Neuropilin-1 regulates renin synthesis in juxtaglomerular cells.

Renin is the key enzyme of the systemic renin-angiotensin-aldosterone system, which plays an essential role in regulating blood pressure and maintaining electrolyte and extracellular volume homeostasis. Renin is mainly produced and secreted by specialized juxtaglomerular (JG) cells in the kidney. In the present study, we report for the first time that the conserved transmembrane receptor neuropilin-1 (NRP1) participates in the development of JG cells and plays a key role in renin production. We used the myelin protein zero-Cre (P0-Cre) to abrogate Nrp1 constitutively in P0-Cre lineage-labelled cells of the kidney. We found that the P0-Cre precursor cells differentiate into renin-producing JG cells. We employed a lineage-tracing strategy combined with RNAscope quantification and metabolic studies to reveal a cell-autonomous role for NRP1 in JG cell function. Nrp1-deficient animals displayed abnormal levels of tissue renin expression and failed to adapt properly to a homeostatic challenge to sodium balance. These findings provide new insights into cell fate decisions and cellular plasticity operating in P0-Cre-expressing precursors and identify NRP1 as a novel key regulator of JG cell maturation. KEY POINTS: Renin is a centrepiece of the renin-angiotensin-aldosterone system and is produced by specialized juxtaglomerular cells (JG) of the kidney. Neuropilin-1 (NRP1) is a conserved membrane-bound receptor that regulates vascular and neuronal development, cancer aggressiveness and fibrosis progression. We used conditional mutagenesis and lineage tracing to show that NRP1 is expressed in JG cells where it regulates their function. Cell-specific Nrp1 knockout mice present with renin paucity in JG cells and struggle to adapt to a homeostatic challenge to sodium balance. The results support the versatility of renin-producing cells in the kidney and may open new avenues for therapeutic approaches.

Energy depletion by cell proliferation sensitizes the kidney epithelial cells to injury.

Acute kidney injury activates both proliferative and antiproliferative pathways, the consequences of which are not fully elucidated. If an initial proliferation of the renal epithelium is necessary for the successful repair, the persistence of proliferation markers is associated with the occurrence of chronic kidney disease. We hypothesized that proliferation in stress conditions impacts cell viability and renal outcomes. We found that proliferation is associated with cell death after various stresses in kidney cells. In vitro, the ATP/ADP ratio oscillates reproducibly throughout the cell cycle, and cell proliferation is associated with a decreased intracellular ATP/ADP ratio. In vivo, transcriptomic data from transplanted kidneys revealed that proliferation was strongly associated with a decrease in the expression of the mitochondria-encoded genes of the oxidative phosphorylation pathway, but not of the nucleus-encoded ones. These observations suggest that mitochondrial function is a limiting factor for energy production in proliferative kidney cells after injury. The association of increased proliferation and decreased mitochondrial function was indeed associated with poor renal outcomes. In summary, proliferation is an energy-demanding process impairing the cellular ability to cope with an injury, highlighting proliferative repair and metabolic recovery as indispensable and interdependent features for successful kidney repair.NEW & NOTEWORTHY ATP depletion is a hallmark of acute kidney injury. Proliferation is instrumental to kidney repair. We show that ATP levels vary during the cell cycle and that proliferation sensitizes renal epithelial cells to superimposed injuries in vitro. More proliferation and less energy production by the mitochondria are associated with adverse outcomes in injured kidney allografts. This suggests that controlling the timing of kidney repair might be beneficial to mitigate the extent of acute kidney injury.

Orphan nuclear receptor COUP-TFII enhances myofibroblast glycolysis leading to kidney fibrosis.

Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.

Immunoadsorption-Based HLA Desensitization in Patients Awaiting Deceased Donor Kidney Transplantation: An Interventional, Non-Randomised, Single Cohort Study.

