Chemically based transmissible ER stress protocols are unsuitable to study cell-to-cell UPR transmission

Renal epithelial cells regulate the destructive activity of macrophages and participate in the progression of kidney diseases. Critically, the Unfolded Protein Response (UPR), which is activated in renal epithelial cells in the course of kidney injury, is required for the optimal differentiation and activation of macrophages. Given that macrophages are key regulators of renal inflammation and fibrosis, we suppose that the identification of mediators that are released by renal epithelial cells under Endoplasmic Reticulum (ER) stress and transmitted to macrophages is a critical issue to address. Signals leading to a paracrine transmission of ER stress (TERS) from a donor cell to a recipient cells could be of paramount importance to understand how ER-stressed cells shape the immune microenvironment. Critically, the vast majority of studies that have examined TERS used thaspigargin as an inducer of ER stress in donor cells in cellular models. By using multiple sources of ER stress, we evaluated if human renal epithelial cells undergoing ER stress can transmit the UPR to human monocyte-derived macrophages and if such TERS can modulate the inflammatory profiles of these cells. Our results indicate that carry-over of thapsigargin is a confounding factor in chemically based TERS protocols classically used to induce ER Stress in donor cells. Hence, such protocols are not suitable to study the TERS phenomenon and to identify its mediators. In addition, the absence of TERS transmission in more physiological models of ER stress indicates that cell-to-cell UPR transmission is not a universal feature in cultured cells

Allelic effects on uromodulin aggregates drive autosomal dominant tubulointerstitial kidney disease

Missense mutations in the uromodulin (UMOD) gene cause autosomal dominant tubulointerstitial kidney disease (ADTKD), one of the most common monogenic kidney diseases. The unknown impact of the allelic and gene dosage effects and fate of mutant uromodulin leaves open the gap between postulated gain-of-function mutations, end-organ damage and disease progression in ADTKD. Based on two prevalent missense UMOD mutations with divergent disease progression, we generated Umod$^{C171Y}$ and Umod$^{R186S}$ knock-in mice that showed strong allelic and gene dosage effects on uromodulin aggregates and activation of ER stress and unfolded protein and immune responses, leading to variable kidney damage. Deletion of the wild-type Umod allele in heterozygous Umod$^{R186S}$ mice increased the formation of uromodulin aggregates and ER stress. Studies in kidney tubular cells confirmed differences in uromodulin aggregates, with activation of mutation-specific quality control and clearance mechanisms. Enhancement of autophagy by starvation and mTORC1 inhibition decreased uromodulin aggregates. These studies substantiate the role of toxic aggregates as driving progression of ADTKD-UMOD, relevant for therapeutic strategies to improve clearance of mutant uromodulin

Autosomal Dominant Tubulointerstitial Kidney Disease with Adult Onset due to a Novel Renin Mutation Mapping in the Mature Protein

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a genetically heterogeneous renal disorder leading to progressive loss of renal function. ADTKD-REN is due to rare mutations in renin, all localized in the protein leader peptide and affecting its co-translational insertion in the endoplasmic reticulum (ER). Through exome sequencing in an adult-onset ADTKD family we identified a new renin variant, p.L381P, mapping in the mature protein. To assess its pathogenicity, we combined genetic data, computational and predictive analysis and functional studies. The L381P substitution affects an evolutionary conserved residue, co-segregates with renal disease, is not found in population databases and is predicted to be deleterious by in silico tools and by structural modelling. Expression of the L381P variant leads to its ER retention and induction of the Unfolded Protein Response in cell models and to defective pronephros development in zebrafish. Our work shows that REN mutations outside of renin leader peptide can cause ADTKD and delineates an adult form of ADTKD-REN, a condition which has usually its onset in childhood. This has implications for the molecular diagnosis and the estimated prevalence of the disease and points at ER homeostasis as a common pathway affected in ADTKD-REN, and possibly more generally in ADTKD

Hepsin-mediated Processing of Uromodulin is Crucial for Salt-sensitivity and Thick Ascending Limb Homeostasis.

