The CTNS-MTORC1 axis couples lysosomal cystine to epithelial cell fate decisions and is a targetable pathway in cystinosis.

Differentiation and fate decisions are critical for the epithelial cells lining the proximal tubule (PT) of the kidney, but the signals involved remain unknown. Defective cystine mobilization from lysosomes through CTNS (cystinosin, lysosomal cystine transporter), which is mutated in cystinosis, triggers the dedifferentiation and dysfunction of the PT cells, causing kidney disease and severe metabolic complications. Using preclinical models and physiologically relevant cellular systems, along with functional assays and a generative artificial intelligence (AI)-powered engine, we found that cystine storage imparted by CTNS deficiency stimulates Ragulator-RRAG GTPase-dependent recruitment of MTORC1 and its constitutive activation. In turn, this diverts the catabolic trajectories and differentiating states of PT cells toward growth and proliferation, disrupting homeostasis and their specialized functions. Therapeutic MTORC1 inhibition by using low doses of rapamycin corrects lysosome function and differentiation downstream of cystine storage and ameliorates PT dysfunction in preclinical models of cystinosis. These discoveries suggest that cystine may act as a lysosomal fasting signal that tailors MTORC1 signaling to direct fate decisions in the kidney PT epithelium, highlighting novel therapeutic paradigms for cystinosis and other lysosome-related disorders.

Drug discovery and therapeutic perspectives for proximal tubulopathies.

The efficient reabsorption of essential nutrients by epithelial cells in the proximal tubule of the kidney is crucial for maintaining homeostasis. This process relies heavily on a complex ecosystem of vesicular trafficking pathways. At the center of this network, the lysosome plays a pivotal role in processing incoming molecules, sensing nutrient availability, sorting receptors and transporters, and balancing differentiation and proliferation in the tubular epithelial cells. Disruptions in these fundamental processes can lead to proximal tubulopathy-a condition characterized by the dysfunction of the tubular cells followed by the presence of low-molecular-weight proteins and solutes in urine. If left untreated, proximal tubulopathy can progress to chronic kidney disease and severe complications. Functional studies of rare inherited disorders affecting the proximal tubule have gleaned actionable insights into fundamental mechanisms of homeostasis while revealing drug targets for therapeutic discovery and development. In this mini review, we explore hereditary proximal tubulopathies as a paradigm of kidney homeostasis disorders, discussing the factors contributing to tubular dysfunction. In addition, we shed light on the current landscape of drug discovery approaches used to identify actionable targets and summarize the preclinical pipeline of potential therapeutic agents. These efforts may ultimately lead to new treatment avenues for proximal tubulopathies, which are currently inadequately tackled by existing therapies. Through this article, our hope is to promote academia-industry partnerships and advocate for research consortia that can accelerate the effective translation of knowledge advances into innovative therapies addressing the huge unmet needs of individuals with these debilitating diseases.

Lysosomal cystine export regulates mTORC1 signaling to guide kidney epithelial cell fate specialization.

Differentiation is critical for cell fate decisions, but the signals involved remain unclear. The kidney proximal tubule (PT) cells reabsorb disulphide-rich proteins through endocytosis, generating cystine via lysosomal proteolysis. Here we report that defective cystine mobilization from lysosomes through cystinosin (CTNS), which is mutated in cystinosis, diverts PT cells towards growth and proliferation, disrupting their functions. Mechanistically, cystine storage stimulates Ragulator-Rag GTPase-dependent recruitment of mechanistic target of rapamycin complex 1 (mTORC1) and its constitutive activation. Re-introduction of CTNS restores nutrient-dependent regulation of mTORC1 in knockout cells, whereas cell-permeant analogues of L-cystine, accumulating within lysosomes, render wild-type cells resistant to nutrient withdrawal. Therapeutic mTORC1 inhibition corrects lysosome and differentiation downstream of cystine storage, and phenotypes in preclinical models of cystinosis. Thus, cystine serves as a lysosomal signal that tailors mTORC1 and metabolism to direct epithelial cell fate decisions. These results identify mechanisms and therapeutic targets for dysregulated homeostasis in cystinosis.

