2-Step-Scores with optional nephropathology for the prediction of adverse outcomes for brain-dead donor kidneys in Eurotransplant.

BACKGROUND AND HYPOTHESIS: The decision for acceptance or discard of the increasingly rare and marginal brain-dead donor kidneys in Eurotransplant (ET) countries has to be made without solid evidence. Thus, we developed and validated flexible clinicopathological scores called 2-Step Scores for the prognosis of delayed graft function (DGF) and one-year death-censored transplant loss (1y-tl) reflecting the current practice of six ET countries including Croatia and Belgium. METHODS: The training set was n=620 for DGF and n=711 for 1y-tl, with validation sets n=158 and n=162. In step 1, stepwise logistic regression models including only clinical predictors were used to estimate the risks. In step 2, risk estimates were updated for statistically relevant intermediate risk percentiles with nephropathology. RESULTS: Step 1 revealed an increased risk of DGF with increased cold ischaemia time, donor and recipient BMI, dialysis vintage, number of HLA-DR mismatches or recipient CMV IgG positivity. On the training and validation set, c-statistics were 0.672 and 0.704, respectively. At a range between 18% and 36%, accuracy of DGF-prognostication improved with nephropathology including number of glomeruli and Banff cv (updated overall c statistics of 0.696 and 0.701, respectively).Risk of 1y-tl increased in recipients with cold ischaemia time, sum of HLA-A. -B, -DR mismatches and donor age. On training and validation sets, c-statistics were 0.700 and 0.769, respectively. Accuracy of 1y-tl prediction improved (c-statistics = 0.706 and 0.765) with Banff ct. Overall, calibration was good on the training, but moderate on the validation set; discrimination was at least as good as established scores when applied to the validation set. CONCLUSION: Our flexible 2-Step Scores with optional inclusion of time-consuming and often unavailable nephropathology should yield good results for clinical practice in ET, and may be superior to established scores. Our scores are adaptable to donation after cardiac death and perfusion pump use.

Early progression of chronic histologic lesions in kidney transplant biopsies is not associated with HLA histocompatibility.

BACKGROUND: Early progression of chronic histologic lesions in kidney allografts represents the main finding in graft attrition. The objective of this retrospective cohort study was to elucidate whether HLA histocompatibility is associated with progression of chronic histologic lesions in the first year post-transplant. Established associations of de novo donor-specific antibody (dnDSA) formation with HLA mismatch and microvascular inflammation (MVI) were calculated to allow for comparability with other study cohorts. METHODS: We included 117 adult kidney transplant recipients, transplanted between 2016 and 2020 from predominantly deceased donors, who had surveillance biopsies at three and twelve months. Histologic lesion scores were assessed according to the Banff classification. HLA mismatch scores (i.e. eplet, predicted indirectly recognizable HLA-epitopes algorithm (PIRCHE-II), HLA epitope mismatch algorithm (HLA-EMMA), HLA whole antigen A/B/DR) were calculated for all transplant pairs. Formation of dnDSAs was quantified by single antigen beads. RESULTS: More than one third of patients exhibited a progression of chronic lesion scores by at least one Banff grade in tubular atrophy (ct), interstitial fibrosis (ci), arteriolar hyalinosis (ah) and inflammation in the area of interstitial fibrosis and tubular atrophy (i-IFTA) from the three to the twelve-month biopsy. Multivariable proportional odds logistic regression models revealed no association of HLA mismatch scores with progression of histologic lesions, except for ah and especially HLA-EMMA DRB1 (OR = 1.10, 95%-CI: 1.03-1.18). Furthermore, the established associations of dnDSA formation with HLA mismatch and MVI (OR = 5.31, 95-% CI: 1.19-22.57) could be confirmed in our cohort. CONCLUSIONS: These data support the association of HLA mismatch and alloimmune response, while suggesting that other factors contribute to early progression of chronic histologic lesions.

Performance of Scores Predicting Adverse Outcomes in Procurement Kidney Biopsies From Deceased Donors With Organs of Lower-Than-Average Quality.

