Spatial transcriptomics: Integrating morphology and molecular mechanisms of kidney diseases.

The recent arrival of high-resolution spatial transcriptomics (ST) technologies is generating a veritable revolution in life sciences, enabling biomolecules to be measured in their native spatial context. By integrating morphology and molecular biology, ST technologies offer the potential of improving the understanding of tissue biology and disease and may also provide meaningful clinical insights. In this review, we describe the main spatial transcriptomics technologies currently available, the computational analysis for data interpretation and visualization, and illustrate their scientific and potential medical interest in the context of kidney disease. Finally, we discuss the perspectives and challenges of these booming new technologies.

Histopathological-based analysis of human kidney spatial transcriptomics data: toward precision pathology.

The application of spatial transcriptomic (ST) technologies is booming, and has already yielded important insights across many different tissues and disease models. In nephrology, ST technologies have helped to decipher the cellular and molecular mechanisms at work in kidney diseases and have allowed the recent creation of spatially anchored human kidney atlases in healthy and diseased kidney tissues. During ST data analysis, the obtained computationally-annotated clusters are often superimposed on a histological image without their initial identification being based on the morphological and spatial analysis of the tissues and lesions. In this study, we conduct a histopathological-based analysis of spatial transcriptomics data on a human kidney sample corresponding as closely as possible to the reality of the interpretation of a kidney biopsy in a healthcare or research context. Our work demonstrates the feasibility of a morphological-based approach to interpreting ST data, helping to improve our understanding of the lesional phenomena at work in CKD, at both cellular and molecular levels. Finally, we show that our newly identified pathological-based clusters can be accurately projected onto other slides from nephrectomy or needle biopsies samples and thus serve as a reference for analyzing other kidney tissues paving the way for the future of molecular microscopy and precision pathology.

Combined germline and somatic human FADD mutations cause autoimmune lymphoproliferative syndrome.

BACKGROUND: The autoimmune lymphoproliferative syndrome (ALPS) is a noninfectious and nonmalignant lymphoproliferative disease frequently associated with autoimmune cytopenia resulting from defective FAS signaling. We previously described germline monoallelic FAS (TNFRSF6) haploinsufficient mutations associated with somatic events, such as loss of heterozygosity on the second allele of FAS, as a cause of ALPS-FAS. These somatic events were identified by sequencing FAS in DNA from double-negative (DN) T cells, the pathognomonic T-cell subset in ALPS, in which the somatic events accumulated. OBJECTIVE: We sought to identify whether a somatic event affecting the FAS-associated death domain (FADD) gene could be related to the disease onset in 4 unrelated patients with ALPS carrying a germline monoallelic mutation of the FADD protein inherited from a healthy parent. METHODS: We sequenced FADD and performed array-based comparative genomic hybridization using DNA from sorted CD4+ or DN T cells. RESULTS: We found homozygous FADD mutations in the DN T cells from all 4 patients, which resulted from uniparental disomy. FADD deficiency caused by germline heterozygous FADD mutations associated with a somatic loss of heterozygosity was a phenocopy of ALPS-FAS without the more complex symptoms reported in patients with germline biallelic FADD mutations. CONCLUSIONS: The association of germline and somatic events affecting the FADD gene is a new genetic cause of ALPS.

The renal inflammatory network of nephronophthisis.

Renal ciliopathies are the leading cause of inherited kidney failure. In autosomal dominant polycystic kidney disease (ADPKD), mutations in the ciliary gene PKD1 lead to the induction of CCL2, which promotes macrophage infiltration in the kidney. Whether or not mutations in genes involved in other renal ciliopathies also lead to immune cells recruitment is controversial. Through the parallel analysis of patients' derived material and murine models, we investigated the inflammatory components of nephronophthisis (NPH), a rare renal ciliopathy affecting children and adults. Our results show that NPH mutations lead to kidney infiltration by neutrophils, macrophages and T cells. Contrary to ADPKD, this immune cell recruitment does not rely on the induction of CCL2 in mutated cells, which is dispensable for disease progression. Through an unbiased approach, we identified a set of inflammatory cytokines that are upregulated precociously and independently of CCL2 in murine models of NPH. The majority of these transcripts is also upregulated in NPH patient renal cells at a level exceeding those found in common non-immune chronic kidney diseases. This study reveals that inflammation is a central aspect in NPH and delineates a specific set of inflammatory mediators that likely regulates immune cell recruitment in response to NPH genes mutations.