Endolysosomal Disorders Affecting the Proximal Tubule of the Kidney: New Mechanistic Insights and Therapeutics.

Epithelial cells that line the proximal tubule of the kidney rely on an intertwined ecosystem of vesicular membrane trafficking pathways to ensure the reabsorption of essential nutrients. To function effectively and to achieve homeostasis, these specialized cells require the sorting and recycling of a wide array of cell surface proteins within the endolysosomal network, including signaling receptors, nutrient transporters, ion channels, and polarity markers. The dysregulation of the endolysosomal system can lead to a generalized proximal tubule dysfunction, ultimately causing severe metabolic complications and kidney disease.In this chapter, we highlight the biological functions of the genes that code endolysosomal proteins from the perspective of understanding - and potentially reversing - the pathophysiology of endolysosomal disorders affecting the proximal tubule of the kidney. These insights might ultimately lead to potential treatments for currently intractable diseases and transform our ability to regulate kidney homeostasis and health.

Mitochondrial Distress in Methylmalonic Acidemia: Novel Pathogenic Insights and Therapeutic Perspectives.

Mitochondria are highly dynamic, double-membrane-enclosed organelles that sustain cellular metabolism and, hence, cellular, and organismal homeostasis. Dysregulation of the mitochondrial network might, therefore, confer a potentially devastating vulnerability to high-energy-requiring cell types, contributing to a broad variety of hereditary and acquired diseases, which include inborn errors of metabolism, cancer, neurodegeneration, and aging-associated adversities. In this Review, we highlight the biological functions of mitochondria-localized enzymes, from the perspective of understanding the pathophysiology of the inherited disorders destroying mitochondrial homeostasis and cellular metabolism. Using methylmalonic acidemia (MMA) as a paradigm of mitochondrial dysfunction, we discuss how mitochondrial-directed signaling pathways sustain the physiological homeostasis of specialized cell types and how these may be disturbed in disease conditions. This Review also provides a critical analysis of molecular underpinnings, through which defects in the autophagy-mediated quality control and surveillance systems contribute to cellular dysfunction, and indicates potential therapeutic strategies for affected tissues. These insights might, ultimately, advance the discovery and development of new therapeutics, not only for methylmalonic acidemia but also for other currently intractable mitochondrial diseases, thus transforming our ability to modulate health and homeostasis.

Too bright for 2 dimensions: recent progress in advanced 3-dimensional microscopy of the kidney.

The kidney is a structurally and functionally complex organ responsible for the control of water, ion, and other solute homeostasis. Moreover, the kidneys excrete metabolic waste products and produce hormones, such as renin and erythropoietin. The functional unit of the kidney is the nephron, which is composed by a serial arrangement of a filter unit called the renal corpuscle and several tubular segments that modulate the filtered fluid by reabsorption and secretion. Within each kidney, thousands of nephrons are closely intermingled and surrounded by an intricate network of blood vessels and various interstitial cell types, including fibroblasts and immune cells. This complex tissue architecture is essential for proper kidney function. In fact, kidney disease is often reflected or even caused by a derangement of the histologic structures. Frequently, kidney histology is studied using microscopic analysis of 2-dimensional tissue sections, which, however, misses important 3-dimensional spatial information. Reconstruction of serial sections tries to overcome this limitation, but is technically challenging, time-consuming, and often inherently linked to sectioning artifacts. In recent years, advances in tissue preparation (e.g., optical clearing) and new light- and electron-microscopic methods have provided novel avenues for 3-dimensional kidney imaging. Combined with novel machine-learning algorithms, these approaches offer unprecedented options for large-scale and automated analysis of kidney structure and function. This review provides a brief overview of these emerging imaging technologies and presents key examples of how these approaches are already used to study the normal and the diseased kidney.

Multisystem involvement, defective lysosomes and impaired autophagy in a novel rat model of nephropathic cystinosis.

