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1.
J Clin Invest ; 2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33591954

RESUMO

Podocytes are key to kidney glomerular filtration barrier by forming slit diaphragm between interdigitating foot processes; however, molecular details and functional importance of protein folding and degradation in the ER remain unknown. Here we show that SEL1L-HRD1 protein complex of endoplasmic reticulum (ER)-associated degradation (ERAD) is required for slit diaphragm formation and glomerular filtration function. SEL1L-HRD1 ERAD is highly expressed in podocytes of both mouse and human kidneys. Mice with podocyte-specific Sel1L deficiency develop podocytopathy and severe congenital nephrotic syndrome shortly after weaning with impaired slit diaphragm, and die prematurely with a median life span of ~3 months. Mechanistically, we show that nephrin, a type-1 membrane protein causally linked to congenital nephrotic syndrome, is an endogenous ERAD substrate. ERAD deficiency attenuates the maturation of nascent nephrin, leading to its retention in the ER. Lastly, we show that various autosomal-recessive nephrin disease mutants are highly unstable and degraded by Sel1L-Hrd1 ERAD, which attenuates the pathogenicity of the mutants towards the wildtype allele. Hence, this study uncovers a critical role of Sel1L-Hrd1 ERAD in glomerular filtration barrier function and provides new insights into the pathogenesis associated with autosomal recessive disease mutants.

2.
J Clin Pathol ; 2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431484

RESUMO

AIMS: Detection of one segmentally sclerosed glomerulus (SSG) identifies patients with focal segmental glomerulosclerosis (FSGS) but rare SSGs may be missed in kidney biopsies. It is unknown whether alterations of unaffected glomeruli in patients with infrequent SSG can be detected by quantitative morphometrics. METHODS: We determined SSG frequency and obtained quantitative morphometrics in glomeruli without a pathologic phenotype in large kidney sections of non-involved kidney tissue from 137 patients undergoing total nephrectomy. We used multivariate modelling to identify morphometrics independently associated with increasing frequency of SSG and Receiver Operator Curve (ROC) analysis to determine the ability of quantitative morphometrics to identify patients with FSGS. We used the geometric distribution to estimate the sensitivity and specificity of a needle biopsy to identify patients with FSGS. RESULTS: In seventy-one patients (51.8%), at least one SSG was observed, and of those, 39 (54.9%) had an SSG lesion in less than 2% of all glomeruli (mean of 249 glomeruli per specimen). Increasing percent of SSG was independently associated with decreasing podocyte density and increasing mesangial index in multivariate modelling. For infrequent SSG lesions (<1% of glomeruli), kidney biopsy could miss FSGS diagnosis more than 74% of the time, and podocyte density had an area under the curve (AUC) of 0.77, and mesangial index, an AUC of 0.79 to identify patients with FSGS. CONCLUSIONS: More than half of patients had FSGS, although 30% had infrequent SSG. Quantitative morphometrics in glomeruli without pathology, such as podocyte density and mesangial index, identified patients with infrequent SSG and may serve as clinical markers to identify patients with FSGS.

3.
Physiol Genomics ; 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33197228

RESUMO

Comprehensive and spatially mapped molecular atlases of organs at a cellular level are a critical resource to gain insights into pathogenic mechanisms and personalized therapies for diseases. The Kidney Precision Medicine Project (KPMP) is an endeavor to generate 3-dimensional (3D) molecular atlases of healthy and diseased kidney biopsies using multiple state-of-the-art OMICS and imaging technologies across several institutions. Obtaining rigorous and reproducible results from disparate methods and at different sites to interrogate biomolecules at a single cell level or in 3D space is a significant challenge that can be a futile exercise if not well controlled. We describe a "follow the tissue" pipeline for generating a reliable and authentic single cell/region 3D molecular atlas of human adult kidney. Our approach emphasizes quality assurance, quality control, validation and harmonization across different OMICS and imaging technologies from sample procurement, processing, storage, shipping to data generation, analysis and sharing. We established benchmarks for quality control, rigor, reproducibility and feasibility across multiple technologies through a pilot experiment using common source tissue that was processed and analyzed at different institutions and different technologies. A peer review system was established to critically review quality control measures and the reproducibility of data generated by each technology before being approved to interrogate clinical biopsy specimens. The process established economizes the use of valuable biopsy tissue for multi-OMICS and imaging analysis with stringent quality control to ensure rigor and reproducibility of results and serves as a model for precision medicine projects across laboratories, institutions and consortia.