Whether immunoadsorption (IADS) as part of desensitization protocols could facilitate deceased donor kidney transplantation (KT) in highly sensitized (HS) patients remains to be proven. We retrospectively analyzed our IADS based desensitization protocol for deceased donor KTs between 2013 and 2018. Fifteen HS patients (age 52 years [40-56]) were included. Waiting time before IADS was 6 years [5-10] and the interval between IADS initiation and KT was 5 months [1-12] for the 14 transplanted patients. Nine patients had prior KT. Calculated panel reactive antibody decreased significantly during the protocol (99.3% [92.5-99.9] vs. 79.4% [56.7-81.9]; p = 0.004). Death-censored graft survival was 85.7% at 1 and 2 years post-transplantation. One-year median plasma creatinine level was 135 µmol/L [111-202]. Six developed active antibody mediated rejection (ABMR) at 1 year, with a median delay of 13 days [11-26]. Eight patients developed severe infections, including two fatal outcomes. Finally, compared to 93% of patients who received desensitization receiving a KT, only 43% of a control with similar characteristics underwent transplantation. However, no difference was found in overall probability of being alive with a functioning graft at the end of follow-up. The results indicate that our IADS-based desensitization strategy was not effective due to a high rate of ABMR and severe infectious complications which pose a challenge to its universalization.

Predictive factors of glomerular filtration rate loss associated with living kidney donation: a single-center retrospective study.

PURPOSE: Living kidney donors (LKD) partially compensate the initial loss of glomerular filtration rate (GFR), a phenomenon known as renal functional reserve (RFR). RFR is reduced in the elderly, a population with increased prevalence of chronic kidney disease. We hypothesized that the selected, healthy population of LKD, would specifically inform about the physiological determinants of the RFR and studied it using measured GFR (mGFR). METHODS: We retrospectively analyzed pre-donation and post-donation mGFR in 76 LKD from Tenon Hospital (Paris, France) between 2002 and 2018. In addition to GFR measurements, we collected pre-donation morphologic parameters, demographic data, and kidney volumes. RESULTS: Mean pre-donation mGFR was 90.11 ± 12.64 mL/min/1.73 m2 and decreased to 61.26 ± 9.57 mL/min/1.73 m2 1 year after donation. Pre-donation mGFR correlated with age (p = 0.0003), total kidney volume (p = 0.0004) and pre-donation serum creatinine (p = 0.0453). Pre-donation mGFR strongly predicted 1-year post-donation mGFR. Mean RFR (increase in GFR of the remnant kidney between pre-donation and post-donation) was 36.67 ± 16.67% 1 year after donation. In the multivariate linear model, RFR was negatively correlated to total kidney volume (p = 0.02) but not with age or pre-donation serum creatinine. CONCLUSIONS: We found that pre-donation mGFR decreases with age and identified low total kidney volume as a predictor of RFR in healthy individuals. This suggests an adaptative and reversible decrease in kidney function rather than age-related damage. Older subjects may have reduced metabolic requirements with subsequent reduction in glomerular filtration and kidney volume and preserved RFR. Therefore, low GFR in older subjects should not preclude kidney donation.

Anaesthesia-Induced Transcriptomic Changes in the Context of Renal Ischemia Uncovered by the Use of a Novel Clamping Device.

Ischemia is a common cause of acute kidney injury worldwide, frequently occurring in patients undergoing cardiac surgery or admitted to the intensive care unit (ICU). Thus, ischemia-reperfusion injury (IRI) remains one of the main experimental models for the study of kidney diseases. However, the classical technique, based on non-traumatic surgical clamps, suffers from several limitations. It does not allow the induction of multiple episodes of acute kidney injury (AKI) in the same animal, which would be relevant from a human perspective. It also requires a deep and long sedation, raising the question of potential anaesthesia-related biases. We designed a vascular occluding device that can be activated remotely in conscious mice. We first assessed the intensity and the reproducibility of the acute kidney injury induced by this new device. We finally investigated the role played by the anaesthesia in the IRI models at the histological, functional and transcriptomic levels. We showed that this technique allows the rapid induction of renal ischemia in a repeatable and reproducible manner, breaking several classical limitations. In addition, we used its unique specificities to highlight the renal protective effect conferred by the anaesthesia, related to the mitigation of the IRI transcriptomic program.