Recessive mutations in the CTNS gene encoding the lysosomal transporter cystinosin cause cystinosis, a lysosomal storage disease leading to kidney failure and multisystem manifestations. A Ctns knockout mouse model recapitulates features of cystinosis, but the delayed onset of kidney manifestations, phenotype variability and strain effects limit its use for mechanistic and drug development studies. To provide a better model for cystinosis, we generated a Ctns knockout rat model using CRISPR/Cas9 technology. The Ctns-/- rats display progressive cystine accumulation and crystal formation in multiple tissues including kidney, liver and thyroid. They show an early onset and progressive loss of urinary solutes, indicating generalized proximal tubule dysfunction, with development of typical swan-neck lesions, tubulointerstitial fibrosis and kidney failure, and decreased survival. The Ctns-/- rats also present crystals in the cornea, and bone and liver defects, as observed in patients. Mechanistically, the loss of cystinosin induces a phenotype switch associating abnormal proliferation and dedifferentiation, loss of apical receptors and transporters, and defective lysosomal activity and autophagy in the cells. Primary cultures of proximal tubule cells derived from the Ctns-/- rat kidneys confirmed the key changes caused by cystine overload, including reduced endocytic uptake, increased proliferation and defective lysosomal dynamics and autophagy. The novel Ctns-/- rat model and derived proximal tubule cell system provide invaluable tools to investigate the pathogenesis of cystinosis and to accelerate drug discovery.

Defective Cystinosin, Aberrant Autophagy-Endolysosome Pathways, and Storage Disease: Towards Assembling the Puzzle.

Epithelial cells that form the kidney proximal tubule (PT) rely on an intertwined ecosystem of vesicular membrane trafficking pathways to ensure the reabsorption of essential nutrients-a key requisite for homeostasis. The endolysosome stands at the crossroads of this sophisticated network, internalizing molecules through endocytosis, sorting receptors and nutrient transporters, maintaining cellular quality control via autophagy, and toggling the balance between PT differentiation and cell proliferation. Dysregulation of such endolysosome-guided trafficking pathways might thus lead to a generalized dysfunction of PT cells, often causing chronic kidney disease and life-threatening complications. In this review, we highlight the biological functions of endolysosome-residing proteins from the perspectives of understanding-and potentially reversing-the pathophysiology of rare inherited diseases affecting the kidney PT. Using cystinosis as a paradigm of endolysosome disease causing PT dysfunction, we discuss how the endolysosome governs the homeostasis of specialized epithelial cells. This review also provides a critical analysis of the molecular mechanisms through which defects in autophagy pathways can contribute to PT dysfunction, and proposes potential interventions for affected tissues. These insights might ultimately accelerate the discovery and development of new therapeutics, not only for cystinosis, but also for other currently intractable endolysosome-related diseases, eventually transforming our ability to regulate homeostasis and health.

Mitochondrial disease, mitophagy, and cellular distress in methylmalonic acidemia.

Mitochondria-the intracellular powerhouse in which nutrients are converted into energy in the form of ATP or heat-are highly dynamic, double-membraned organelles that harness a plethora of cellular functions that sustain energy metabolism and homeostasis. Exciting new discoveries now indicate that the maintenance of this ever changing and functionally pleiotropic organelle is particularly relevant in terminally differentiated cells that are highly dependent on aerobic metabolism. Given the central role in maintaining metabolic and physiological homeostasis, dysregulation of the mitochondrial network might therefore confer a potentially devastating vulnerability to high-energy requiring cell types, contributing to a broad variety of hereditary and acquired diseases. In this Review, we highlight the biological functions of mitochondria-localized enzymes from the perspective of understanding-and potentially reversing-the pathophysiology of inherited disorders affecting the homeostasis of the mitochondrial network and cellular metabolism. Using methylmalonic acidemia as a paradigm of complex mitochondrial dysfunction, we discuss how mitochondrial directed-signaling circuitries govern the homeostasis and physiology of specialized cell types and how these may be disturbed in disease. This Review also provides a critical analysis of affected tissues, potential molecular mechanisms, and novel cellular and animal models of methylmalonic acidemia which are being used to develop new therapeutic options for this disease. These insights might ultimately lead to new therapeutics, not only for methylmalonic acidemia, but also for other currently intractable mitochondrial diseases, potentially transforming our ability to regulate homeostasis and health.

