Podocytes maintain high basal levels of autophagy independent of mTOR signaling

While constant basal levels of macroautophagy/autophagy are a prerequisite to preserve long-lived podocytes at the filtration barrier, MTOR regulates at the same time podocyte size and compensatory hypertrophy. Since MTOR is known to generally suppress autophagy, the apparently independent regulation of these two key pathways of glomerular maintenance remained puzzling. We now report that long-term genetic manipulation of MTOR activity does in fact not influence high basal levels of autophagy in podocytes either in vitro or in vivo. Instead we present data showing that autophagy in podocytes is mainly controlled by AMP-activated protein kinase (AMPK) and ULK1 (unc-51 like kinase 1). Pharmacological inhibition of MTOR further shows that the uncoupling of MTOR activity and autophagy is time dependent. Together, our data reveal a novel and unexpected cell-specific mechanism, which permits concurrent MTOR activity as well as high basal autophagy rates in podocytes. Thus, these data indicate manipulation of the AMPK-ULK1 axis rather than inhibition of MTOR as a promising therapeutic intervention to enhance autophagy and preserve podocyte homeostasis in glomerular diseases

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9–mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.publishedVersio

BECLIN1 Is Essential for Podocyte Secretory Pathways Mediating VEGF Secretion and Podocyte-Endothelial Crosstalk

Vascular endothelial growth factor A (VEGFA) secretion from podocytes is crucial for maintaining endothelial integrity within the glomerular filtration barrier. However, until now, the molecular mechanisms underlying podocyte secretory function remained unclear. Through podocyte-specific deletion of BECLIN1 (ATG6 or Becn1), a key protein in autophagy initiation, we identified a major role for this molecule in anterograde Golgi trafficking. The Becn1-deficient podocytes displayed aberrant vesicle formation in the trans-Golgi network (TGN), leading to dramatic vesicle accumulation and complex disrupted patterns of intracellular vesicle trafficking and membrane dynamics. Phenotypically, podocyte-specific deletion of Becn1 resulted in early-onset glomerulosclerosis, which rapidly progressed and dramatically reduced mouse life span. Further, in vivo and in vitro studies clearly showed that VEGFA secretion, and thereby endothelial integrity, greatly depended on BECLIN1 availability and function. Being the first to demonstrate the importance of a secretory pathway for podocyte integrity and function, we identified BECLIN1 as a key component in this complex cellular process. Functionally, by promoting VEGFA secretion, a specific secretory pathway emerged as an essential component for the podocyte-endothelial crosstalk that maintains the glomerular filtration barrier

Cisplatin Nephrotoxicity Is Critically Mediated by the Availability of BECLIN1

Cisplatin nephrotoxicity is a critical limitation of solid cancer treatment. Until now, the complex interplay of various pathophysiological mechanisms leading to proximal tubular cell apoptosis after cisplatin exposure has not been fully understood. In our study, we assessed the role of the autophagy-related protein BECLIN1 (ATG6) in cisplatin-induced acute renal injury (AKI)—a candidate protein involved in autophagy and with putative impact on apoptosis by harboring a B-cell lymphoma 2 (BCL2) interaction site of unknown significance. By using mice with heterozygous deletion of Becn1, we demonstrate that reduced intracellular content of BECLIN1 does not impact renal function or autophagy within 12 months. However, these mice were significantly sensitized towards cisplatin-induced AKI, and by using Becn1+/−;Sglt2-Cre;Tomato/EGFP mice with subsequent primary cell analysis, we confirmed that nephrotoxicity depends on proximal tubular BECLIN1 content. Mechanistically, BECLIN1 did not impact autophagy or primarily the apoptotic pathway. In fact, a lack of BECLIN1 sensitized mice towards cisplatin-induced ER stress. Accordingly, the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) blunted cisplatin-induced cell death in Becn1 heterozygosity. In conclusion, our data first highlight a novel role of BECLIN1 in protecting against cellular ER stress independent from autophagy. These novel findings open new therapeutic avenues to intervene in this important intracellular stress response pathway with a promising impact on future AKI management

BECLIN1 Is Essential for Podocyte Secretory Pathways Mediating VEGF Secretion and Podocyte-Endothelial Crosstalk