Uromodulin is a zona pellucida-type protein essentially produced in the thick ascending limb (TAL) of the mammalian kidney. It is the most abundant protein in normal urine. Defective uromodulin processing is associated with various kidney disorders. The luminal release and subsequent polymerization of uromodulin depend on its cleavage mediated by the serine protease hepsin. The biological relevance of a proper cleavage of uromodulin remains unknown. Here we combined in vivo testing on hepsin-deficient mice, ex vivo analyses on isolated tubules and in vitro studies on TAL cells to demonstrate that hepsin influence on uromodulin processing is an important modulator of salt transport via the sodium cotransporter NKCC2 in the TAL. At baseline, hepsin-deficient mice accumulate uromodulin, along with hyperactivated NKCC2, resulting in a positive sodium balance and a better adaptation to water deprivation. In conditions of high salt intake, defective uromodulin processing predisposes hepsin-deficient mice to a salt-wasting phenotype, with a decreased salt sensitivity. These modifications are associated with intracellular accumulation of uromodulin, endoplasmic reticulum-stress and signs of tubular damage. These studies expand the physiological role of hepsin and uromodulin and highlight the importance of hepsin-mediated processing of uromodulin for kidney tubule homeostasis and salt sensitivity

Uromodulin is expressed in renal primary cilia and UMOD mutations result in decreased ciliary uromodulin expression

Uromodulin (UMOD) mutations are responsible for three autosomal dominant tubulo-interstitial nephropathies including medullary cystic kidney disease type 2 (MCKD2), familial juvenile hyperuricemic nephropathy and glomerulocystic kidney disease. Symptoms include renal salt wasting, hyperuricemia, gout, hypertension and end-stage renal disease. MCKD is part of the ‘nephronophthisis-MCKD complex', a group of cystic kidney diseases. Both disorders have an indistinguishable histology and renal cysts are observed in either. For most genes mutated in cystic kidney disease, their proteins are expressed in the primary cilia/basal body complex. We identified seven novel UMOD mutations and were interested if UMOD protein was expressed in the primary renal cilia of human renal biopsies and if mutant UMOD would show a different expression pattern compared with that seen in control individuals. We demonstrate that UMOD is expressed in the primary cilia of renal tubules, using immunofluorescent studies in human kidney biopsy samples. The number of UMOD-positive primary cilia in UMOD patients is significantly decreased when compared with control samples. Additional immunofluorescence studies confirm ciliary expression of UMOD in cell culture. Ciliary expression of UMOD is also confirmed by electron microscopy. UMOD localization at the mitotic spindle poles and colocalization with other ciliary proteins such as nephrocystin-1 and kinesin family member 3A is demonstrated. Our data add UMOD to the group of proteins expressed in primary cilia, where mutations of the gene lead to cystic kidney diseas

A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing

The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in ∼12days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 ± 0.8mV/cm2) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease

Association of Estimated Glomerular Filtration Rate and Urinary Uromodulin Concentrations with Rare Variants Identified by UMOD Gene Region Sequencing

Background: Recent genome-wide association studies (GWAS) have identified common variants in the UMOD region associated with kidney function and disease in the general population. To identify novel rare variants as well as common variants that may account for this GWAS signal, the exons and 4 kb upstream region of UMOD were sequenced. Methodology/Principal Findings Individuals (n = 485) were selected based on presence of the GWAS risk haplotype and chronic kidney disease (CKD) in the ARIC Study and on the extremes of of the UMOD gene product, uromodulin, in urine (Tamm Horsfall protein, THP) in the Framingham Heart Study (FHS). Targeted sequencing was conducted using capillary based Sanger sequencing (3730 DNA Analyzer). Variants were tested for association with THP concentrations and estimated glomerular filtration rate (eGFR), and identified non-synonymous coding variants were genotyped in up to 22,546 follow-up samples. Twenty-four and 63 variants were identified in the 285 ARIC and 200 FHS participants, respectively. In both studies combined, there were 33 common and 54 rare (MAF<0.05) variants. Five non-synonymous rare variants were identified in FHS; borderline enrichment of rare variants was found in the extremes of THP (SKAT p-value = 0.08). Only V458L was associated with THP in the FHS general-population validation sample (p = 9*10$^{−3}$, n = 2,522), but did not show direction-consistent and significant association with eGFR in both the ARIC (n = 14,635) and FHS (n = 7,520) validation samples. Pooling all non-synonymous rare variants except V458L together showed non-significant associations with THP and eGFR in the FHS validation sample. Functional studies of V458L revealed no alternations in protein trafficking. Conclusions/Significance: Multiple novel rare variants in the UMOD region were identified, but none were consistently associated with eGFR in two independent study samples. Only V458L had modest association with THP levels in the general population and thus could not account for the observed GWAS signal