Several scores have been devised for providing a prognosis of outcomes after kidney transplantation. This study is a comprehensive test of these scores in a cohort of deceased donors with kidneys of lower-than-average quality and procurement biopsies. In total, 15 scores were tested on a retrospective cohort consisting of 221 donors, 223 procurement biopsies, and 223 recipient records for performance on delayed graft function, graft function, or death-censored graft loss. The best-performing score for DGF was the purely clinical Chapal score (AUC 0.709), followed by the Irish score (AUC 0.684); for graft function, the Nyberg score; and for transplant loss, the Snoeijs score (AUC 0.630) and the Leuven scores (AUCs 0.637 and 0.620). The only score with an acceptable performance was the Chapal score. Its disadvantage is that knowledge of the cold ischemia time is required, which is not known at allocation. None of the other scores performed acceptably. The scores fared better in discarded kidneys than in transplanted kidneys. Our study shows an unmet need for practical prognostic scores useful at the time of a decision about discarding or accepting deceased donor kidneys of lower-than-average quality in the Eurotransplant consortium.

Effect of creatinine metrics on outcome after transplantation of marginal donor kidneys.

INTRODUCTION: Predicting outcome after transplantation of marginal kidneys is a challenging task. Donor creatinine or estimated glomerular filtration rate (eGFR) are integral components of the respective risk scores. However, there is uncertainty on which of their values obtained successively during procurement is the most suitable. MATERIAL AND METHODS: This is a retrospective study of 221 adult brain death donors with marginal kidneys, transplanted in 223 recipients. We applied logistic regression analysis to investigate the association between initial (at hospital admission), nadir (lowest), zenith (highest) and terminal (at recovery) donor eGFR with primary non-function (PNF), delayed graft function (DGF), 3- and 12-month graft function and 1- and 3-year patient- and death-censored graft survival. RESULTS: In the multivariate analysis, admission, terminal, and the lowest donor eGFR could most accurately predict DGF. The respective ORs [95% CI] were: 0.875 [0.771-0.993], 0.818 [95% CI: 0.726-0.922] and 0.793 [0.689-0.900]. Although not being significant for DGF (OR 0.931 [95% CI: 0.817-1.106]), the highest eGFR was the best predictor of 3-month graft function (adjusted b coefficient 1.161 [95% CI: 0.355-1.968]). Analysis of primary nonfunction showed that determination of initial and the highest eGFR proved to be the best predictors. The respective ORs [95% CI] were: 0.804 [0.667-0.968] and 0.750 [0.611-0.919]. There were no differences in the risk associations of each of the four eGFR recordings with patient- and graft survival. CONCLUSION: The various eGFR recordings determined during the procurement process of marginal donors can predict PNF, DGF and 3- and 12-month graft function. Regarding short-term patient- and graft survival, there appears to be impacted by recipient factors rather than donor kidney function.

PodoSighter: A Cloud-Based Tool for Label-Free Podocyte Detection in Kidney Whole-Slide Images.

BACKGROUND: Podocyte depletion precedes progressive glomerular damage in several kidney diseases. However, the current standard of visual detection and quantification of podocyte nuclei from brightfield microscopy images is laborious and imprecise. METHODS: We have developed PodoSighter, an online cloud-based tool, to automatically identify and quantify podocyte nuclei from giga-pixel brightfield whole-slide images (WSIs) using deep learning. Ground-truth to train the tool used immunohistochemically or immunofluorescence-labeled images from a multi-institutional cohort of 122 histologic sections from mouse, rat, and human kidneys. To demonstrate the generalizability of our tool in investigating podocyte loss in clinically relevant samples, we tested it in rodent models of glomerular diseases, including diabetic kidney disease, crescentic GN, and dose-dependent direct podocyte toxicity and depletion, and in human biopsies from steroid-resistant nephrotic syndrome and from human autopsy tissues. RESULTS: The optimal model yielded high sensitivity/specificity of 0.80/0.80, 0.81/0.86, and 0.80/0.91, in mouse, rat, and human images, respectively, from periodic acid-Schiff-stained WSIs. Furthermore, the podocyte nuclear morphometrics extracted using PodoSighter were informative in identifying diseased glomeruli. We have made PodoSighter freely available to the general public as turnkey plugins in a cloud-based web application for end users. CONCLUSIONS: Our study demonstrates an automated computational approach to detect and quantify podocyte nuclei in standard histologically stained WSIs, facilitating podocyte research, and enabling possible future clinical applications.