The phosphoinositide 3-kinase inhibitor alpelisib restores actin organization and improves proximal tubule dysfunction in vitro and in a mouse model of Lowe syndrome and Dent disease.

Loss-of-function mutations in the OCRL gene, which encodes the phosphatidylinositol [PI] 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase OCRL, cause defective endocytosis and proximal tubule dysfunction in Lowe syndrome and Dent disease 2. The defect is due to increased levels of PI(4,5)P2 and aberrant actin polymerization, blocking endosomal trafficking. PI 3-phosphate [PI(3)P] has been recently identified as a coactivator with PI(4,5)P2 in the actin pathway. Here, we tested the hypothesis that phosphoinositide 3-kinase (PI3K) inhibitors may rescue the endocytic defect imparted by OCRL loss, by rebalancing phosphoinositide signals to the actin machinery. The broad-range PI3K inhibitor copanlisib and class IA p110α PI3K inhibitor alpelisib reduced aberrant actin polymerization in OCRL-deficient human kidney cells in vitro. Levels of PI 3,4,5-trisphosphate, PI(4,5)P2 and PI(3)P were all reduced with alpelisib treatment, and siRNA knockdown of the PI3K catalytic subunit p110α phenocopied the actin phenotype. In a humanized OcrlY/- mouse model, alpelisib reduced endosomal actin staining while restoring stress fiber architecture and levels of megalin at the plasma membrane of proximal tubule cells, reflected by improved endocytic uptake of low molecular weight proteins in vivo. Thus, our findings support the link between phosphoinositide lipids, actin polymerization and endocytic trafficking in the proximal tubule and represent a proof-of-concept for repurposing alpelisib in Lowe syndrome/Dent disease 2.

Mitochondria, mitophagy, and metabolic disease: towards assembling the puzzle.

Dysregulation of the mitochondrial network in terminally differentiated cells contributes to a broad spectrum of disorders. Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of intermediary metabolism caused by the deficiency of methylmalonyl-CoA mutase (MMUT) - a mitochondrial enzyme that mediates the degradation of certain amino acids and lipids. The loss of MMUT activity triggers an accumulation of toxic endogenous metabolites causing severe organ dysfunctions and life-threatening complications. How MMUT deficiency instigates mitochondrial distress and tissue damage remains poorly understood. Using cell and animal-based models, we recently discovered that MMUT deficiency disables the PINK1-induced translocation of PRKN/Parkin to MMA-damaged mitochondria, impeding their delivery and subsequent dismantling by macroautophagy/autophagy-lysosome degradation systems (Luciani et al. Nat Commun. 11(1):970). This promotes an accumulation of damaged and/or dysfunctional mitochondria that spark epithelial distress and tissue damage. Using a systems biology approach based on drug-disease network perturbation modeling, we predicted targetable pathways, whose modulation repairs mitochondrial dysfunctions in patient-derived kidney cells and ameliorates disease-relevant phenotypes in mmut-deficient zebrafish. These results unveil a link between primary MMUT deficiency, defective mitophagy, and cell distress, offering promising therapeutic avenues for MMA and other mitochondria-related diseases.

Induced pluripotent stem cells provide mega insights into kidney disease.

Rare mutations in the LRP2 gene encoding for the endocytic receptor megalin cause developmental abnormalities and kidney disease. However, the mechanisms governing the dysfunction of mutant megalin remain unclear. A new study utilizing patient-derived induced pluripotent stem cells is now putting the endolysosomal system into the spotlight, as it is proposed to play a central role in the regulation of megalin in health and disease.

Cell-Based Phenotypic Drug Screening Identifies Luteolin as Candidate Therapeutic for Nephropathic Cystinosis.

