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.

A reciprocal regulation of spermidine and autophagy in podocytes maintains the filtration barrier.

Podocyte maintenance and stress resistance are exquisitely based on high basal rates of autophagy making these cells a unique model to unravel mechanisms of autophagy regulation. Polyamines have key cellular functions such as proliferation, nucleic acid biosynthesis and autophagy. Here we test whether endogenous spermidine signaling is a driver of basal and dynamic autophagy in podocytes by using genetic and pharmacologic approaches to interfere with different steps of polyamine metabolism. Translational studies revealed altered spermidine signaling in focal segmental glomerulosclerosis in vivo and in vitro. Exogenous spermidine supplementation emerged as new treatment strategy by successfully activating autophagy in vivo via inhibition of EP300, a protein with an essential role in controlling cell growth, cell division and prompting cells to differentiate to take on specialized functions. Surprisingly, gas chromatography-mass spectroscopy based untargeted metabolomics of wild type and autophagy deficient primary podocytes revealed a positive feedback mechanism whereby autophagy itself maintains polyamine metabolism and spermidine synthesis. The transcription factor MAFB acted as an upstream regulator of polyamine metabolism. Thus, our data highlight a novel positive feedback loop of autophagy and spermidine signaling allowing maintenance of high basal levels of autophagy as a key mechanism to sustain the filtration barrier. Hence, spermidine supplementation may emerge as a new therapeutic to restore autophagy in glomerular disease.

Targeting mTOR Signaling Can Prevent the Progression of FSGS.

Mammalian target of rapamycin (mTOR) signaling is involved in a variety of kidney diseases. Clinical trials administering mTOR inhibitors to patients with FSGS, a prototypic podocyte disease, led to conflicting results, ranging from remission to deterioration of kidney function. Here, we combined complex genetic titration of mTOR complex 1 (mTORC1) levels in murine glomerular disease models, pharmacologic studies, and human studies to precisely delineate the role of mTOR in FSGS. mTORC1 target genes were significantly induced in microdissected glomeruli from both patients with FSGS and a murine FSGS model. Furthermore, a mouse model with constitutive mTORC1 activation closely recapitulated human FSGS. Notably, the complete knockout of mTORC1 by induced deletion of both Raptor alleles accelerated the progression of murine FSGS models. However, lowering mTORC1 signaling by deleting just one Raptor allele ameliorated the progression of glomerulosclerosis. Similarly, low-dose treatment with the mTORC1 inhibitor rapamycin efficiently diminished disease progression. Mechanistically, complete pharmacologic inhibition of mTOR in immortalized podocytes shifted the cellular energy metabolism toward reduced rates of oxidative phosphorylation and anaerobic glycolysis, which correlated with increased production of reactive oxygen species. Together, these data suggest that podocyte injury and loss is commonly followed by adaptive mTOR activation. Prolonged mTOR activation, however, results in a metabolic podocyte reprogramming leading to increased cellular stress and dedifferentiation, thus offering a treatment rationale for incomplete mTOR inhibition.

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.

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.

Targeting transcription factor Stat4 uncovers a role for interleukin-18 in the pathogenesis of severe lupus nephritis in mice.

Polymorphisms in the transcription factor Stat4 gene have been implicated as risk factors for systemic lupus erythematosus. Although some polymorphisms have a strong association with autoantibodies and nephritis, their impact on pathophysiology is still unknown. To explore this further we used signal transducers and activators of transcription 4 (Stat4) knockout MRL/MpJ-Fas(lpr)/Fas(lpr) (MRL-Fas(lpr)) mice and found that they did not differ in survival or renal function from Stat4-intact MRL-Fas(lpr) mice. Circulating interleukin (IL)-18 levels, however, were elevated in Stat4-deficient compared to Stat4-intact mice, suggesting that this interleukin might contribute to the progression of lupus nephritis independent of Stat4. In a second approach, Stat4 antisense or missense oligonucleotides or vehicle were given to MRL-Fas(lpr) mice with advanced nephritis. Each of these treatments temporarily ameliorated disease, although IL-18 was increased in each setting. Based on these findings, studies using gene transfer to overexpress IL-18 in MRL-Fas(lpr) and IL-12p40/IL-23 knockout MRL-Fas(lpr) mice reveal a critical role for IL-18 in mediating disease. Thus, the Stat4 and IL-12 (an activator of Stat4)-independent factor, IL-18, can drive autoimmune lupus nephritis in MRL-Fas(lpr) mice. Temporarily blocking Stat4 during advanced nephritis ameliorates disease, suggesting a time-dependent compensatory proinflammatory mechanism.