Differential expression of microRNA miR-150-5p in IgA nephropathy as a potential mediator and marker of disease progression.

Understanding why certain patients with IgA nephropathy progress to kidney failure while others maintain normal kidney function remains a major unanswered question. To help answer this, we performed miRNome profiling by next generation sequencing of kidney biopsies in order to identify microRNAs specifically associated with the risk of IgA nephropathy progression. Following sequencing and validation in independent cohorts, four microRNAs (-150-5p, -155-5p, -146b-5p, -135a-5p) were found to be differentially expressed in IgA nephropathy progressors compared to non-progressors, and patients with thin membrane nephropathy, lupus nephritis and membranous nephropathy, and correlated with estimated glomerular filtration rate, proteinuria, and the Oxford MEST-C scores (five histological features that are independent predictors of clinical outcome). Each individual microRNA increased the discrimination score of the International IgAN Prediction Tool, although due to the small number of samples the results did not reach statistical significance. miR-150-5p exhibited the largest amplitude of expression between cohorts and displayed the best discrimination between IgA nephropathy progressors and non-progressors by receiver operating curve analysis (AUC: 0.8). However, expression was similarly upregulated in kidneys with established fibrosis and low estimated glomerular filtration rates at the time of biopsy. Consistent with a more generic role in kidney fibrosis, in situ hybridization revealed that miR-150-5p was found in lymphoid infiltrates, and areas of proliferation and fibrosis consistent with the known drivers of progression. Thus, miR-150-5p may be a potential functional mediator of kidney fibrosis that may add value in predicting risk of progression in IgA nephropathy and other kidney diseases.

Artificial intelligence and machine learning in nephropathology.

Artificial intelligence (AI) for the purpose of this review is an umbrella term for technologies emulating a nephropathologist's ability to extract information on diagnosis, prognosis, and therapy responsiveness from native or transplant kidney biopsies. Although AI can be used to analyze a wide variety of biopsy-related data, this review focuses on whole slide images traditionally used in nephropathology. AI applications in nephropathology have recently become available through several advancing technologies, including (i) widespread introduction of glass slide scanners, (ii) data servers in pathology departments worldwide, and (iii) through greatly improved computer hardware to enable AI training. In this review, we explain how AI can enhance the reproducibility of nephropathology results for certain parameters in the context of precision medicine using advanced architectures, such as convolutional neural networks, that are currently the state of the art in machine learning software for this task. Because AI applications in nephropathology are still in their infancy, we show the power and potential of AI applications mostly in the example of oncopathology. Moreover, we discuss the technological obstacles as well as the current stakeholder and regulatory concerns about developing AI applications in nephropathology from the perspective of nephropathologists and the wider nephrology community. We expect the gradual introduction of these technologies into routine diagnostics and research for selective tasks, suggesting that this technology will enhance the performance of nephropathologists rather than making them redundant.

The Banff 2019 Kidney Meeting Report (I): Updates on and clarification of criteria for T cell- and antibody-mediated rejection.

The XV. Banff conference for allograft pathology was held in conjunction with the annual meeting of the American Society for Histocompatibility and Immunogenetics in Pittsburgh, PA (USA) and focused on refining recent updates to the classification, advances from the Banff working groups, and standardization of molecular diagnostics. This report on kidney transplant pathology details clarifications and refinements to the criteria for chronic active (CA) T cell-mediated rejection (TCMR), borderline, and antibody-mediated rejection (ABMR). The main focus of kidney sessions was on how to address biopsies meeting criteria for CA TCMR plus borderline or acute TCMR. Recent studies on the clinical impact of borderline infiltrates were also presented to clarify whether the threshold for interstitial inflammation in diagnosis of borderline should be i0 or i1. Sessions on ABMR focused on biopsies showing microvascular inflammation in the absence of C4d staining or detectable donor-specific antibodies; the potential value of molecular diagnostics in such cases and recommendations for use of the latter in the setting of solid organ transplantation are presented in the accompanying meeting report. Finally, several speakers discussed the capabilities of artificial intelligence and the potential for use of machine learning algorithms in diagnosis and personalized therapeutics in solid organ transplantation.

Utility of immunohistochemistry with C3d in C3 glomerulopathy.