4.
Kidney Int ; 98(6): 1502-1518, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33038424

RESUMO

COVID-19 morbidity and mortality are increased via unknown mechanisms in patients with diabetes and kidney disease. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) for entry into host cells. Because ACE2 is a susceptibility factor for infection, we investigated how diabetic kidney disease and medications alter ACE2 receptor expression in kidneys. Single cell RNA profiling of kidney biopsies from healthy living donors and patients with diabetic kidney disease revealed ACE2 expression primarily in proximal tubular epithelial cells. This cell-specific localization was confirmed by in situ hybridization. ACE2 expression levels were unaltered by exposures to renin-angiotensin-aldosterone system inhibitors in diabetic kidney disease. Bayesian integrative analysis of a large compendium of public -omics datasets identified molecular network modules induced in ACE2-expressing proximal tubular epithelial cells in diabetic kidney disease (searchable at hb.flatironinstitute.org/covid-kidney) that were linked to viral entry, immune activation, endomembrane reorganization, and RNA processing. The diabetic kidney disease ACE2-positive proximal tubular epithelial cell module overlapped with expression patterns seen in SARS-CoV-2-infected cells. Similar cellular programs were seen in ACE2-positive proximal tubular epithelial cells obtained from urine samples of 13 hospitalized patients with COVID-19, suggesting a consistent ACE2-coregulated proximal tubular epithelial cell expression program that may interact with the SARS-CoV-2 infection processes. Thus SARS-CoV-2 receptor networks can seed further research into risk stratification and therapeutic strategies for COVID-19-related kidney damage.

5.
Kidney Int ; 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32835732

RESUMO

The application of deep learning for automated segmentation (delineation of boundaries) of histologic primitives (structures) from whole slide images can facilitate the establishment of novel protocols for kidney biopsy assessment. Here, we developed and validated deep learning networks for the segmentation of histologic structures on kidney biopsies and nephrectomies. For development, we examined 125 biopsies for Minimal Change Disease collected across 29 NEPTUNE enrolling centers along with 459 whole slide images stained with Hematoxylin & Eosin (125), Periodic Acid Schiff (125), Silver (102), and Trichrome (107)] divided into training, validation and testing sets (ratio 6:1:3). Histologic structures were manually segmented (30048 total annotations) by five nephropathologists. Twenty deep learning models were trained with optimal digital magnification across the structures and stains. Periodic Acid Schiff-stained whole slide images yielded the best concordance between pathologists and deep learning segmentation across all structures (F-scores: 0.93 for glomerular tufts, 0.94 for glomerular tuft plus Bowman's capsule, 0.91 for proximal tubules, 0.93 for distal tubular segments, 0.81 for peritubular capillaries, and 0.85 for arteries and afferent arterioles). Optimal digital magnifications were 5X for glomerular tuft/tuft plus Bowman's capsule, 10X for proximal/distal tubule, arteries and afferent arterioles, and 40X for peritubular capillaries. Silver stained whole slide images yielded the worst deep learning performance. Thus, this largest study to date adapted deep learning for the segmentation of kidney histologic structures across multiple stains and pathology laboratories. All data used for training and testing and a detailed online tutorial will be publicly available.

6.
medRxiv ; 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32511461

RESUMO

COVID-19 morbidity and mortality is significantly increased in patients with diabetes and kidney disease via unknown mechanisms. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) for entry into human host cells, and ACE2 levels in target cells may influence SARS-CoV-2 susceptibility. We investigated how pre-existing conditions and drug treatments alter receptor expression in kidney tissue. Using single cell RNA profiling (scRNAseq) to assess ACE2 and associated SARS-CoV-2 proteases in healthy living donors (LD) kidneys, diabetic kidney disease (DKD), and in kidney injury during viral infection, ACE2 expression was primarily associated with proximal tubular epithelial cells (PTEC). ACE2 mRNA expression levels were significantly upregulated in DKD versus LD, however, ACE2 levels were not altered by exposures to renin angiotensin aldosterone system (RAAS) inhibitors. ACE2+ expression signatures were defined by differential expression analysis and characterized by Bayesian integrative analysis of a large compendium of public -omics datasets, resulting in the identification of network modules induced in ACE2 positive PTEC in DKD and BK virus nephropathy. These ACE2 upregulated cell programs were linked to viral entry, immune activation, endomembrane reorganization, and RNA processing and overlapped significantly with the cellular responses induced by SARS-CoV-2 infection. Similar cellular programs were activated in ACE2-positive PTEC isolated in a urine sample from a COVID19 patient with acute kidney injury, suggesting a consistent ACE2-coregulated expression program that may interact with SARS-Cov-2 infection processes. The SARS-CoV-2 receptor associated gene signatures could seed further research into therapeutic strategies for COVID-19. Functional networks of gene expression signatures are available for further exploration to researchers at HumanBase (hb.flatironinstitute.org/covid-kidney).