Liver resection vs radiofrequency ablation in single hepatocellular carcinoma of posterosuperior segments in elderly patients

Background: Liver resection and radiofrequency ablation are considered curative options for hepatocellular carcinoma. The choice between these techniques is still controversial especially in cases of hepatocellular carcinoma affecting posterosuperior segments in elderly patients. Aim: To compare post-operative outcomes between liver resection and radiofrequency ablation in elderly with single hepatocellular carcinoma located in posterosuperior segments. Methods: A retrospective multicentric study was performed enrolling 77 patients age ≥ 70-years-old with single hepatocellular carcinoma (≤ 30 mm), located in posterosuperior segments (4a, 7, 8). Patients were divided into liver resection and radiofrequency ablation groups and preoperative, peri-operative and long-term outcomes were retrospectively analyzed and compared using a 1:1 propensity score matching. Results: After propensity score matching, twenty-six patients were included in each group. Operative time and overall postoperative complications were higher in the resection group compared to the ablation group (165 min vs 20 min, P &lt; 0.01; 54% vs 19% P = 0.02 respectively). A median hospital stay was significantly longer in the resection group than in the ablation group (7.5 d vs 3 d, P &lt; 0.01). Ninety-day mortality was comparable between the two groups. There were no significant differences between resection and ablation group in terms of overall survival and disease free survival at 1, 3, and 5 years. Conclusion: Radiofrequency ablation in posterosuperior segments in elderly is safe and feasible and ensures a short hospital stay, better quality of life and does not modify the overall and disease-free survival

An intermediate-effect size variant in UMOD confers risk for chronic kidney disease

The kidney-specific gene UMOD encodes for uromodulin, the most abundant protein excreted in normal urine. Rare large-effect variants in UMOD cause autosomal dominant tubulointerstitial kidney disease (ADTKD), while common low-impact variants strongly associate with kidney function and the risk of chronic kidney disease (CKD) in the general population. It is unknown whether intermediate-effect variants in UMOD contribute to CKD. Here, candidate intermediate-effect UMOD variants were identified using large-population and ADTKD cohorts. Biological and phenotypical effects were investigated using cell models, in silico simulations, patient samples, and international databases and biobanks. Eight UMOD missense variants reported in ADTKD are present in the Genome Aggregation Database (gnomAD), with minor allele frequency (MAF) ranging from 10(−5) to 10(−3). Among them, the missense variant p.Thr62Pro is detected in ∼1/1,000 individuals of European ancestry, shows incomplete penetrance but a high genetic load in familial clusters of CKD, and is associated with kidney failure in the 100,000 Genomes Project (odds ratio [OR] = 3.99 [1.84 to 8.98]) and the UK Biobank (OR = 4.12 [1.32 to 12.85). Compared with canonical ADTKD mutations, the p.Thr62Pro carriers displayed reduced disease severity, with slower progression of CKD and an intermediate reduction of urinary uromodulin levels, in line with an intermediate trafficking defect in vitro and modest induction of endoplasmic reticulum (ER) stress. Identification of an intermediate-effect UMOD variant completes the spectrum of UMOD-associated kidney diseases and provides insights into the mechanisms of ADTKD and the genetic architecture of CKD