C3-dominance by immunofluorescence is a defining feature in the diagnosis of C3 glomerulopathy. Most pathologists stain for C3c, which has been reported as a trace/negative even in otherwise clear-cut cases of dense deposit disease. We investigated the usefulness of C3d immunohistochemistry in biopsies with C3 glomerulopathy as an ancillary diagnostic tool. All biopsies from patients diagnosed with C3 glomerulopathy in the period January 2005 to June 2017 in the Erasmus MC, Rotterdam were included (n = 14; 10 C3 glomerulonephritis, 4 dense deposit disease). The staining pattern of C3d and C4d by immunohistochemistry was analyzed. As controls, biopsies from patients with immune complex membranoproliferative glomerulonephritis (n = 2), infection-associated glomerulonephritis (n = 6), pauci-immune crescentic glomerulonephritis (n = 7), tubulointerstitial nephritis (n = 7) and chronic-active antibody-mediated rejection (n = 9) were included. All 14 biopsies with C3 glomerulopathy showed a C3d score of ≥2, including two clear-cut biopsies with C3 glomerulopathy originally showing a trace/negative staining for C3c. In the control group, a C3d score ≥2 was observed in 11 biopsies (35%; 2 with immune complex membranoproliferative glomerulonephritis (100%), 6 with infection-associated glomerulonephritis (100%), 1 with pauci-immune crescentic glomerulonephritis (14%), 1 with tubulointerstitial nephritis (14%) and 1 with chronic-active antibody-mediated rejection (11%)). C4d was positive in 71% of the biopsies with C3 glomerulopathy (10/14). In conclusion, C3d immunohistochemistry is a valuable tool in the diagnosis of C3 glomerulopathy, especially in cases in which C3c immunofluorescence shows a trace/negative. We recommend the use of C3d in addition to C3c in cases suspicious for C3 glomerulopathy.

Mitochondria Permeability Transition versus Necroptosis in Oxalate-Induced AKI.

BACKGROUND: Serum oxalate levels suddenly increase with certain dietary exposures or ethylene glycol poisoning and are a well known cause of AKI. Established contributors to oxalate crystal-induced renal necroinflammation include the NACHT, LRR and PYD domains-containing protein-3 (NLRP3) inflammasome and mixed lineage kinase domain-like (MLKL) protein-dependent tubule necroptosis. These studies examined the role of a novel form of necrosis triggered by altered mitochondrial function. METHODS: To better understand the molecular pathophysiology of oxalate-induced AIK, we conducted in vitro studies in mouse and human kidney cells and in vivo studies in mice, including wild-type mice and knockout mice deficient in peptidylprolyl isomerase F (Ppif) or deficient in both Ppif and Mlkl. RESULTS: Crystals of calcium oxalate, monosodium urate, or calcium pyrophosphate dihydrate, as well as silica microparticles, triggered cell necrosis involving PPIF-dependent mitochondrial permeability transition. This process involves crystal phagocytosis, lysosomal cathepsin leakage, and increased release of reactive oxygen species. Mice with acute oxalosis displayed calcium oxalate crystals inside distal tubular epithelial cells associated with mitochondrial changes characteristic of mitochondrial permeability transition. Mice lacking Ppif or Mlkl or given an inhibitor of mitochondrial permeability transition displayed attenuated oxalate-induced AKI. Dual genetic deletion of Ppif and Mlkl or pharmaceutical inhibition of necroptosis was partially redundant, implying interlinked roles of these two pathways of regulated necrosis in acute oxalosis. Similarly, inhibition of mitochondrial permeability transition suppressed crystal-induced cell death in primary human tubular epithelial cells. PPIF and phosphorylated MLKL localized to injured tubules in diagnostic human kidney biopsies of oxalosis-related AKI. CONCLUSIONS: Mitochondrial permeability transition-related regulated necrosis and necroptosis both contribute to oxalate-induced AKI, identifying PPIF as a potential molecular target for renoprotective intervention.