Effects of hemorrhagic shock and rhabdomyolysis on renal microcirculation, oxygenation and function in a female swine model.

BACKGROUND: Hemorrhagic shock (HS) and rhabdomyolysis (RM) are two important risk factors of acute kidney injury (AKI) after severe trauma, however the effects of the combination of RM and HS on kidney function are unknown. The purpose of this study was to determine the impact of RM and HS on renal function, oxygenation, perfusion and morphology, in a pig model. METHODS: Forty-seven female pigs were divided into 5 groups: sham, RM, HS, HS and moderate RM (RM4/HS), HS and severe RM (RM8/HS). RM was induced by intramuscular injection of Glycerol 50% with a moderate dose (4 ml/kg for RM4/HS group) or a high dose (8 ml/kg for RM and RM8/HS groups). Among animals with HS, after 90 min of hemorrhage, animals were resuscitated with fluid followed by transfusion of the withdrawn blood. Animals were followed for 48 hours. Macro and microcirculatory parameters measurements were performed. RESULTS: RM alone induced a decrease in creatinine clearance at 48 hours (19 (0-41) vs 102 (56-116) ml/min for RM and SHAM respectively; p = 0.0006) without alteration in renal perfusion and oxygenation. HS alone impaired temporarily renal microcirculation, function and oxygenation that were restored with fluid resuscitation. RM4/HS and RM8/HS groups induced greater impairment of renal microcirculation and function than HS alone at the end of blood spoliation that were not improved by fluid resuscitation. Mortality was increased in RM8/HS and RM4/HS groups in the first 48 hours (73% vs 56% vs 9% for RM8/HS, RM4/HS and HS groups respectively). CONCLUSIONS: The combination of HS and RM induced an early deleterious effect on renal microcirculation, function and oxygenation with decreased response to resuscitation and transfusion compared with HS or RM alone.

Comparative histological analysis of spleens in pediatric patients with hemolytic anemias: Insights into the pathophysiological mechanisms of spleen destruction in sickle cell anemia.

While sickle cell anemia (SCA) and hereditary spherocytosis (HS) share common features of increased spleen erythrophagocytosis due to increased red blood cell (RBC) turnover, SCA is specifically characterized by susceptibility to infections. In this study, histological lesions in the spleens of pediatric patients with SCA were analyzed, in close correlation with past clinical history and comparatively to HS, healthy and transfused ß-thalassemia patients (TDT). An evaluation of red pulp elementary lesions (red pulp fibrosis, iron deposition, number of Gandy-Gamna, and RBC trapping) combined into a severity score was established, as well as B-cell follicles analysis. Quantification on digitalized slides of iron deposition, RBC trapping, and red pulp fibrosis was additionally performed. Spleens from 22 children with SCA, eight with HS, eight with TDT, and three healthy controls (HC) were analyzed. Median age at splenectomy was not different between SCA and HS patients, 6.05 years (range: 4.5-16.0) versus 4.75 (range: 2.2-9.5). Marked heterogeneity was found in SCA spleens in contrast to other conditions. Contrary to previous reports, B-cell follicles were generally preserved in SCA. While RBC trapping was significantly increased in both SCA and HS (compared to TDT and HC), quantitative fibrosis and overall red pulp severity score were significantly increased in SCA spleens compared to other conditions. Moreover, there was an inverse correlation between quantitative fibrosis and number of B-cell follicles, linking these two compartments as well as spleen fibrosis to infectious susceptibility in SCA, potentially through impaired red pulp macrophage scavenging and B-cell subpopulations defects.

The spectrum of glomerular and vascular kidney pathology associated with myeloproliferative neoplasms.