7.
J Am Soc Nephrol ; 31(8): 1883-1904, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32561683

RESUMO

BACKGROUND: The mechanisms leading to extracellular matrix (ECM) replacement of areas of glomerular capillaries in histologic variants of FSGS are unknown. This study used proteomics to test the hypothesis that glomerular ECM composition in collapsing FSGS (cFSGS) differs from that of other variants. METHODS: ECM proteins in glomeruli from biopsy specimens of patients with FSGS not otherwise specified (FSGS-NOS) or cFSGS and from normal controls were distinguished and quantified using mass spectrometry, verified and localized using immunohistochemistry (IHC) and confocal microscopy, and assessed for gene expression. The analysis also quantified urinary excretion of ECM proteins and peptides. RESULTS: Of 58 ECM proteins that differed in abundance between cFSGS and FSGS-NOS, 41 were more abundant in cFSGS and 17 in FSGS-NOS. IHC showed that glomerular tuft staining for cathepsin B, cathepsin C, and annexin A3 in cFSGS was significantly greater than in other FSGS variants, in minimal change disease, or in membranous nephropathy. Annexin A3 colocalized with cathepsin B and C, claudin-1, phosphorylated ERK1/2, and CD44, but not with synaptopodin, in parietal epithelial cells (PECs) infiltrating cFSGS glomeruli. Transcripts for cathepsins B and C were increased in FSGS glomeruli compared with normal controls, and urinary excretion of both cathepsins was significantly greater in cFSGS compared with FSGS-NOS. Urinary excretion of ECM-derived peptides was enhanced in cFSGS, although in silico analysis did not identify enhanced excretion of peptides derived from cathepsin B or C. CONCLUSIONS: ECM differences suggest that glomerular sclerosis in cFSGS differs from that in other FSGS variants. Infiltration of activated PECs may disrupt ECM remodeling in cFSGS. These cells and their cathepsins may be therapeutic targets.

8.
J Inherit Metab Dis ; 43(5): 1131-1142, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32233035

RESUMO

Sphingosine-1-phosphate (S1P) lyase is a vitamin B6-dependent enzyme that degrades sphingosine-1-phosphate in the final step of sphingolipid metabolism. In 2017, a new inherited disorder was described caused by mutations in SGPL1, which encodes sphingosine phosphate lyase (SPL). This condition is referred to as SPL insufficiency syndrome (SPLIS) or alternatively as nephrotic syndrome type 14 (NPHS14). Patients with SPLIS exhibit lymphopenia, nephrosis, adrenal insufficiency, and/or neurological defects. No targeted therapy for SPLIS has been reported. Vitamin B6 supplementation has therapeutic activity in some genetic diseases involving B6-dependent enzymes, a finding ascribed largely to the vitamin's chaperone function. We investigated whether B6 supplementation might have activity in SPLIS patients. We retrospectively monitored responses of disease biomarkers in patients supplemented with B6 and measured SPL activity and sphingolipids in B6-treated patient-derived fibroblasts. In two patients, disease biomarkers responded to B6 supplementation. S1P abundance and activity levels increased and sphingolipids decreased in response to B6. One responsive patient is homozygous for an SPL R222Q variant present in almost 30% of SPLIS patients. Molecular modeling suggests the variant distorts the dimer interface which could be overcome by cofactor supplementation. We demonstrate the first potential targeted therapy for SPLIS and suggest that 30% of SPLIS patients might respond to cofactor supplementation.