The proteome microenvironment determines the protective effect of preconditioning in cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) leads to significant morbidity and mortality; unfortunately, strategies to prevent or treat AKI are lacking. In recent years, several preconditioning protocols have been shown to be effective in inducing organ protection in rodent models. Here, we characterized two of these interventions-caloric restriction and hypoxic preconditioning-in a mouse model of cisplatin-induced AKI and investigated the underlying mechanisms by acquisition of multi-layered omic data (transcriptome, proteome, N-degradome) and functional parameters in the same animals. Both preconditioning protocols markedly ameliorated cisplatin-induced loss of kidney function, and caloric restriction also induced lipid synthesis. Bioinformatic analysis revealed mRNA-independent proteome alterations affecting the extracellular space, mitochondria, and transporters. Interestingly, our analyses revealed a strong dissociation of protein and RNA expression after cisplatin treatment that showed a strong correlation with the degree of damage. N-degradomic analysis revealed that most posttranscriptional changes were determined by arginine-specific proteolytic processing. This included a characteristic cisplatin-activated complement signature that was prevented by preconditioning. Amyloid and acute-phase proteins within the cortical parenchyma showed a similar response. Extensive analysis of disease-associated molecular patterns suggested that transcription-independent deposition of amyloid P-component serum protein may be a key component in the microenvironmental contribution to kidney damage. This proof-of-principle study provides new insights into the pathogenesis of cisplatin-induced AKI and the molecular mechanisms underlying organ protection by correlating phenotypic and multi-layered omics data.

Case report: a peculiar glomerulopathy in a patient suffering from nephrotic syndrome.

BACKGROUND: Podocyte infolding glomerulopathy (PIG) is a rare histopathologic finding with global infolding of the podocytes into the glomerular basement membrane (GBM), accompanied by microstructures underneath. Described in 2002 for the first time, PIG was proposed as a new pathological entity in 2008 based on the largest case series so far. Yet all of the described cases derive from Asian countries. We report a case from Germany fulfilling the diagnostic criteria of PIG. Considering the scarcity of data on this entity especially in Western countries, collecting cases like ours and multicentric meta-analyses will be crucial to obtain a better understanding of PIG, its causes, clinical course and potential treatment options. CASE PRESENTATION: A 56-year-old Caucasian woman with a history of rheumatoid arthritis (RA), no other comorbidities and no known renal disease was admitted to the hospital with acute kidney injury (AKI) and nephrotic syndrome. Physical examination was unremarkable except for anasarca. Renal ultrasound revealed no abnormalities. Laboratory and urine analyses were consistent with the nephrotic syndrome and renal failure. Serological studies regarding ANA, ANCA, anti-PLA2R autoantibodies, complement, virus infections, immunofixation and quantitative light chain analysis were unremarkable. A renal biopsy was performed. Light microscopic examination showed flattened tubular epithelium consistent with acute tubular damage, no infiltrates and unremarkable glomeruli except diffuse and global holes in the GBM (Fig. 1a) and negative staining for immunoglobulin heavy-chains, light-chains and complement split products. Electron microscopy revealed a rare correlate for these holes: global peculiar infolding of podocyte cytoplasm into the GBM. Most of these infoldings were accompanied by condensation of the GBM underneath. No such condensation or electron dense deposits were found without these infoldings or outside the GBM. CONCLUSION: Here we report the first case of PIG outside of Asia. Since there are only few reports about this specific finding, we feel there is a need to share information in an attempt to accumulate knowledge about this possible new entity and potential treatment options.

Pathological manifestations of Farber disease in a new mouse model.

Farber disease (FD) is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments are clinically available and affected patients have a severely shortened lifespan. Due to the low incidence, the pathogenesis of FD is still poorly understood. Here, we report a novel acid ceramidase mutant mouse model that enables the study of pathogenic mechanisms of FD and ceramide accumulation. Asah1tmEx1 mice were generated by deletion of the acid ceramidase signal peptide sequence. The effects on lysosomal targeting and activity of the enzyme were assessed. Ceramide and sphingomyelin levels were quantified by liquid chromatography tandem-mass spectrometry (LC-MS/MS) and disease manifestations in several organ systems were analyzed by histology and biochemistry. We show that deletion of the signal peptide sequence disrupts lysosomal targeting and enzyme activity, resulting in ceramide and sphingomyelin accumulation. The affected mice fail to thrive and die early. Histiocytic infiltrations were observed in many tissues, as well as lung inflammation, liver fibrosis, muscular disease manifestations and mild kidney injury. Our new mouse model mirrors human FD and thus offers further insights into the pathogenesis of this disease. In the future, it may also facilitate the development of urgently needed therapies.