BACKGROUND: Mutations in the gene that encodes the lysosomal cystine transporter cystinosin cause the lysosomal storage disease cystinosis. Defective cystine transport leads to intralysosomal accumulation and crystallization of cystine. The most severe phenotype, nephropathic cystinosis, manifests during the first months of life, as renal Fanconi syndrome. The cystine-depleting agent cysteamine significantly delays symptoms, but it cannot prevent progression to ESKD and does not treat Fanconi syndrome. This suggests the involvement of pathways in nephropathic cystinosis that are unrelated to lysosomal cystine accumulation. Recent data indicate that one such potential pathway, lysosome-mediated degradation of autophagy cargoes, is compromised in cystinosis. METHODS: To identify drugs that reduce levels of the autophagy-related protein p62/SQSTM1 in cystinotic proximal tubular epithelial cells, we performed a high-throughput screening on the basis of an in-cell ELISA assay. We then tested a promising candidate in cells derived from patients with, and mouse models of, cystinosis, and in preclinical studies in cystinotic zebrafish. RESULTS: Of 46 compounds identified as reducing p62/SQSTM1 levels in cystinotic cells, we selected luteolin on the basis of its efficacy, safety profile, and similarity to genistein, which we previously showed to ameliorate other lysosomal abnormalities of cystinotic cells. Our data show that luteolin improves the autophagy-lysosome degradative pathway, is a powerful antioxidant, and has antiapoptotic properties. Moreover, luteolin stimulates endocytosis and improves the expression of the endocytic receptor megalin. CONCLUSIONS: Our data show that luteolin improves defective pathways of cystinosis and has a good safety profile, and thus has potential as a treatment for nephropathic cystinosis and other renal lysosomal storage diseases.

Methylmalonyl acidemia: from mitochondrial metabolism to defective mitophagy and disease.

Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of metabolism due to the deficiency of mitochondrial MMUT (methylmalonyl-CoA mutase) - an enzyme that mediates the cellular breakdown of certain amino acids and lipids. The loss of MMUT leads to the accumulation of toxic organic acids causing severe organ dysfunctions and life-threatening complications. The mechanisms linking MMUT deficiency, mitochondrial alterations and cell toxicity remain uncharacterized. Using cell and animal-based models, we recently unveiled that MMUT deficiency impedes the PINK1-induced translocation of PRKN/Parkin to MMA-damaged mitochondria, thereby halting their delivery and subsequent degradation by macroautophagy/autophagy-lysosome systems. In turn, this defective mitophagy process instigates the accumulation of dysfunctional mitochondria that spark epithelial distress and tissue damage. Correction of PINK1-directed mitophagy defects or mitochondrial dysfunctions rescues epithelial distress in MMA cells and alleviates disease-relevant phenotypes in mmut‒deficient zebrafish. Our findings suggest a link between primary MMUT deficiency and diseased mitochondria, mitophagy dysfunction and cell distress, offering potential therapeutic perspectives for MMA and other metabolic diseases.

Author Correction: Impaired mitophagy links mitochondrial disease to epithelial stress in methylmalonyl-CoA mutase deficiency.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transgenic zebrafish modeling low-molecular-weight proteinuria and lysosomal storage diseases.

Epithelial cells lining the proximal tubule of the kidney reabsorb and metabolize most of the filtered low-molecular-weight proteins through receptor-mediated endocytosis and lysosomal processing. Congenital and acquired dysfunctions of the proximal tubule are consistently reflected by the inappropriate loss of solutes including low-molecular-weight proteins in the urine. The zebrafish pronephros shares individual functional segments with the human nephron, including lrp2a/megalin-dependent endocytic transport processes of the proximal tubule. Although the zebrafish has been used as a model organism for toxicological studies and drug discovery, there is no available assay that allows large-scale assessment of proximal tubule function in larval or adult stages. Here we establish a transgenic Tg(lfabp::½vdbp-mCherry) zebrafish line expressing in the liver the N-terminal region of vitamin D-binding protein coupled to the acid-insensitive, red monomeric fluorescent protein mCherry (½vdbp-mCherry). This low-molecular-weight protein construct is secreted into the bloodstream, filtered through the glomerulus, reabsorbed by receptor-mediated endocytosis and processed in the lysosomes of proximal tubule cells of the fish. Thus, our proof-of-concept studies using zebrafish larvae knockout for lrp2a and clcn7 or exposed to known nephrotoxins (gentamicin and cisplatin) demonstrate that this transgenic line is useful to monitor low-molecular-weight proteinuria and lysosomal processing. This represents a powerful new model organism for drug screening and studies of nephrotoxicity.