Microvasculature partial endothelial mesenchymal transition in early posttransplant biopsy with acute tubular necrosis identifies poor recovery renal allografts.

Acute tubular necrosis (ATN), a frequent histopathological feature in the early post-renal transplant biopsy, affects long-term graft function. Appropriate markers to identify patients at risk of no or incomplete recovery after delayed graft function are lacking. In this study, we first included 41 renal transplant patients whose biopsy for cause during the first month after transplantation showed ATN lesions. Using partial microvasculature endothelial (fascin, vimentin) and tubular epithelial (vimentin) to mesenchymal transition markers, detected by immunohistochemistry, we found a significant association between partial endothelial to mesenchymal transition and poor graft function recovery (Spearman's rho = -0.55, P = .0005). Transforming growth factor-ß1 was strongly expressed in these phenotypic changed endothelial cells. Extent of ATN was also correlated with short- and long-term graft dysfunction. However, the association of extensive ATN with long-term graft dysfunction (24 months posttransplant) was observed only in patients with partial endothelial to mesenchymal transition marker expression in their grafts (Spearman's rho = -0.64, P = .003), but not in those without. The association of partial endothelial to mesenchymal transition with worse renal graft outcome was confirmed on 34 other early biopsies with ATN from a second transplant center. Our results suggest that endothelial cell activation at the early phase of renal transplantation plays a detrimental role.

Predictive value of mixed antigen screen beads in pre-transplant assessment of HLA immunization in solid organ transplant recipients.

Pre-transplant serum screening of anti-HLA antibodies is recommended for solid organ transplantations. Many laboratories use the less expensive bead-based screening assay as the main technique and, if positive, turn to single-antigen beads (SAB). We studied the correlations between these two immunoassays. We re-analyzed the raw data of the two assays in 3030 first organ transplant recipients, explored with the two tests. We performed a ROC curve analysis of the screening ratio to predict a positive SAB assay. The AUC were 0.72 and 0.64 for class I and class II. The optimal thresholds of screening ratios were 3.28 (class I) and 2.11 (class II). Whatever the class, the negative predictive value was low, around 40%, with 36% of discordant sera, as defined by negative screening and positive SAB. Testing class I discordant sera on acid-treated SAB showed that 54% of antibodies reacted against denatured HLA molecules. However, these screening-negative sera may contain donor-specific antibodies in 13.9% and 28.7% of cases for class I and class II, respectively, involved in antibody-mediated rejection with the same frequency as non-discordant sera. Given the low predictive value of screening, both assays should be performed at least once on the same serum before transplantation.

Increased Fatty Acid Oxidation in Differentiated Proximal Tubular Cells Surviving a Reversible Episode of Acute Kidney Injury.

BACKGROUND/AIMS: Fatty acid oxidation (FAO), the main source of energy produced by tubular epithelial cells in the kidney, was found to be defective in tubulo-interstitial samples dissected out in kidney biopsies from patients with chronic kidney disease (CKD). Experimental data indicated that this decrease was a strong determinant of renal fibrogenesis, hence a focus for therapeutic interventions. Nevertheless, whether persistently differentiated renal tubules, surviving in a pro-fibrotic environment, also suffer from a decrease in FAO, is currently unknown. METHODS: To address this question, we isolated proximal tubules captured ex vivo on the basis of the expression of an intact brush border antigen (Prominin-1) in C57BL6/J mice subjected to a controlled, two-hit model of renal fibrosis (reversible ischemic acute kidney injury (AKI) or sham surgery, followed by angiotensin 2 administration). A transcriptomic high throughput sequencing was performed on total mRNA from these cells, and on whole kidneys. RESULTS: In contrast to mice subjected to sham surgery, mice with a history of AKI displayed histologically more renal fibrosis when exposed to angiotensin 2. High throughput RNA sequencing, principal component analysis and clustering showed marked consistency within experimental groups. As expected, FAO transcripts were decreased in whole fibrotic kidneys. Surprisingly, however, up- rather than down-regulation of metabolic pathways (oxidative phosphorylation, fatty acid metabolism, glycolysis, and PPAR signalling pathway) was a hallmark of the differentiated tubules captured from fibrotic kidneys. Immunofluorescence co-staining analysis confirmed that the expression of FAO enzymes was dependent of tubular trophicity. CONCLUSIONS: These data suggest that in differentiated proximal tubules energetic hyperactivity is promoted concurrently with organ fibrogenesis.