Mycophenolate mofetil following glucocorticoid treatment in Henoch-Schönlein purpura nephritis: the role of early initiation and therapeutic drug monitoring.

BACKGROUND: Henoch-Schönlein purpura (HSP) is the most common vasculitis in childhood and traditionally considered as a self-limiting disease. However, renal involvement can unfavorably determine long-term prognosis. The reported regimens to treat HSP nephritis (HSPN) are diverse, indicating that the most effective treatment remains controversial. METHODS: This retrospective, single-center study involved 18 patients presenting with HSPN and nephrotic-range proteinuria. We aimed to investigate the efficacy and safety of mycophenolate mofetil (MMF) and identify a cut-off level for estimated mycophenolic acid area under the curve (eMPA-AUC0-12h) values, which can predict complete remission with high sensitivity. RESULTS: Despite prior insufficient therapeutic response to corticosteroids, 89% of patients showed a significant decrease in proteinuria after 1 month of MMF treatment. None of them relapsed during treatment; however, two children relapsed after discontinuation. Based on results of a receiver operating characteristic (ROC) analysis, an eMPA-AUC0-12h >56.4 mg*h/l was a predictor for complete remission within 3 months (80% sensitivity, 83.3% specificity, p = 0.035). During MMF administration, we encountered no adverse event requiring discontinuation of treatment. CONCLUSION: Our study demonstrates that MMF is a safe and potentially effective secondary treatment option for children with HSPN to achieve and maintain long-term remission without serious side effects. To achieve complete remission within 3 months, resolve severe inflammatory glomerular lesions, and avoid progression to chronic kidney disease, we propose timely diagnosis and early initiation of MMF with an eMPA-AUC0-12h value of 56.4 mg*h/l.

A Multicenter Study of the Predictive Value of Crescents in IgA Nephropathy.

The Oxford Classification of IgA nephropathy does not account for glomerular crescents. However, studies that reported no independent predictive role of crescents on renal outcomes excluded individuals with severe renal insufficiency. In a large IgA nephropathy cohort pooled from four retrospective studies, we addressed crescents as a predictor of renal outcomes and determined whether the fraction of crescent-containing glomeruli associates with survival from either a ≥50% decline in eGFR or ESRD (combined event) adjusting for covariates used in the original Oxford study. The 3096 subjects studied had an initial mean±SD eGFR of 78±29 ml/min per 1.73 m2 and median (interquartile range) proteinuria of 1.2 (0.7-2.3) g/d, and 36% of subjects had cellular or fibrocellular crescents. Overall, crescents predicted a higher risk of a combined event, although this remained significant only in patients not receiving immunosuppression. Having crescents in at least one sixth or one fourth of glomeruli associated with a hazard ratio (95% confidence interval) for a combined event of 1.63 (1.10 to 2.43) or 2.29 (1.35 to 3.91), respectively, in all individuals. Furthermore, having crescents in at least one fourth of glomeruli independently associated with a combined event in patients receiving and not receiving immunosuppression. We propose adding the following crescent scores to the Oxford Classification: C0 (no crescents); C1 (crescents in less than one fourth of glomeruli), identifying patients at increased risk of poor outcome without immunosuppression; and C2 (crescents in one fourth or more of glomeruli), identifying patients at even greater risk of progression, even with immunosuppression.

Regardless of etiology, progressive renal disease causes ultrastructural and functional alterations of peritubular capillaries.

