Hydroxychloroquine-Induced Renal Phospholipidosis: Case Report and Review of Differential Diagnoses.

Introduction: Renal phospholipidosis describes the accumulation of phospholipids in the lysosomes of kidney cells, in particular podocytes. Originally, this was described primarily in the context of the lysosomal storage disorder Fabry disease. It is now known that a variety of drugs can lead to the accumulation of lysosomal phospholipids. Case Presentation: We present the case of a 69-year-old female patient suffering chronic kidney disease and systemic lupus erythematosus who underwent a kidney biopsy because of a further increase in serum creatinine levels. There was no evidence of lupus nephritis, but electron microscopy showed zebra bodies as a morphological sign of phospholipidosis. This was most likely drug-induced after 25 years of continuous medication with hydroxychloroquine. A renal biopsy 2 years and 6 months earlier, when the renal function of the patient was distinctively better, showed no signs of renal phospholipidosis. Afterward, medication with hydroxychloroquine was discontinued, and renal function parameters remained stable in the 1-year course. Conclusion: This case raises the question of how severely impaired renal function affects the risk of hydroxychloroquine-induced renal phospholipidosis and underlines that hydroxychloroquine should be administered with caution in patients with kidney insufficiency. Moreover, we provide a review of the causes of renal phospholipidosis, which have been described in the literature and give an overview of possible differential diagnoses in cases with histologically proven phospholipidosis in renal biopsies.

Plasma protein signatures reflect systemic immunity and allograft function in kidney transplantation.

Kidney transplantation causes large perturbations of the immune system. While many studies focus on the allograft, insights into systemic effects are largely missing. Here, we analyzed the systemic immune response in 3 cohorts of kidney transplanted patients. Using serum proteomics, laboratory values, mass cytometry, histological and clinical parameters, inter-patient heterogeneity was leveraged for multi-omic co-variation analysis. We identified circulating immune modules (CIM) that describe extra-renal signatures of co-regulated plasma proteins. CIM are present in nontransplanted controls, in transplant conditions and during rejection. They are enriched in pathways linked to kidney function, extracellular matrix, signaling, and cellular activation. A complex leukocyte response in the blood during allograft quiescence and rejection is associated with CIM activity and CIM-specific cytokines. CIM activity correlates with kidney function including a 2-month prediction. Together, the data suggest a systemic and multi-layered response of transplant immunity that might be insightful for understanding allograft dysfunction and developing translational biomarkers.

Perfusion and T2 Relaxation Time as Predictors of Severity and Outcome in Sepsis-Associated Acute Kidney Injury: A Preclinical MRI Study.

BACKGROUND: Preventing sepsis-associated acute kidney injury (S-AKI) can be challenging because it develops rapidly and is often asymptomatic. Probability assessment of disease progression for therapeutic follow-up and outcome are important to intervene and prevent further damage. PURPOSE: To establish a noninvasive multiparametric MRI (mpMRI) tool, including T1 , T2 , and perfusion mapping, for probability assessment of the outcome of S-AKI. STUDY TYPE: Preclinical randomized prospective study. ANIMAL MODEL: One hundred and forty adult female SD rats (65 control and 75 sepsis). FIELD STRENGTH/SEQUENCE: 9.4T; T1 and perfusion map (FAIR-EPI) and T2 map (multiecho RARE). ASSESSMENT: Experiment 1: To identify renal injury in relation to sepsis severity, serum creatinine levels were determined (31 control and 35 sepsis). Experiment 2: Animals underwent mpMRI (T1 , T2 , perfusion) 18 hours postsepsis. A subgroup of animals was immediately sacrificed for histology examination (nine control and seven sepsis). Result of mpMRI in follow-up subgroup (25 control and 33 sepsis) was used to predict survival outcomes at 96 hours. STATISTICAL TESTS: Mann-Whitney U test, Spearman/Pearson correlation (r), P < 0.05 was considered statistically significant. RESULTS: Severely ill septic animals exhibited significantly increased serum creatinine levels compared to controls (70 ± 30 vs. 34 ± 9 µmol/L, P < 0.0001). Cortical perfusion (480 ± 80 vs. 330 ± 140 mL/100 g tissue/min, P < 0.005), and cortical and medullary T2 relaxation time constants were significantly reduced compared to controls (41 ± 4 vs. 37 ± 5 msec in cortex, P < 0.05, 52 ± 7 vs. 45 ± 6 msec in medulla, P < 0.05). The combination of cortical T2 relaxation time constants and perfusion results at 18 hours could predict survival outcomes at 96 hours with high sensitivity (80%) and specificity (73%) (area under curve of ROC = 0.8, Jmax = 0.52). DATA CONCLUSION: This preclinical study suggests combined T2 relaxation time and perfusion mapping as first line diagnostic tool for treatment planning. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2.

Disruption of the Rab7-Dependent Final Common Pathway of Endosomal and Autophagic Processing Results in a Severe Podocytopathy.