A high prevalence of chronic kidney disease (CKD) occurs in patients with myeloproliferative neoplasms (MPN). However, MPN-related glomerulopathy (MPN-RG) may not account for the entirety of CKD risk in this population. The systemic vasculopathy encountered in these patients raises the hypothesis that vascular nephrosclerosis may be a common pattern of injury in patients with MPN and with CKD. In an exhaustive, retrospective, multicenter study of MPN kidney biopsies in four different pathology departments, we now describe glomerular and vascular lesions and establish clinicopathologic correlations. Our study encompassed 47 patients with MPN who underwent a kidney biopsy that included 16 patients with chronic myeloid leukemia (CML) and 31 patients with non-CML MPN. Fourteen cases met a proposed definition of MPN-RG based on mesangial sclerosis and hypercellularity, as well as glomerular thrombotic microangiopathy. MPN-RG was significantly associated with both myelofibrosis and poorer kidney survival. Thirty-three patients had moderate-to-severe arteriosclerosis while 39 patients had moderate-to-severe arteriolar hyalinosis. Multivariable models that included 188 adult native kidney biopsies as controls revealed an association between MPN and chronic kidney vascular damage, which was independent of established risk factors such as age, diabetes mellitus and hypertension. Therefore, MPN-RG is associated with myelofibrosis and has a poor kidney prognosis. Thus, our findings suggest that the kidney vasculature is a target during MPN-associated vasculopathy and establish a new link between MPN and CKD. Hence, these results may raise new hypotheses regarding the pathophysiology of vascular nephrosclerosis in the general population.

Stiripentol and Lumasiran as a Rescue Therapy for Oxalate Nephropathy Recurrence After Kidney Transplantation in an Adult Patient With Primary Hyperoxaluria Type 1.

Primary hyperoxaluria type 1 is a rare cause of kidney failure. Stiripentol, an inhibitor of lactate dehydrogenase A, and lumasiran, a small interfering RNA targeting glycolate oxidase, have been proposed as therapeutic options, but clinical data are scarce, especially in adults and transplanted patients. We describe the case of a 51-year-old patient with a biopsy-proven recurrence of oxalate nephropathy after a kidney-only transplantation. He received stiripentol and lumasiran without adverse events. Fourteen months after transplantation, graft function, serum, and urinary oxalate levels have remained stable, and kidney biopsy showed a complete regression of oxalate crystals. Further studies are needed to assess whether this strategy is effective and could replace liver-kidney transplantation.

Renal Microcirculation and Function in a Pig Model of Hemorrhagic Shock Resuscitation with Norepinephrine.

Rationale: Norepinephrine (NE) is commonly used in combination with fluid during resuscitation of hemorrhagic shock, but its impact on kidney microcirculation, oxygenation, and function is still unknown in this setting. Objectives: During hemorrhagic shock resuscitation, does a combination of fluid and NE affect kidney oxygenation tension, kidney microcirculatory perfusion, and 48-hour kidney function, as compared with fluid alone? Methods: Hemorrhagic shock was induced in 24 pigs, and 8 pigs were included as a sham group. Resuscitation of hemorrhagic shock was performed, using a closed-loop device, either by fluid alone (0.9% NaCl; fluid group) or associated with the administration of NE at two doses (moderate dose: mean rate of 0.64 µg ⋅ kg-1 ⋅ min-1; high dose: mean rate of 1.57 µg ⋅ kg-1 ⋅ min-1) to obtain a target systolic arterial pressure of 80 to 90 mm Hg. Resuscitation was followed by transfusion of the withdrawn blood. Measurements and Main Results: The amount of fluid required to reach the target systolic arterial pressure was lower in the NE groups than in the fluid group, with subsequently less hemodilution. NE restored kidney microcirculation, oxygenation, and function in a manner comparable to that achieved with fluid resuscitation alone. There were no histologic differences between animals resuscitated with fluid and those resuscitated with NE. Conclusions: In pigs with hemorrhagic shock, resuscitation with a combination of NE and fluid restored kidney microcirculation and oxygenation, as well as renal function, in a manner comparable to fluid resuscitation alone and without differences between the two NE doses. NE administration led to a fluid volume-sparing effect with subsequently less hemodilution.

[Immunopathology of the small intestine]. / Immunopathologie de l'intestin grêle.

The gastrointestinal tract is the site of exciting immunological interactions between the epithelium and the mucosa-associated lymphoid tissue, leading to the immune response to food and microbial antigens in the digestive lumen. The objective of this review is to present the main dysimmune pathologies of the digestive tract leading to an enteropathy. As examples, we describe celiac and non-celiac enteropathies to clarify a florid diagnostic framework, by identifying a spectrum of elementary lesions, which must be confronted with the clinico biological context of the patient to orient the diagnosis. The microscopic lesions observed are most often non-specific and may be encountered in several diagnostic settings. Moreover, it is a set of elementary lesions in each clinical context that will orient the diagnostic framework. Celiac disease is the main etiology of enteropathy with villous atrophy, its diagnosis is multidisciplinary and there are many differential diagnoses. We will discuss celiac disease lymphomatous complications as enteropathy associated T-cell lymphoma including refractory sprue type 2. We will then present the non-celiac enteropathies. Among these, enteropathies of unknown etiology may be associated with a primary immune deficiency that may be reflected by florid lymphoid hyperplasia of the gastrointestinal tract and/or be associated with an infectious etiology that should also be constantly sought. Finally, we will discuss of induced enteropathy by new immunomodulatory treatments.