Progressive renal diseases are associated with rarefaction of peritubular capillaries, but the ultrastructural and functional alterations of the microvasculature are not well described. To study this, we analyzed different time points during progressive kidney damage and fibrosis in 3 murine models of different disease etiologies. These models were unilateral ureteral obstruction, unilateral ischemia-reperfusion injury, and Col4a3-deficient mice, we analyzed ultrastructural alterations in patient biopsy specimens. Compared with kidneys of healthy mice, we found a significant and progressive reduction of peritubular capillaries in all models analyzed. Ultrastructurally, compared with the kidneys of control mice, focal widening of the subendothelial space and higher numbers of endothelial vacuoles and caveolae were found in fibrotic kidneys. Quantitative analysis showed that peritubular capillary endothelial cells in fibrotic kidneys had significantly and progressively reduced numbers of fenestrations and increased thickness of the cell soma and lamina densa of the capillary basement membrane. Similar ultrastructural changes were also observed in patient's kidney biopsy specimens. Compared with healthy murine kidneys, fibrotic kidneys had significantly increased extravasation of Evans blue dye in all 3 models. The extravasation could be visualized using 2-photon microscopy in real time in living animals and was mainly localized to capillary branching points. Finally, fibrotic kidneys in all models exhibited a significantly greater degree of interstitial deposition of fibrinogen. Thus, peritubular capillaries undergo significant ultrastructural and functional alterations during experimental progressive renal diseases, independent of the underlying injury. Analyses of these alterations could provide read-outs for the evaluation of therapeutic approaches targeting the renal microvasculature.

Regulation of Arthritis Severity by the Acid Sphingomyelinase.

BACKGROUND/AIMS: Rheumatoid arthritis is a chronic autoimmune disease hallmarked by inflammation in synovial joints. Treatment is hampered by the lack of a cure and current disease-modifying drugs are associated with potentially severe toxicities. METHODS: We investigated arthritis severity by measuring joint swelling and pro-inflammatory cytokine production in a murine experimental model of inflammatory arthritis (antigen-induced arthritis). We analyzed acid sphingomyelinase knock-out mice and wild-type littermates, as well as mice treated with the pharmacological acid sphingomyelinase inhibitor amitriptyline. RESULTS: Genetic ablation or pharmacological inhibition of acid sphingomyelinase reduced joint swelling and levels of pro-inflammatory cytokines in the arthritic joint. CONCLUSION: We identified acid sphingomyelinase as a novel druggable target in rheumatoid arthritis. Functional inhibitors of acid sphingomyelinase have been clinically used for decades, are well tolerated and suitable for long-term treatment. They would be immediately available for clinical development as a novel rheumatoid arthritis therapy.

Current status of pediatric renal transplant pathology.

Histopathology is still an indispensable tool for the diagnosis of kidney transplant dysfunction in adult and pediatric patients. This review presents consolidated knowledge, recent developments and future prospects on the biopsy procedure, the diagnostic work-up, classification schemes, the histopathology of rejection, including antibody-mediated forms, ABO-incompatible transplants, protocol biopsies, recurrent and de novo disease, post-transplant lymphoproliferative disorder, infectious complications and drug-induced toxicity. It is acknowledged that frequently the correct diagnosis can only be reached in consensus with clinical, serological, immunogenetical, bacteriological and virological findings. This review shall enhance the understanding of the pediatric nephrologist for the thought processes of nephropathologists with the aim to facilitate teamwork between these specialist groups for the benefit of the patient.

TRPC6 G757D Loss-of-Function Mutation Associates with FSGS.

FSGS is a CKD with heavy proteinuria that eventually progresses to ESRD. Hereditary forms of FSGS have been linked to mutations in the transient receptor potential cation channel, subfamily C, member 6 (TRPC6) gene encoding a nonselective cation channel. Most of these TRPC6 mutations cause a gain-of-function phenotype, leading to calcium-triggered podocyte cell death, but the underlying molecular mechanisms are unclear. We studied the molecular effect of disease-related mutations using tridimensional in silico modeling of tetrameric TRPC6. Our results indicated that G757 is localized in a domain forming a TRPC6-TRPC6 interface and predicted that the amino acid exchange G757D causes local steric hindrance and disruption of the channel complex. Notably, functional characterization of model interface domain mutants suggested a loss-of-function phenotype. We then characterized 19 human FSGS-related TRPC6 mutations, the majority of which caused gain-of-function mutations. However, five mutations (N125S, L395A, G757D, L780P, and R895L) caused a loss-of-function phenotype. Coexpression of wild-type TRPC6 and TRPC6 G757D, mimicking heterozygosity observed in patients, revealed a dominant negative effect of TRPC6 G757D. Our comprehensive analysis of human disease-causing TRPC6 mutations reveals loss of TRPC6 function as an additional concept of hereditary FSGS and provides molecular insights into the mechanism responsible for the loss-of-function phenotype of TRPC6 G757D in humans.