9.
J Am Soc Nephrol ; 31(4): 841-854, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32086276

RESUMO

BACKGROUND: The analysis and reporting of glomerular features ascertained by electron microscopy are limited to few parameters with minimal predictive value, despite some contributions to disease diagnoses. METHODS: We investigated the prognostic value of 12 electron microscopy histologic and ultrastructural changes (descriptors) from the Nephrotic Syndrome Study Network (NEPTUNE) Digital Pathology Scoring System. Study pathologists scored 12 descriptors in NEPTUNE renal biopsies from 242 patients with minimal change disease or FSGS, with duplicate readings to evaluate reproducibility. We performed consensus clustering of patients to identify unique electron microscopy profiles. For both individual descriptors and clusters, we used Cox regression models to assess associations with time from biopsy to proteinuria remission and time to a composite progression outcome (≥40% decline in eGFR, with eGFR<60 ml/min per 1.73 m2, or ESKD), and linear mixed models for longitudinal eGFR measures. RESULTS: Intrarater and interrater reproducibility was >0.60 for 12 out of 12 and seven out of 12 descriptors, respectively. Individual podocyte descriptors such as effacement and microvillous transformation were associated with complete remission, whereas endothelial cell and glomerular basement membrane abnormalities were associated with progression. We identified six descriptor-based clusters with distinct electron microscopy profiles and clinical outcomes. Patients in a cluster with more prominent foot process effacement and microvillous transformation had the highest rates of complete proteinuria remission, whereas patients in clusters with extensive loss of primary processes and endothelial cell damage had the highest rates of the composite progression outcome. CONCLUSIONS: Systematic analysis of electron microscopic findings reveals clusters of findings associated with either proteinuria remission or disease progression.


Assuntos
Membrana Basal Glomerular/ultraestrutura , Glomerulosclerose Segmentar e Focal/patologia , Nefrose Lipoide/patologia , Podócitos/ultraestrutura , Proteinúria/patologia , Adolescente , Adulto , Progressão da Doença , Feminino , Taxa de Filtração Glomerular , Glomerulosclerose Segmentar e Focal/complicações , Humanos , Masculino , Microscopia Eletrônica , Nefrose Lipoide/complicações , Valor Preditivo dos Testes , Prognóstico , Proteinúria/etiologia , Reprodutibilidade dos Testes , Adulto Jovem
10.
JCI Insight ; 5(6)2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32107344

RESUMO

To define cellular mechanisms underlying kidney function and failure, the KPMP analyzes biopsy tissue in a multicenter research network to build cell-level process maps of the kidney. This study aimed to establish a single cell RNA sequencing strategy to use cell-level transcriptional profiles from kidney biopsies in KPMP to define molecular subtypes in glomerular diseases. Using multiple sources of adult human kidney reference tissue samples, 22,268 single cell profiles passed KPMP quality control parameters. Unbiased clustering resulted in 31 distinct cell clusters that were linked to kidney and immune cell types using specific cell markers. Focusing on endothelial cell phenotypes, in silico and in situ hybridization methods assigned 3 discrete endothelial cell clusters to distinct renal vascular beds. Transcripts defining glomerular endothelial cells (GEC) were evaluated in biopsies from patients with 10 different glomerular diseases in the NEPTUNE and European Renal cDNA Bank (ERCB) cohort studies. Highest GEC scores were observed in patients with focal segmental glomerulosclerosis (FSGS). Molecular endothelial signatures suggested 2 distinct FSGS patient subgroups with α-2 macroglobulin (A2M) as a key downstream mediator of the endothelial cell phenotype. Finally, glomerular A2M transcript levels associated with lower proteinuria remission rates, linking endothelial function with long-term outcome in FSGS.

11.
JCI Insight ; 4(8)2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30996136

RESUMO

Autoimmune disease is 4 times more common in women than men. This bias is largely unexplained. Female skin is "autoimmunity prone," showing upregulation of many proinflammatory genes, even in healthy women. We previously identified VGLL3 as a putative transcription cofactor enriched in female skin. Here, we demonstrate that skin-directed overexpression of murine VGLL3 causes a severe lupus-like rash and systemic autoimmune disease that involves B cell expansion, autoantibody production, immune complex deposition, and end-organ damage. Excess epidermal VGLL3 drives a proinflammatory gene expression program that overlaps with both female skin and cutaneous lupus. This includes increased B cell-activating factor (BAFF), the only current biologic target in systemic lupus erythematosus (SLE); IFN-κ, a key inflammatory mediator in cutaneous lupus; and CXCL13, a biomarker of early-onset SLE and renal involvement. Our results demonstrate that skin-targeted overexpression of the female-biased factor VGLL3 is sufficient to drive cutaneous and systemic autoimmune disease that is strikingly similar to SLE. This work strongly implicates VGLL3 as a pivotal orchestrator of sex-biased autoimmunity.


