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

Dent's disease

Dent's disease is a renal tubular disorder characterized by manifestations of proximal tubule dysfunction, including low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, nephrocalcinosis, and progressive renal failure. These features are generally found in males only, and may be present in early childhood, whereas female carriers may show a milder phenotype. Prevalence is unknown; the disorder has been reported in around 250 families to date. Complications such as rickets or osteomalacia may occur. The disease is caused by mutations in either the CLCN5 (Dent disease 1) or OCRL1 (Dent disease 2) genes that are located on chromosome Xp11.22 and Xq25, respectively. CLCN5 encodes the electrogenic Cl-/H+ exchanger ClC-5, which belongs to the CLC family of Cl- channels/transporters. OCRL1 encodes a phosphatidylinositol bisphosphate (PIP2) 5-phosphatase and mutations are also associated with Lowe Syndrome. The phenotype of Dent's disease is explained by the predominant expression of ClC-5 in the proximal tubule segments of the kidney. No genotype-phenotype correlation has been described thus far, and there is considerable intra-familial variability in disease severity. A few patients with Dent's disease do not harbour mutations in CLCN5 and OCRL1, pointing to the involvement of other genes. Diagnosis is based on the presence of all three of the following criteria: low-molecular-weight proteinuria, hypercalciuria and at least one of the following: nephrocalcinosis, kidney stones, hematuria, hypophosphatemia or renal insufficiency. Molecular genetic testing confirms the diagnosis. The differential diagnosis includes other causes of generalized dysfunction of the proximal tubules (renal Fanconi syndrome), hereditary, acquired, or caused by exogenous substances. Antenatal diagnosis and pre-implantation genetic testing is not advised. The care of patients with Dent's disease is supportive, focusing on the treatment of hypercalciuria and the prevention of nephrolithiasis. The vital prognosis is good in the majority of patients. Progression to end-stage renal failure occurs between the 3rd and 5th decades of life in 30-80% of affected males

Vasopressin-2 receptor antagonists in autosomal dominant polycystic kidney disease: from man to mouse and back

nephropathy, with an esti-mated prevalence of 1:1000. The disease is characterized by the development of multiple cysts from all nephron segments leading to the enlargement of both kidneys and replacement of normal parenchyma (see [1]). Change in total kidney volume over time is the strongest predictor of renal function decline in ADPKD [2]. Glomerular filtra-tion rate remains preserved up to the age of 40 years in most patients because glomerular hyperfiltration in functioning nephrons compensates for the ongoing loss of renal tissue, until end-stage renal failure ensues in>50 % of patients, usually in their fifth decade. Mutations in the PKD1 gene account for ~85 % of the affected families, whereas the remaining cases are caused by mutations in PKD2. PKD1 encodes polycystin-1, an integral membrane protein with a large extracellular domain that probably functions as a re-ceptor and/or an adhesion molecule, whereas PKD2 enco-des polycystin-2, a non-selective cation channel belonging to the family of transient receptor potential channels. The polycystins are located in the primary cilium and interact to form a mechanosensory complex that is involved in intra-cellular Ca21 homeostasis and various signalling pathways. Disruption of the complex leads to cyst development and enlargement resulting from tubular cell proliferation and transepithelial fluid secretion. The progressive understand-ing of these pathways has led to spectacular advances in the prospective treatment for ADPKD, including the blockade of vasopressin 2 receptor (V2R) to decrease the intracellu-lar level of 3#-5#-cyclic adenosine monophosphate (cAMP) in cyst-lining tubular cells [1]

Regulated acid-base transport in the collecting duct

The renal collecting system serves the fine-tuning of renal acid-base secretion. Acid-secretory type-A intercalated cells secrete protons via a luminally expressed V-type H+-ATPase and generate new bicarbonate released by basolateral chloride/bicarbonate exchangers including the AE1 anion exchanger. Efficient proton secretion depends both on the presence of titratable acids (mainly phosphate) and the concomitant secretion of ammonia being titrated to ammonium. Collecting duct ammonium excretion requires the Rhesus protein RhCG as indicated by recent KO studies. Urinary acid secretion by type-A intercalated cells is strongly regulated by various factors among them acid-base status, angiotensin II and aldosterone, and the Calcium-sensing receptor. Moreover, urinary acidification by H+-ATPases is modulated indirectly by the activity of the epithelial sodium channel ENaC. Bicarbonate secretion is achieved by non-type-A intercalated cells characterized by the luminal expression of the chloride/bicarbonate exchanger pendrin. Pendrin activity is driven by H+-ATPases and may serve both bicarbonate excretion and chloride reabsorption. The activity and expression of pendrin is regulated by different factors including acid-base status, chloride delivery, and angiotensin II and may play a role in NaCl retention and blood pressure regulation. Finally, the relative abundance of type-A and non-type-A intercalated cells may be tightly regulated. Dysregulation of intercalated cell function or abundance causes various syndromes of distal renal tubular acidosis underlining the importance of these processes for acid-base homeostasi

Targeting chloride transport in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent inherited kidney disease. Transepithelial fluid secretion is one of the key factors of cystogenesis in ADPKD. Multiple studies have suggested that fluid secretion across ADPKD cyst-lining cells is driven by the secretion of chloride, essentially mediated by the CFTR channel and stimulated by increased intracellular levels of 3',5'-cyclic adenosine monophosphate. This review focuses on the pathophysiology of fluid secretion in ADPKD based on the pioneering studies of Jared Grantham and colleagues, and on the follow-up investigations from the molecular level to the potential applications in ADPKD patients. Altogether, the studies of fluid and chloride transport in ADPKD paved the way for innovative therapeutic targets to prevent cyst volume expansion and thus, kidney disease progression.Peer reviewe