Vascular endothelial growth factor A (VEGFA) secretion from podocytes is crucial for maintaining endothelial integrity within the glomerular filtration barrier. However, until now, the molecular mechanisms underlying podocyte secretory function remained unclear. Through podocyte-specific deletion of BECLIN1 (ATG6 or Becn1), a key protein in autophagy initiation, we identified a major role for this molecule in anterograde Golgi trafficking. The Becn1-deficient podocytes displayed aberrant vesicle formation in the trans-Golgi network (TGN), leading to dramatic vesicle accumulation and complex disrupted patterns of intracellular vesicle trafficking and membrane dynamics. Phenotypically, podocyte-specific deletion of Becn1 resulted in early-onset glomerulosclerosis, which rapidly progressed and dramatically reduced mouse life span. Further, in vivo and in vitro studies clearly showed that VEGFA secretion, and thereby endothelial integrity, greatly depended on BECLIN1 availability and function. Being the first to demonstrate the importance of a secretory pathway for podocyte integrity and function, we identified BECLIN1 as a key component in this complex cellular process. Functionally, by promoting VEGFA secretion, a specific secretory pathway emerged as an essential component for the podocyte-endothelial crosstalk that maintains the glomerular filtration barrier

Impact of Diabetic Stress Conditions on Renal Cell Metabolome

Diabetic kidney disease is a major complication in diabetes mellitus, and the most common reason for end-stage renal disease. Patients suffering from diabetes mellitus encounter glomerular damage by basement membrane thickening, and develop albuminuria. Subsequently, albuminuria can deteriorate the tubular function and impair the renal outcome. The impact of diabetic stress conditions on the metabolome was investigated by untargeted gas chromatography–mass spectrometry (GC-MS) analyses. The results were validated by qPCR analyses. In total, four cell lines were tested, representing the glomerulus, proximal nephron tubule, and collecting duct. Both murine and human cell lines were used. In podocytes, proximal tubular and collecting duct cells, high glucose concentrations led to global metabolic alterations in amino acid metabolism and the polyol pathway. Albumin overload led to the further activation of the latter pathway in human proximal tubular cells. In the proximal tubular cells, aldo-keto reductase was concordantly increased by glucose, and partially increased by albumin overload. Here, the combinatorial impact of two stressful agents in diabetes on the metabolome of kidney cells was investigated, revealing effects of glucose and albumin on polyol metabolism in human proximal tubular cells. This study shows the importance of including highly concentrated albumin in in vitro studies for mimicking diabetic kidney disease

Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis

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The Rapamycin-Sensitive Complex of Mammalian Target of Rapamycin Is Essential to Maintain Male Fertility

The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin and its analogs are being increasingly used in solid-organ transplantation. A commonly reported side effect is male subfertility to infertility, yet the precise mechanisms of mTOR interference with male fertility remain obscure. With the use of a conditional mouse genetic approach we demonstrate that deficiency of mTORC1 in the epithelial derivatives of the Wolffian duct is sufficient to cause male infertility. Analysis of spermatozoa from Raptor fl/fl*KspCre mice revealed an overall decreased motility pattern. Both epididymis and seminal vesicles displayed extensive organ regression with increasing age. Histologic and ultrastructural analyses demonstrated increased amounts of destroyed and absorbed spermatozoa in different segments of the epididymis. Mechanistically, genetic and pharmacologic mTORC1 inhibition was associated with an impaired cellular metabolism and a disturbed protein secretion of epididymal epithelial cells. Collectively, our data highlight the role of mTORC1 to preserve the function of the epididymis, ductus deferens, and the seminal vesicles. We thus reveal unexpected new insights into the frequently observed mTORC1 inhibitor side effect of male infertility in transplant recipients

CXCL12 and MYC control energy metabolism to support adaptive responses after kidney injury.

Kidney injury is a common complication of severe disease. Here, we report that injuries of the zebrafish embryonal kidney are rapidly repaired by a migratory response in 2-, but not in 1-day-old embryos. Gene expression profiles between these two developmental stages identify cxcl12a and myca as candidates involved in the repair process. Zebrafish embryos with cxcl12a, cxcr4b, or myca deficiency display repair abnormalities, confirming their role in response to injury. In mice with a kidney-specific knockout, Cxcl12 and Myc gene deletions suppress mitochondrial metabolism and glycolysis, and delay the recovery after ischemia/reperfusion injury. Probing these observations in zebrafish reveal that inhibition of glycolysis slows fast migrating cells and delays the repair after injury, but does not affect the slow cell movements during kidney development. Our findings demonstrate that Cxcl12 and Myc facilitate glycolysis to promote fast migratory responses during development and repair, and potentially also during tumor invasion and metastasis