In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Progress has been made in our understanding of the mechanisms of endoplasmic reticulum (ER) proteostasis, ER stress and the unfolded protein response (UPR), as well as ER stress-induced autophagy, in the kidney. Experimental models have revealed that disruption of the UPR, including a protein that senses misfolded proteins (namely, inositol-requiring enzyme 1α) in mouse podocytes causes podocyte injury and albuminuria as mice age. Protein misfolding and ER stress are evident in various renal diseases, including primary glomerulonephritides, glomerulopathies associated with genetic mutations, diabetic nephropathy, acute kidney injury, chronic kidney disease and renal fibrosis. The induction of ER stress may be cytoprotective, or it may be cytotoxic by activating apoptosis. The UPR may interact in a coordinated manner with autophagy to alleviate protein misfolding and its consequences. Monitoring the excretion of ER chaperones into the urine can potentially serve as a biomarker of renal ER stress. In specific kidney diseases, the treatment of experimental animals with chemical chaperones that improve protein folding or with chaperone inducers has alleviated kidney injury. Given the limited availability of mechanism-based therapies for kidney diseases, normalization of ER stress using pharmacological agents represents a promising therapeutic approach towards preventing or arresting the progression of kidney disease.

Endoplasmic reticulum stress, the unfolded protein response and autophagy in kidney diseases

Loss-of-function diseases are often caused by a mutation in a protein traversing the secretory pathway that compromises the normal balance between protein folding, trafficking, and degradation. We demonstrate that the innate cellular protein homeostasis, or proteostasis, capacity can be enhanced to fold mutated enzymes that would otherwise misfold and be degraded, using small molecule proteostasis regulators. Two proteostasis regulators are reported that alter the composition of the proteostasis network in the endoplasmic reticulum through the unfolded protein response, increasing the mutant folded protein concentration that can engage the trafficking machinery, restoring function to two nonhomologous mutant enzymes associated with distinct lysosomal storage diseases. Coapplication of a pharmacologic chaperone and a proteostasis regulator exhibits synergy because of the former's ability to further increase the concentration of trafficking-competent mutant folded enzymes. It may be possible to ameliorate loss-of-function diseases by using proteostasis regulators alone or in combination with a pharmacologic chaperone.

Chemical and biological approaches synergize to ameliorate protein-folding diseases

对于膜性肾病患者,我们首先要根据不同的危险度给予对应治疗[11,31].尿蛋白 8g/24h或肾功能不全患者,应立即接受使用ACEI±ARB,限制蛋白饮食,控制血压≤125/75mmHg,观察时间≤6月,可选择CsA治疗.如果无效,可使用细胞毒加激素治疗.当一种治疗出现副作用时,可以使用CsA、FK506或MMF等替代.对于肾功能不全(Scr>354μmol/L)或肾活检示Ⅳ期、广泛间质纤维化的患者,ACEI类药物和控制血压在125/75 mm Hg仍是首选的治疗.对于持续存在肾综的患者,还应注意高脂血症、高凝倾向及免疫治疗带来副作用。

Membranous nephropathy

Acute Kidney Injury and Progression of Diabetic Kidney Disease.

Inositol-requiring enzyme-1α (IRE1α) is an endoplasmic reticulum (ER)-transmembrane endoribonuclease kinase that plays an essential function in extraembryonic tissues during normal development and is activated during ER stress. To address the functional role of IRE1α in glomerular podocytes, we produced podocyte-specific IRE1α-deletion mice. In male mice, deletion of IRE1α in podocytes resulted in albuminuria beginning at 5 mo of age and worsening with time. Electron microscopy revealed focal podocyte foot-process effacement in 9-mo-old male IRE1α-deletion mice, as well as microvillous transformation of podocyte plasma membranes. Compared with control, glomerular cross-sectional and capillary lumenal areas were greater in deletion mice, and there was relative podocyte depletion. Levels of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II expression and c-Jun N-terminal kinase-1 phosphorylation were decreased in IRE1α-deletion glomeruli, in keeping with reduced autophagy. Deletion of IRE1α exacerbated glomerular injury in anti-glomerular basement membrane nephritis. In cell culture, IRE1α dominant-negative mutants reduced the physiological (basal) accumulation of LC3B-II and the size of autophagic vacuoles but did not affect ER-associated degradation. Thus IRE1α is essential for maintaining podocyte and glomerular integrity as mice age and in glomerulonephritis. The mechanism is related, at least in part, to the maintenance of autophagy in podocytes.

Deletion of Inositol-Requiring Enzyme-1α in Podocytes Disrupts Glomerular Capillary Integrity and Autophagy

https://digitalcommons.wustl.edu/cgi/viewcontent.cgi?article=6516&context=open_access_pubs

Genetic Ablation of Calcium-independent Phospholipase A2γ Induces Glomerular Injury in Mice.

Mutations in α-actinin-4 (actinin-4) result in hereditary focal segmental glomerulosclerosis (FSGS) in humans. Actinin-4 mutants induce podocyte injury because of dysregulation of the cytoskeleton and proteotoxicity. Injury may be associated with endoplasmic reticulum (ER) stress and polyubiquitination of proteins. We assessed if the chemical chaperone 4-phenylbutyrate (4-PBA) can ameliorate the proteotoxicity of an actinin-4 mutant. Actinin-4 K255E, which causes FSGS in humans (K256E in the mouse), showed enhanced ubiquitination, accelerated degradation, aggregate formation, and enhanced association with filamentous (F)-actin in glomerular epithelial cells (GECs). The mutant disrupted ER function and stimulated autophagy. 4-PBA reduced actinin-4 K256E aggregation and its tight association with F-actin. Transgenic mice that express actinin-4 K256E in podocytes develop podocyte injury, proteinuria, and FSGS in association with glomerular ER stress. Treatment of these mice with 4-PBA in the drinking water over a 10-wk period significantly reduced albuminuria and ER stress. Another drug, celastrol, which enhanced expression of ER and cytosolic chaperones in GECs, tended to reduce actinin-4 aggregation but did not decrease the tight association of actinin-4 K256E with F-actin and did not reduce albuminuria in actinin-4 K256E transgenic mice. Thus, chemical chaperones, such as 4-PBA, may represent a novel therapeutic approach to certain hereditary glomerular diseases.

Daniel R. Kaufman

Papers

Deletion of Inositol-Requiring Enzyme-1α in Podocytes Disrupts Glomerular Capillary Integrity and Autophagy

Genetic Ablation of Calcium-independent Phospholipase A2γ Induces Glomerular Injury in Mice.

Proteostasis as a therapeutic target in glomerular injury associated with mutant α-actinin-4.