Temporal virus serological profiling of kidney graft recipients using VirScan.

At this time, pretransplant viral screening of donors and recipients is based on serological status and limited to certain viruses. After transplantation, patient follow-up is based on a monitoring strategy using ELISA or PCR. Such approaches exclude other emerging viruses that can affect the transplant outcome. Recently, a multiplex unbiased array, VirScan, was developed. This tool allows the detection of antibodies against viruses, using a synthetic human virome, with minimal serum and cost. We decided to test the value of VirScan in the follow-up of a cohort of transplant recipients. We enrolled 45 kidney transplant recipients and performed virus serological profiling at day 0 and day +365, using VirScan. We compared the results obtained with ELISA/PCR assays. We detected antibody responses to 39 of the 206 species of virus present in the VirScan library, with an average of 12 species of virus per sample. VirScan gave similar results to PCR/ELISA screening tests. Using VirScan, we found that anti-viral antibody responses were largely conserved in patients during the first year after transplantation, regardless of immunosuppressive treatment. Our study suggests VirScan offers an unprecedented opportunity to screen and monitor posttransplant virus infection in a cost-effective, easy, and unbiased manner.

REST and Stress Resistance in the Aging Kidney.

BACKGROUND: CKD is associated with the loss of functional nephr ons, leading to increased mechanical and metabolic stress in the remaining cells, particularly for cells constituting the filtration barrier, such as podocytes. The failure of podocytes to mount an adequate stress response can lead to further nephron loss and disease progression. However, the mechanisms that regulate this degenerative process in the kidney are unknown. METHODS: We combined in vitro, in vivo, and organ-on-chip approaches to identify the RE1-silencing transcription factor (REST), a repressor of neuronal genes during embryonic development, as a central regulator of podocyte adaptation to injury and aging. RESULTS: Mice with a specific deletion of REST in podocytes exhibit albuminuria, podocyte apoptosis, and glomerulosclerosis during aging, and exhibit increased vulnerability to renal injury. This phenotype is mediated, in part, by the effects of REST on the podocyte cytoskeleton that promote resistance to mechanical stressors and augment podocyte survival. Finally, REST expression is upregulated in human podocytes during aging, consistent with a conserved mechanism of stress resistance. CONCLUSIONS: These results suggest REST protects the kidney from injury and degeneration during aging, with potentially important therapeutic implications.

Dissociation of humoral and cellular immune responses in kidney transplant recipients with EBV mucocutaneous ulcer.

EBV-positive mucocutaneous ulcer (EBV-MCU) is a rare EBV-positive B-cell lymphoproliferative disorder occurring in immunocompromised patients such as patients with solid organ or hematopoietic stem cells transplantation. EBV-MCU often consists of an isolated and circumscribed cutaneous or mucosal ulcerative lesion with a self-limited growth potential and a high regression rate upon immunosuppressive treatment withdrawal or rituximab therapy. Nevertheless, the pathophysiology of this latent infection leading to clonal lymphoproliferation is not well established. We report here two cases of EBV-MCU in kidney transplant recipients with a dissociated immune response to EBV with the absence of EBV-related antibodies and a positive T-cell response to EBV suggesting a potential specific oncogenic mechanism in this lymphoproliferative disorder.

[Pathology of heart transplantation: Where are we now?] / Pathologie de la transplantation cardiaque : où en est-on 15 ans et 7 ans après les documents de consensus sur le rejet cellulaire et humoral.

Pathology is still the gold standard for the diagnosis of rejection in heart transplantation. During the last decade, molecular pathology has emerged as a powerful tool for the understanding of the processes implicated in allograft rejection. Transcriptomic analysis of the allograft may also help the pathologist for diagnosis and accurate classification of rejection. This review will describe the recent advances and perspectives of molecular pathology in the field of heart transplantation.