Stress Response Gene Nupr1 Alleviates Cyclosporin A Nephrotoxicity In Vivo.

Acute tubular damage is a major cause of renal failure, especially at the early phase of kidney transplant when ischemia-reperfusion injury and cyclosporin A toxicity may coexist. The mechanisms of the latter are largely unknown. Using an mRNA microarray on microdissected tubules from a rat model of cyclosporin A toxicity to describe the related epithelial-specific transcriptional signature in vivo, we found that cyclosporin A induces pathways dependent on the transcription factor ATF4 and identified nuclear protein transcriptional regulator 1 (Nupr1), a stress response gene induced by ATF4, as the gene most strongly upregulated. Upon cyclosporin A treatment, Nupr1-deficient mice exhibited worse renal tubular lesions than wild-type mice. In primary cultures treated with cyclosporin A, renal tubular cells isolated from Nupr1-deficient mice exhibited more apoptosis and ATP depletion than cells from wild-type mice. Furthermore, cyclosporin A decreased protein synthesis and abolished proliferation in wild-type tubular cells, but only reduced proliferation in Nupr1-deficient cells. Compared with controls, mouse models of ischemia-reperfusion injury, urinary obstruction, and hypertension exhibited upregulated expression of renal NUPR1, and cyclosporin A induced Nupr1 expression in cultured human tubular epithelial cells. Finally, immunohistochemical analysis revealed strong expression of NUPR1 in the nuclei of renal proximal tubules of injured human kidney allografts, but not in those of stable allografts. Taken together, these results suggest that epithelial expression of NUPR1 has a protective role in response to injury after renal transplant and, presumably, in other forms of acute tubular damage.

Rapid Occurrence of Chronic Kidney Disease in Patients Experiencing Reversible Acute Kidney Injury after Cardiac Surgery.

BACKGROUND: There is recent evidence to show that patients suffering from acute kidney injury are at increased risk of developing chronic kidney disease despite the fact that surviving tubular epithelial cells have the capacity to fully regenerate renal tubules and restore renal function within days or weeks. The aim of the study was to investigate the impact of acute kidney injury on de novo chronic kidney disease. METHODS: The authors conducted a retrospective population-based cohort study of patients initially free from chronic kidney disease who were scheduled for elective cardiac surgery with cardiopulmonary bypass and who developed an episode of acute kidney injury from which they recovered. The study was conducted at two French university hospitals between 2005 and 2015. These individuals were matched with patients without acute kidney injury according to a propensity score for developing acute kidney injury. RESULTS: Among the 4,791 patients meeting the authors' inclusion criteria, 1,375 (29%) developed acute kidney injury and 685 fully recovered. Propensity score matching was used to balance the distribution of covariates between acute kidney injury and non- acute kidney injury control patients. Matching was possible for 597 cases. During follow-up, 34 (5.7%) had reached a diagnosis of chronic kidney disease as opposed to 17 (2.8%) in the control population (hazard ratio, 2.3; bootstrapping 95% CI, 1.9 to 2.6). CONCLUSIONS: The authors' data consolidate the recent paradigm shift, reporting acute kidney injury as a strong risk factor for the rapid development of chronic kidney disease.

Evaluation of the ability of bone marrow derived cells to engraft the kidney and promote renal tubular regeneration in mice following exposure to cisplatin.

