured podocytes in vitro and in podocytes in mice. In cultured podocytes, activation of autophagy with rapamycin led to the suppression of adriamycin-induced apoptosis, whereas inhibition of autophagy with chloroquine enhanced podocyte apoptosis during adriamycin treatment. To determine the role of autophagy in vivo, we established an inducible podocyte-specific autophagy-related gene 7 knockout mouse model (Podo-Atg7-KO). Compared with wild-type littermates, Podo-Atg7-KO mice showed higher levels of podocyte injury, glomerulopathy, and proteinuria during adriamycin treatment. Together, these observations support an important role of autophagy in protecting podocytes under the pathological conditions of glomerular disease, suggesting the therapeutic potential of autophagy induction.

Autophagy in Podocytes

Background: Podocyte injury and loss, caused by a variety of insults, is a common and determining factor for the progression of glomerular diseases. Being postmitotic and highly differentiated, podocytes have no or a very limited ability to proliferate or regenerate. Thus, understanding the mechanisms that help maintain and defend podocytes is of great importance. Autophagy is a cellular process by which cytoplasmic cargos are sequestered within autophagosomes and then delivered to lysosomes for degradation and turnover. Recent research has highlighted an important role of autophagy in the maintenance of cellular homeostasis under physiological and pathological conditions. Summary: A high basal level of autophagy is a characteristic hallmark of differentiated podocytes, which may contribute critically to the structural and functional integrity of the cells. Autophagy is also essential for long-term podocyte homeostasis in aging, protecting podocytes against cellular degeneration and age-related kidney diseases. In glomerular diseases, alteration of autophagic activity may be an adaptive and protective mechanism against podocyte injury and disease progression. Recent studies have further suggested an intriguing and unique interplay between mTOR signaling and autophagy in podocytes. Key Messages: Altogether, these findings unveil an emerging role of autophagy in the physiology and pathology of podocytes. Further work should elucidate the regulatory mechanism of autophagy in podocytes, determine its pathophysiological role in various kidney diseases, and test autophagy-targeting therapies.

microRNA-668 represses MTP18 to preserve mitochondrial dynamics in ischemic acute kidney injury

The pathogenesis of ischemic diseases remains unclear. Here we demonstrate the induction of microRNA-668 (miR-668) in ischemic acute kidney injury (AKI) in human patients, mice, and renal tubular cells. The induction was HIF-1 dependent, as HIF-1 deficiency in cells and kidney proximal tubules attenuated miR-668 expression. We further identified a functional HIF-1 binding site in the miR-668 gene promoter. Anti–miR-668 increased apoptosis in renal tubular cells and enhanced ischemic AKI in mice, whereas miR-668 mimic was protective. Mechanistically, anti–miR-668 induced mitochondrial fragmentation, whereas miR-668 blocked mitochondrial fragmentation during hypoxia. We analyzed miR-668 target genes through immunoprecipitation of microRNA-induced silencing complexes followed by RNA deep sequencing and identified 124 protein-coding genes as likely targets of miR-668. Among these genes, only mitochondrial protein 18 kDa (MTP18) has been implicated in mitochondrial dynamics. In renal cells and mouse kidneys, miR-668 mimic suppressed MTP18, whereas anti–miR-668 increased MTP18 expression. Luciferase microRNA target reporter assay further verified MTP18 as a direct target of miR-668. In renal tubular cells, knockdown of MTP18 suppressed mitochondrial fragmentation and apoptosis. Together, the results suggest that miR-668 is induced via HIF-1 in ischemic AKI and that, upon induction, miR-668 represses MTP18 to preserve mitochondrial dynamics for renal tubular cell survival and kidney protection.