Assuntos
Autoimunidade/genética , Regulação da Expressão Gênica/imunologia , Lúpus Eritematoso Cutâneo/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fatores de Transcrição/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Lúpus Eritematoso Cutâneo/genética , Lúpus Eritematoso Cutâneo/patologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Masculino , Camundongos , Camundongos Transgênicos , Fatores Sexuais , Pele/imunologia , Pele/patologia , Fatores de Transcrição/genética
12.
JCI Insight ; 4(1)2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30626756

RESUMO

Podocyte injury is central to many forms of kidney disease, but transcriptional signatures reflecting podocyte injury and compensation mechanisms are challenging to analyze in vivo. Human kidney organoids derived from pluripotent stem cells (PSCs), a potentially new model for disease and regeneration, present an opportunity to explore the transcriptional plasticity of podocytes. Here, transcriptional profiling of more than 12,000 single cells from human PSC-derived kidney organoid cultures was used to identify robust and reproducible cell lineage gene expression signatures shared with developing human kidneys based on trajectory analysis. Surprisingly, the gene expression signature characteristic of developing glomerular epithelial cells was also observed in glomerular tissue from a kidney disease cohort. This signature correlated with proteinuria and inverse eGFR, and it was confirmed in an independent podocytopathy cohort. Three genes in particular were further characterized as potentially novel components of the glomerular disease signature. We conclude that cells in human PSC-derived kidney organoids reliably recapitulate the developmental transcriptional program of podocytes and other cell lineages in the human kidney and that transcriptional profiles seen in developing podocytes are reactivated in glomerular disease. Our findings demonstrate an approach to identifying potentially novel molecular programs involved in the pathogenesis of glomerulopathies.


Assuntos
Nefropatias/genética , Glomérulos Renais/metabolismo , Organoides/metabolismo , Transcriptoma , Adulto , Células-Tronco Embrionárias , Humanos , Nefropatias/metabolismo , Nefropatias/patologia , Glomérulos Renais/patologia , Organoides/patologia , Células-Tronco Pluripotentes/citologia , Podócitos/metabolismo , Análise de Célula Única , Técnicas de Cultura de Tecidos
13.
Front Immunol ; 9: 1322, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29942314

RESUMO

Objectives: CD39 and CD73 are surface enzymes that jut into the extracellular space where they mediate the step-wise phosphohydrolysis of the autocrine and paracrine danger signals ATP and ADP into anti-inflammatory adenosine. Given the role of vascular and immune cells' "purinergic halo" in maintaining homeostasis, we hypothesized that the ectonucleotidases CD39 and CD73 might play a protective role in lupus. Methods: Lupus was modeled by intraperitoneal administration of pristane to three groups of mice: wild-type (WT), CD39-/-, and CD73-/-. After 36 weeks, autoantibodies, endothelial function, kidney disease, splenocyte activation/polarization, and neutrophil activation were characterized. Results: As compared with WT mice, CD39-/- mice developed exaggerated splenomegaly in response to pristane, while both groups of ectonucleotidase-deficient mice demonstrated heightened anti-ribonucleoprotein production. The administration of pristane to WT mice triggered only subtle dysfunction of the arterial endothelium; however, both CD39-/- and CD73-/- mice demonstrated striking endothelial dysfunction following induction of lupus, which could be reversed by superoxide dismutase. Activated B cells and plasma cells were expanded in CD73-/- mice, while deficiency of either ectonucleotidase led to expansion of TH17 cells. CD39-/- and CD73-/- mice demonstrated exaggerated neutrophil extracellular trap release, while CD73-/- mice additionally had higher levels of plasma cell-free DNA. Conclusion: These data are the first to link ectonucleotidases with lupus autoimmunity and vascular disease. New therapeutic strategies may harness purinergic nucleotide dissipation or signaling to limit the damage inflicted upon organs and blood vessels by lupus.

14.
J Immunol ; 201(2): 393-405, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29884703

RESUMO

Systemic lupus erythematosus is an autoimmune disease characterized by increased type I IFNs, autoantibodies, and inflammatory-mediated multiorgan damage. TLR7 activation is an important contributor to systemic lupus erythematosus pathogenesis, but the mechanisms by which type I IFNs participate in TLR7-driven pathologic conditions remain uncertain. In this study, we examined the requirement for type I IFNs in TLR7-stimulated lupus nephritis. Lupus-prone NZM2328, INZM (which lack a functional type I IFN receptor), and NZM2328 IL-1ß-/- mice were treated at 10 wk of age on the right ear with R848 (TLR7 agonist) or control (DMSO). Autoantibody production and proteinuria were assessed throughout treatment. Multiorgan inflammation was assessed at the time of decline in health. Renal infiltrates and mRNA expression were also examined after 14 d of treatment. Both NZM2328 and INZM mice exhibited a decline in survival after 3-4 wk of R848 but not vehicle treatment. Development of splenomegaly and liver inflammation were dependent on type I IFN. Interestingly, autoantibody production, early renal infiltration of dendritic cells, upregulation of IL-1ß, and lupus nephritis occurred independent of type I IFN signaling. Development of TLR7-driven lupus nephritis was not abolished by the deletion of IL-1ß. Thus, although IFN-α is sufficient to induce nephritis acceleration, our data emphasize a critical role for IFN-independent signaling in TLR7-mediated lupus nephritis. Further, despite upregulation of IL-1ß after TLR7 stimulation, deletion of IL-1ß is not sufficient to reduce lupus nephritis development in this model.