Parvalbumin: calcium and magnesium buffering in the distal nephron

Parvalbumin (PV) is a classical member of the EF-hand protein superfamily that has been described as a Ca2+ buffer and Ca2+ transporter/shuttle protein and may also play an additional role in Mg2+ handling. PV is exclusively expressed in the early part of the distal convoluted tubule in the human and mouse kidneys. Recent studies in Pvalb knockout mice revealed a role of PV in the distal handling of electrolytes: the lack of PV was associated with a mild salt-losing phenotype with secondary aldosteronism, salt craving and stronger bones compared with controls. A link between the Ca2+-buffering capacity of PV and the expression of the thiazide-sensitive Na+-Cl− cotransporter was established, which could be relevant to the regulation of sodium transport in the distal nephron. Variants in the PVALB gene that encodes PV have been described, but their relevance to kidney function has not been established. PV is also considered a reliable marker of chromophobe carcinoma and oncocytoma, two neoplasms deriving from the distal nephron. The putative role of PV in tumour genesis remains to be investigate

Dedifferentiation and aberrations of the endolysosomal compartment characterize the early stage of nephropathic cystinosis

Nephropathic cystinosis, a lysosomal storage disease caused by mutations in the CTNS gene encoding the lysosomal cystine transporter cystinosin, is characterized by generalized proximal tubule (PT) dysfunction that progresses, if untreated, to end-stage renal disease. The pathogenesis of defective PT cellular transport in nephropathic cystinosis remains unclear. We characterized a recently generated line of C57BL/6 Ctns mice and analyzed endocytic uptake, lysosome function, and dedifferentiation and proliferation markers using primary cultures of PT epithelial cells derived from Ctns−/− and Ctns+/+ littermates. Metabolic studies revealed that Ctns−/− mice show a progressive PT dysfunction characterized by low-molecular-weight (LMW) proteinuria, glucosuria and phosphaturia, before structural damage and in the absence of renal failure. These changes are related to decreased expression of the multi-ligand receptors megalin and cubilin and to increased dedifferentiation (ZONAB transcription factor) and proliferation (PCNA and Cyclin D1) rates. Studies on PT cells derived from Ctns−/− kidneys confirmed cystine overload, with accumulation of enlarged, dysfunctional lysosomes and reduced expression of endocytic receptors reflected by decreased uptake of specific ligands. These changes were related to a loss of integrity of tight junctions with a nuclear translocation of ZONAB and increased proliferation, as observed in Ctns−/− kidneys. These data reveal that the absence of cystinosin in PT cells triggers aberrations of the endolysosomal compartment, transport defects and an abnormal transcription program in the early stage of nephropathic cystinosis. Insights into the early manifestations of cystinosis may offer new targets for intervention, before irreversible renal damag

Decreased renal accumulation of aminoglycoside reflects defective receptor-mediated endocytosis in cystic fibrosis and Dent's disease

The clinical use of aminoglycoside (AG) antibiotics is limited by their renal toxicity, which is caused by drug accumulation in proximal tubule (PT) cells. Clinical studies reported that renal clearance of AG is enhanced in cystic fibrosis (CF) patients, which might reflect the role of CFTR in PT cell endocytosis. In order to assess the role of chloride transporters on the renal handling of AG, we investigated gentamicin uptake and renal accumulation in mice lacking functional CFTR (Cftr ∆F/∆F) or knock-out for the Cl−/H+ exchanger ClC-5 (Clcn5 Y/− ). The latter represent a paradigm of PT dysfunction and defective receptor-mediated endocytosis. As compared with controls, Cftr ∆F/∆F and Clcn5 Y/− mice showed a 15% to 85% decrease in gentamicin accumulation in the kidney, respectively, in absence of renal failure. Studies on primary cultures of Cftr ∆F/∆F and Clcn5 Y/− mouse PT cells confirmed the reduction in gentamicin uptake, although colocalization with endosomes and lysosomes was maintained. Quantification of endocytosis in PT cells revealed that gentamicin, similar to albumin, preferentially binds to megalin. The functional loss of ClC-5 or CFTR was reflected by a decrease of the endocytic uptake of gentamicin, with a more pronounced effect in cells lacking ClC-5. These results support the concept that CFTR, as well as ClC-5, plays a relevant role in PT cell endocytosis. They also demonstrate that the functional loss of these two chloride transporters is associated with impaired uptake of AG in PT cells, reflected by a decreased renal accumulation of the dru

Determination of uromodulin in human urine: influence of storage and processing

Background Uromodulin (Tamm-Horsfall protein) is the most abundant protein excreted in the urine under physiological conditions. It is exclusively produced in the kidney and secreted into the urine via proteolytic cleavage. The involvement of UMOD, the gene that encodes uromodulin, in rare autosomal dominant diseases, and its robust genome-wide association with the risk of chronic kidney disease suggest that the level of uromodulin in urine could represent a critical biomarker for kidney function. The structure of uromodulin is complex, with multiple disulfide bonds and typical domains of extracellular proteins. Methods Thus far, the conditions influencing stability and measurement of uromodulin in human urine have not been systematically investigated, giving inconsistent results. In this study, we used a robust, in-house ELISA to characterize the conditions of sampling and storage necessary to provide a faithful dosage of uromodulin in the urine. Results The levels of uromodulin in human urine were significantly affected by centrifugation and vortexing, as well as by the conditions and duration of storage. Conclusions These results validate a simple, low-cost ELISA and document the optimal conditions of processing and storage for measuring uromodulin in human urin

Paradoxical response to furosemide in uromodulin-associated kidney disease

The mechanism by which uromodulin mutations lead to urine concentrating defect in humans remains to be better elucidated .Labriola et al show original data exploring the tubular function of a patient suffering UAKD during the early phase of the diseas