It has been suggested that bone marrow derived stem cells have the ability to engraft the kidney and improve the outcome of severe acute kidney injury (AKI) in mice exposed to high doses of cisplatin, providing hope for cancer patients in whom irreversible renal damage occasionally occurs following the use of this highly effective anti-tumor drug. We tested the therapeutic potential of bone marrow derived cells injected during the acute phase (day 3 after cisplatin administration) of experimentally-induced AKI in C57Bl6/J mice, characterized by massive tubular necrosis, apoptosis, and a low proliferation capacity. We failed to show any benefit of bone marrow derived cells versus a regular homogenate of intact renal cells, or normal saline. Using cell tracers and flow cytometry, we demonstrated that bone marrow derived cells did indeed home to the bone marrow of the recipients but failed to settle in the kidney. Conversely, renal cells homed to injured kidneys. However, neither cell therapy protected the animals against cisplatin-induced death. We therefore question the short-term efficacy of bone marrow derived cells used to repair established injuries of the tubular epithelium.

Ex vivo analysis of renal proximal tubular cells.

BACKGROUND: Experimental models are inevitably a compromise between accurately reproducing a pathological situation and schematically simplifying it, which is intended to provide both relevance and conclusiveness. In-vivo models are very relevant, but multiple cell-types undergoing various changes may hinder the observation of individual molecular events. RESULTS: Here, we describe a method for analyzing and isolating specific cell types from the kidney and studying the phenotype they have acquired in vivo. Using flow cytometry, immunofluorescence, and RT-PCR, we show that our method is suitable for studying and isolating proximal tubular cells with an anti Prominin-1 antibody. Kidneys are subjected to mechanical dissociation followed by flow-cytometry analysis. Hundreds of thousands of proximal tubular cells are then isolated by magnetic separation followed by direct analysis or primary cell culture. Using our method, we detect phenotypic changes in the proximal tubular cells after renal ischemia reperfusion, and we isolate the proximal tubular cells, with a purity over 80%. CONCLUSIONS: This method is efficient, quick, simple, and cheap, and should be useful for studying cell-type specific parameters after in vivo experimental studies. It is also a simple method to obtain a specific primary cell culture from any animal strain.

Epigenetic changes during sepsis: on your marks!

Epigenetics is the study of how cells, organs, and even individuals utilize their genes over specific periods of time, and under specific environmental constraints. Very importantly, epigenetics is now expanding into the field of medicine and hence should provide new information for the development of drugs. Bomsztyk and colleagues have detected major epigenetic changes occurring in several organs as early as 6 h after the onset of a mouse model of multiple organ dysfunction syndrome induced by Staphylococcus aureus lung injury. Decrease in mRNA of key genes involved in endothelial function was found to be associated with (and potentially explained by) a decrease in permissive histone marks, while repressive marks were unchanged. We discuss here the limitations of a whole-organ as opposed to a cell-specific approach, the nature of the controls that were chosen, and the pitfalls of histone modifications as a cause of the eventual phenotype. While the use of 'epidrugs' is definitely welcome in the clinic, how and when they will be used in sepsis-related multiple organ dysfunction will require further experimental studies.

[The epithelial-mesenchymal transition and fibrosis of the renal transplant]. / La transition épithélio-mésenchymateuse et la fibrose du transplant rénal.

Epithelial-mesenchymal transition (EMT) is a process by which differentiated epithelial cells undergo a phenotypic conversion and acquire a mesenchymal phenotype, including elongated morphology, enhanced migratory and invasion capacity, and greatly increased production of extracellular matrix (ECM) components. This phenomenon plays a pivotal role in embryonic development, wound healing and tissue regeneration. It has also been involved in organ fibrosis. Some studies suggest that following injury, renal tubular epithelial cells undergo reprograming in mesenchymal cells, and thus constitute an important source of de novo myofibroblasts invading the renal interstitium and contributing to fibrosis. However, an increasing number of studies raise doubts about the existence of this process in vivo. The role of EMT in the development of renal fibrosis remains a matter of intense debate and may depend on the model studied. In this review, we describe the role of EMT in the development of fibrosis of renal graft, and then we propose approaches for detecting and treating renal fibrogenesis by targeting TEM.