Assuntos
Interferon Tipo I/imunologia , Nefrite Lúpica/imunologia , Glicoproteínas de Membrana/imunologia , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Autoanticorpos/imunologia , Células Dendríticas/imunologia , Feminino , Inflamação/metabolismo , Interleucina-1beta/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , RNA Mensageiro/imunologia
15.
Arch Pathol Lab Med ; 142(5): 613-625, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29457738

RESUMO

Context Testing reproducibility is critical for the development of methodologies for morphologic assessment. Our previous study using the descriptor-based Nephrotic Syndrome Study Network Digital Pathology Scoring System (NDPSS) on glomerular images revealed variable reproducibility. Objective To test reproducibility and feasibility of alternative scoring strategies for digital morphologic assessment of glomeruli and explore use of alternative agreement statistics. Design The original NDPSS was modified (NDPSS1 and NDPSS2) to evaluate (1) independent scoring of each individual biopsy level, (2) use of continuous measures, (3) groupings of individual descriptors into classes and subclasses prior to scoring, and (4) indication of pathologists' confidence/uncertainty for any given score. Three and 5 pathologists scored 157 and 79 glomeruli using the NDPSS1 and NDPSS2, respectively. Agreement was tested using conventional (Cohen κ) and alternative (Gwet agreement coefficient 1 [AC1]) agreement statistics and compared with previously published data (original NDPSS). Results Overall, pathologists' uncertainty was low, favoring application of the Gwet AC1. Greater agreement was achieved using the Gwet AC1 compared with the Cohen κ across all scoring methodologies. Mean (standard deviation) differences in agreement estimates using the NDPSS1 and NDPSS2 compared with the single-level original NDPSS were -0.09 (0.17) and -0.17 (0.17), respectively. Using the Gwet AC1, 79% of the original NDPSS descriptors had good or excellent agreement. Pathologist feedback indicated the NDPSS1 and NDPSS2 were time-consuming. Conclusions The NDPSS1 and NDPSS2 increased pathologists' scoring burden without improving reproducibility. Use of alternative agreement statistics was strongly supported. We suggest using the original NDPSS on whole slide images for glomerular morphology assessment and for guiding future automated technologies.


Assuntos
Diagnóstico por Imagem/métodos , Síndrome Nefrótica/diagnóstico por imagem , Patologia Clínica/métodos , Algoritmos , Estudos de Viabilidade , Humanos , Variações Dependentes do Observador , Reprodutibilidade dos Testes
16.
Nephrol Dial Transplant ; 33(2): 310-318, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28339906

RESUMO

Background: Interstitial fibrosis (IF), tubular atrophy (TA) and interstitial inflammation (II) are known determinants of progression of renal disease. Standardized quantification of these features could add value to current classification of glomerulopathies. Methods: We studied 315 participants in the Nephrotic Syndrome Study Network (NEPTUNE) study, including biopsy-proven minimal change disease (MCD = 98), focal segmental glomerulosclerosis (FSGS = 121), membranous nephropathy (MN = 59) and IgA nephropathy (IgAN = 37). Cortical IF, TA and II were quantified (%) on digitized whole-slide biopsy images, by five pathologists with high inter-reader agreement (intra-class correlation coefficient >0.8). Tubulointerstitial messenger RNA expression was measured in a subset of patients. Multivariable Cox proportional hazards models were fit to assess association of IF with the composite of 40% decline in estimated glomerular filtration rate (eGFR) and end-stage renal disease (ESRD) and separately as well, and with complete remission (CR) of proteinuria. Results: IF was highly correlated with TA (P < 0.001) and II (P < 0.001). Median IF varied by diagnosis: FSGS 17, IgAN 21, MN 7, MCD 1 (P < 0.001). IF was strongly correlated with baseline eGFR (P < 0.001) and proteinuria (P = 0.002). After adjusting for clinical pathologic diagnosis, age, race, global glomerulosclerosis, baseline proteinuria, eGFR and medications, each 10% increase in IF was associated with a hazard ratio of 1.29 (P < 0.03) for ESRD/40% eGFR decline, but was not significantly associated with CR. A total of 981 genes were significantly correlated with IF (|r| > 0.4, false discovery rate (FDR) < 0.01), including upstream regulators such as tumor necrosis factor, interferon gamma (IFN-gamma), and transforming growth factor beta 1 (TGF-B1), and signaling pathways for antigen presentation and hepatic fibrosis. Conclusions: The degree of IF is associated with risk of eGFR decline across different types of proteinuric glomerulopathy, correlates with inflammatory and fibrotic gene expression, and may have predictive value in assessing risk of progression.