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. Abbreviations: AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy related; BW: body weight; Cq: chloroquine; ER: endoplasmic reticulum; ESRD: end stage renal disease; FACS: fluorescence activated cell sorting; GFP: green fluorescent protein; i.p.: intra peritoneal; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NPHS1: nephrosis 1, nephrin; NPHS2: nephrosis 2, podocin; PLA: proximity-ligation assay; PRKAA: 5'-AMP-activated protein kinase catalytic subunit alpha; RPTOR/RAPTOR: regulatory associated protein of MTOR, complex 1; RFP: red fluorescent protein; TSC1: tuberous sclerosis 1; ULK1: unc-51 like kinase 1.

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.

Anaerobic Glycolysis Maintains the Glomerular Filtration Barrier Independent of Mitochondrial Metabolism and Dynamics.

The cellular responses induced by mitochondrial dysfunction remain elusive. Intrigued by the lack of almost any glomerular phenotype in patients with profound renal ischemia, we comprehensively investigated the primary sources of energy of glomerular podocytes. Combining functional measurements of oxygen consumption rates, glomerular metabolite analysis, and determination of mitochondrial density of podocytes in vivo, we demonstrate that anaerobic glycolysis and fermentation of glucose to lactate represent the key energy source of podocytes. Under physiological conditions, we could detect neither a developmental nor late-onset pathological phenotype in podocytes with impaired mitochondrial biogenesis machinery, defective mitochondrial fusion-fission apparatus, or reduced mtDNA stability and transcription caused by podocyte-specific deletion of Pgc-1α, Drp1, or Tfam, respectively. Anaerobic glycolysis represents the predominant metabolic pathway of podocytes. These findings offer a strategy to therapeutically interfere with the enhanced podocyte metabolism in various progressive kidney diseases, such as diabetic nephropathy or focal segmental glomerulosclerosis (FSGS).

mTOR-mediated podocyte hypertrophy regulates glomerular integrity in mice and humans.

The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin-mediated (mTOR-mediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation-related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis - all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.

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.

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

The glomerulus is a highly specialized capillary tuft, which under pressure filters large amounts of water and small solutes into the urinary space, while retaining albumin and large proteins. The glomerular filtration barrier (GFB) is a highly specialized filtration interface between blood and urine that is highly permeable to small and midsized solutes in plasma but relatively impermeable to macromolecules such as albumin. The integrity of the GFB is maintained by molecular interplay between its 3 layers: the glomerular endothelium, the glomerular basement membrane and podocytes, which are highly specialized postmitotic pericytes forming the outer part of the GFB. Abnormalities of glomerular ultrafiltration lead to the loss of proteins in urine and progressive renal insufficiency, underlining the importance of the GFB. Indeed, albuminuria is strongly predictive of the course of chronic nephropathies especially that of diabetic nephropathy (DN), a leading cause of renal insufficiency. We found that high glucose concentrations promote autophagy flux in podocyte cultures and that the abundance of LC3B II in podocytes is high in diabetic mice. Deletion of Atg5 specifically in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky GFB and glomerulosclerosis. Strikingly, genetic alteration of autophagy on the other side of the GFB involving the endothelial-specific deletion of Atg5 also resulted in capillary rarefaction and accelerated DN. Thus autophagy is a key protective mechanism on both cellular layers of the GFB suggesting autophagy as a promising new therapeutic strategy for DN.