Expression of the transcriptional regulator snail1 in kidney transplants displaying epithelial-to-mesenchymal transition features.

BACKGROUND: The epithelial response to injury is stereotypical and reminiscent of epithelial-to-mesenchymal transitions (EMTs), such as those observed during embryogenesis and tumour metastasis. In the context of solid organ transplantation, EMT-like features are often acquired by epithelial cells and are predictive of graft fibrosis. Here, we studied the possible involvement of several major transcriptional regulators, including snail1, phospho-Smad 2/3 and zeb1, in EMT induction in human renal grafts. METHODS: We used immunohistochemistry to detect the presence of these EMT transcriptional regulators along with that of two validated EMT markers (intra-cytoplasmic translocation of ß-catenin, de novo expression of vimentin), in 103 renal graft biopsy samples taken for routine surveillance or for a clinical indication. RESULTS: We observed the nuclear accumulation of snail1 and phospho-smad2/3 in tubular cells displaying EMT. The level of snail1 was significantly correlated with the scores of EMT markers (ß-catenin: ρ = 0.94, P < 0.0001; vimentin: ρ = 0.93, P < 0.0001) and with deteriorated graft function and proteinuria at the time of biopsy. Furthermore, intense staining for both snail1 and vimentin in tubular cells (≥10% of tubules) was predictive of graft dysfunction 21 months post-biopsy, independently of the other known risk factor for long-term graft dysfunction. In contrast, in both normal and diseased graft, zeb1 expression was detected exclusively in the endothelial cells of glomeruli and peritubular capillaries. CONCLUSION: This study suggests that snail1 is closely related to the fibrogenic, EMT-like response of the tubular epithelium in human renal grafts and predictive of graft function loss.

Unrecognized sequence homologies may confound genome-wide association studies.

Genome-wide association studies (GWAS) have become a preferred method to identify new genetic susceptibility loci. This technique aims to understanding the molecular etiology of common diseases, but in many cases, it has led to the identification of loci with no obvious biological relevance. Herein, we show that previously unrecognized sequence homologies have caused single-nucleotide polymorphism (SNP) microarrays to incorrectly associate a phenotype to a given locus when in fact the linkage is to another distant locus. Using genetic differences between male and female subjects as a model to study the effect of one specific genomic region on the whole SNP microarray, we provide strong evidence that the use of standard methods for GWAS can be misleading. We suggest a new systematic quality control step in the biological interpretation of previous and future GWAS.

Effect of Cardiac Arrest in Brain-dead Donors on Kidney Graft Function.

BACKGROUND: Cardiac arrest (CA) causes renal ischemia in one-third of brain-dead kidney donors before procurement. We hypothesized that the graft function depends on the time interval between CA and organ procurement. METHODS: We conducted a retrospective population-based study on a prospectively curated database. We included 1469 kidney transplantations from donors with a history of resuscitated CA in 2015-2017 in France. CA was the cause of death (primary CA) or an intercurrent event (secondary CA). The main outcome was the percentage of delayed graft function, defined by the use of renal replacement therapy within the first week posttransplantation. RESULTS: Delayed graft function occurred in 31.7% of kidney transplantations and was associated with donor function, vasopressors, cardiovascular history, donor and recipient age, body mass index, cold ischemia time, and time to procurement after primary cardiac arrest. Short cold ischemia time, perfusion device use, and the absence of cardiovascular comorbidities were protected by multivariate analysis, whereas time <3 d from primary CA to procurement was associated with delayed graft function (odds ratio 1.38). CONCLUSIONS: This is the first description of time to procurement after a primary CA as a risk factor for delayed graft function. Delaying procurement after CA should be evaluated in interventional studies.