Assuntos
Diagnóstico por Imagem/métodos , Fibrose/patologia , Glomerulonefrite/diagnóstico , Nefrite Intersticial/patologia , Patologia Clínica/métodos , Proteinúria/diagnóstico , Adulto , Biópsia , Progressão da Doença , Feminino , Fibrose/diagnóstico por imagem , Taxa de Filtração Glomerular , Glomerulonefrite/cirurgia , Humanos , Masculino , Nefrite Intersticial/diagnóstico por imagem , Prognóstico , Proteinúria/cirurgia , Taxa de Sobrevida , Adulto Jovem
17.
JCI Insight ; 2(21)2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29093269

RESUMO

The efficacy of B cell depletion therapies in diseases such as nephrotic syndrome and rheumatoid arthritis suggests a broader role in B cells in human disease than previously recognized. In some of these diseases, such as the minimal change disease subtype of nephrotic syndrome, pathogenic antibodies and immune complexes are not involved. We hypothesized that B cells, activated in the kidney, might produce cytokines capable of directly inducing cell injury and proteinuria. To directly test our hypothesis, we targeted a model antigen to the kidney glomerulus and showed that transfer of antigen-specific B cells could induce glomerular injury and proteinuria. This effect was mediated by IL-4, as transfer of IL-4-deficient B cells did not induce proteinuria. Overexpression of IL-4 in mice was sufficient to induce kidney injury and proteinuria and could be attenuated by JAK kinase inhibitors. Since IL-4 is a specific activator of STAT6, we analyzed kidney biopsies and demonstrated STAT6 activation in up to 1 of 3 of minimal change disease patients, suggesting IL-4 or IL-13 exposure in these patients. These data suggest that the role of B cells in nephrotic syndrome could be mediated by cytokines.


Assuntos
Linfócitos B/metabolismo , Interleucina-4/metabolismo , Podócitos/metabolismo , Proteinúria/imunologia , Animais , Complexo Antígeno-Anticorpo , Biópsia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Células HEK293 , Células HeLa , Humanos , Interleucina-13/metabolismo , Interleucina-4/genética , Janus Quinase 3 , Janus Quinases , Rim/imunologia , Rim/lesões , Rim/patologia , Nefropatias/imunologia , Nefropatias/patologia , Glomérulos Renais , Camundongos , Camundongos Knockout , Síndrome Nefrótica/imunologia , Síndrome Nefrótica/patologia , Proteinúria/patologia , Rituximab/farmacologia , Fator de Transcrição STAT6/metabolismo
18.
Curr Opin Nephrol Hypertens ; 26(6): 450-459, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28858910

RESUMO

PURPOSE OF REVIEW: In this review, we will discuss (i) how the recent advancements in digital technology and computational engineering are currently applied to nephropathology in the setting of clinical research, trials, and practice; (ii) the benefits of the new digital environment; (iii) how recognizing its challenges provides opportunities for transformation; and (iv) nephropathology in the upcoming era of kidney precision and predictive medicine. RECENT FINDINGS: Recent studies highlighted how new standardized protocols facilitate the harmonization of digital pathology database infrastructure and morphologic, morphometric, and computer-aided quantitative analyses. Digital pathology enables robust protocols for clinical trials and research, with the potential to identify previously underused or unrecognized clinically useful parameters. The integration of digital pathology with molecular signatures is leading the way to establishing clinically relevant morpho-omic taxonomies of renal diseases. SUMMARY: The introduction of digital pathology in clinical research and trials, and the progressive implementation of the modern software ecosystem, opens opportunities for the development of new predictive diagnostic paradigms and computer-aided algorithms, transforming the practice of renal disease into a modern computational science.


Assuntos
Nefropatias/patologia , Rim/patologia , Nefrologia/métodos , Patologia/métodos , Algoritmos , Pesquisa Biomédica , Biotecnologia/educação , Biotecnologia/legislação & jurisprudência , Ensaios Clínicos como Assunto , Computadores , Humanos , Processamento de Imagem Assistida por Computador , Nefropatias/genética , Nefropatias/metabolismo , Microscopia/métodos , Medicina de Precisão , Software , Telemedicina
19.
J Am Soc Nephrol ; 28(10): 2931-2945, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28720684

RESUMO

Glomerular sclerotic lesions develop when the glomerular filtration surface area exceeds the availability of podocyte foot process coverage, but the mechanisms involved are incompletely characterized. We evaluated potential mechanisms using a transgenic (podocin promoter-AA-4E-BP1) rat in which podocyte capacity for hypertrophy in response to growth factor/nutrient signaling is impaired. FSGS lesions resembling human FSGS developed spontaneously by 7 months of age, and could be induced earlier by accelerating kidney hypertrophy by nephrectomy. Early segmental glomerular lesions occurred in the absence of a detectable reduction in average podocyte number per glomerulus and resulted from the loss of podocytes in individual glomerular capillary loops. Parietal epithelial cell division, accumulation on Bowman's capsule, and tuft invasion occurred at these sites. Three different interventions that prevented kidney growth and glomerular enlargement (calorie intake reduction, inhibition of mammalian target of rapamycin complex, and inhibition of angiotensin-converting enzyme) protected against FSGS lesion development, even when initiated late in the process. Ki67 nuclear staining and unbiased transcriptomic analysis identified increased glomerular (but not podocyte) cell cycling as necessary for FSGS lesion development. The rat FSGS-associated transcriptomic signature correlated with human glomerular transcriptomes associated with disease progression, compatible with similar processes occurring in man. We conclude that FSGS lesion development resulted from glomerular growth that exceeded the capacity of podocytes to adapt and adequately cover some parts of the filtration surface. Modest modulation of the growth side of this equation significantly ameliorated FSGS progression, suggesting that glomerular growth is an underappreciated therapeutic target for preservation of renal function.


Assuntos
Glomerulosclerose Segmentar e Focal/etiologia , Glomérulos Renais/crescimento & desenvolvimento , Adaptação Fisiológica , Animais , Peso Corporal , Ciclo Celular , Enalapril , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Glomérulos Renais/patologia , Masculino , Tamanho do Órgão , Podócitos/fisiologia , Distribuição Aleatória , Ratos Endogâmicos F344 , Estresse Fisiológico , Transcriptoma
20.
Kidney Int ; 92(4): 909-921, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28554737

RESUMO

Activation of JAK-STAT signaling has been implicated in the pathogenesis of diabetic kidney disease. An increased expression of JAK-STAT genes was found in kidney glomerular cells, including podocytes, in patients with early diabetic kidney disease. However, it is not known whether increased expression of JAK or STAT isoforms in glomerular cells can lead to worsening nephropathy in the setting of diabetes. Therefore, we overexpressed JAK2 mRNA specifically in glomerular podocytes of 129S6 mice to determine whether this change alone could worsen diabetic kidney disease. A 2-3 fold increase in glomerular JAK2 expression, an increase similar to that found in humans with early diabetic kidney disease, led to substantial and statistically significant increases in albuminuria, mesangial expansion, glomerulosclerosis, glomerular fibronectin accumulation, and glomerular basement membrane thickening, and a significant reduction in podocyte density in diabetic mice. Treatment with a specific JAK1/2 inhibitor for 2 weeks partly reversed the major phenotypic changes of diabetic kidney disease and specifically normalized expression of a number of downstream STAT3-dependent genes implicated in diabetic kidney disease progression. Thus, moderate increases in podocyte JAK2 expression at levels similar to those in patients with early diabetic kidney disease can lead directly to phenotypic and other alterations of progressive diabetic glomerulopathy. Hence, inhibition of these changes by treatment with a JAK1/2 inhibitor suggests that such treatment may help retard progression of early diabetic kidney disease in patients.


Assuntos
Nefropatias Diabéticas/patologia , Membrana Basal Glomerular/patologia , Janus Quinase 2/metabolismo , Podócitos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Albuminúria/tratamento farmacológico , Albuminúria/patologia , Animais , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/urina , Modelos Animais de Doenças , Progressão da Doença , Fibronectinas/metabolismo , Membrana Basal Glomerular/citologia , Humanos , Janus Quinase 2/antagonistas & inibidores , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Inibidores de Proteínas Quinases/uso terapêutico , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos
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