SIGNIFICANCE STATEMENT: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates and complex morphology, such as podocytes. To improve our understanding on how disturbances of these trafficking pathways are linked to podocyte depletion and slit diaphragm (SD) injury, the authors explored the role of the small GTPase Rab7, which is linked to endosomal, lysosomal, and autophagic pathways, using as model systems mice and Drosophila with podocyte-specific or nephrocyte-specific loss of Rab7, and a human podocyte cell line depleted for Rab7. Their findings point to maturation and fusion events during endolysosomal and autophagic maturation as key processes for podocyte homeostasis and function and identify altered lysosomal pH values as a putative novel mechanism for podocytopathies. BACKGROUND: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood. METHODS: To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, we focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. We generated mouse and Drosophila in vivo models lacking Rab7 exclusively in podocytes or nephrocytes, and performed histologic and ultrastructural analyses. To further investigate Rab7 function on lysosomal and autophagic structures, we used immortalized human cell lines depleted for Rab7. RESULTS: Depletion of Rab7 in mice, Drosophila , and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, Rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro , Rab7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins. CONCLUSIONS: Disruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease.

Interleukin 24 promotes cell death in renal epithelial cells and is associated with acute renal injury.

Ischemia-reperfusion injury is a major cause of acute kidney injury. Many cytokines are involved in the pathogenesis of renal ischemia-reperfusion injury. IL24 is a member of the IL10 family and has gained importance because of its apoptosis-inducing effects in tumor disease besides its immunoregulative function. Littles is known about the role of IL24 in kidney disease. Using a mouse model, we found that IL24 is upregulated in the kidney after renal ischemia-reperfusion injury and that tubular epithelial cells and infiltrating inflammatory cells are the source of IL24. Mice lacking IL24 are protected from renal injury and inflammation. Cell culture studies showed that IL24 induces apoptosis in renal tubular epithelial cells, which is accompanied by an increased endoplasmatic reticulum stress response. Moreover, IL24 induces robust expression of endogenous IL24 in tubular cells, fostering ER-stress and apoptosis. In kidney transplant recipients with delayed graft function and patients at high risk to develop acute kidney injury after cardiac surgery IL24 is upregulated in the kidney and serum. Taken together, IL24 can serve as a biomarker, plays an important mechanistic role involving both extracellular and intracellular targets, and is a promising therapeutic target in patients at risk of or with ischemia-induced acute kidney injury.

Bryostatin-1 Attenuates Ischemia-Elicited Neutrophil Transmigration and Ameliorates Graft Injury after Kidney Transplantation.

Ischemia reperfusion injury (IRI) is a form of sterile inflammation whose severity determines short- and long-term graft fates in kidney transplantation. Neutrophils are now recognized as a key cell type mediating early graft injury, which activates further innate immune responses and intensifies acquired immunity and alloimmunity. Since the macrolide Bryostatin-1 has been shown to block neutrophil transmigration, we aimed to determine whether these findings could be translated to the field of kidney transplantation. To study the effects of Bryostatin-1 on ischemia-elicited neutrophil transmigration, an in vitro model of hypoxia and normoxia was equipped with human endothelial cells and neutrophils. To translate these findings, a porcine renal autotransplantation model with eight hours of reperfusion was used to study neutrophil infiltration in vivo. Graft-specific treatment using Bryostatin-1 (100 nM) was applied during static cold storage. Bryostatin-1 dose-dependently blocked neutrophil activation and transmigration over ischemically challenged endothelial cell monolayers. When applied to porcine renal autografts, Bryostatin-1 reduced neutrophil graft infiltration, attenuated histological and ultrastructural damage, and improved renal function. Our novel findings demonstrate that Bryostatin-1 is a promising pharmacological candidate for graft-specific treatment in kidney transplantation, as it provides protection by blocking neutrophil infiltration and attenuating functional graft injury.

A Novel Homozygous Mutation in the COL4A4 Gene (Gly1436del) Causing Alport Syndrome Exposed by Pregnancy: A Case Report and Review of the Literature.

BACKGROUND: Alport syndrome results from a hereditary defect of collagen IV synthesis. This causes progressive glomerular disease, ocular abnormalities, and inner ear impairment. Case Presentation. Herein, we present a case of Alport syndrome in a 28-year-old woman caused by a novel mutation (Gly1436del) in the COL4A4 gene that was not unveiled until her first pregnancy. Within the 29th pregnancy week, our patient presented with massive proteinuria and nephrotic syndrome. Light microscopic examination of a kidney biopsy showed typical histological features of segmental sclerosis, and electron microscopy revealed extensive podocyte alterations as well as thickness of glomerular basement membranes with splitting of the lamina densa. One and a half years after childbirth, renal function deteriorated to a preterminal stage, whereas nephrotic syndrome subsided quickly after delivery. CONCLUSION: This case report highlights the awareness of atypical AS courses and emphasizes the importance of genetic testing in such cases.

α-Galactosidase a Deficiency in Fabry Disease Leads to Extensive Dysregulated Cellular Signaling Pathways in Human Podocytes.

Fabry disease (FD) is caused by mutations in the α-galactosidase A (GLA) gene encoding the lysosomal AGAL enzyme. Loss of enzymatic AGAL activity and cellular accumulation of sphingolipids (mainly globotriaosylcermide) may lead to podocyturia and renal loss of function with increased cardiovascular morbidity and mortality in affected patients. To identify dysregulated cellular pathways in FD, we established a stable AGAL-deficient podocyte cell line to perform a comprehensive proteome analysis. Imbalanced protein expression and function were analyzed in additional FD cell lines including endothelial, epithelial kidney, patient-derived urinary cells and kidney biopsies. AGAL-deficient podocytes showed dysregulated proteins involved in thermogenesis, lysosomal trafficking and function, metabolic activity, cell-cell interactions and cell cycle. Proteins associated with neurological diseases were upregulated in AGAL-deficient podocytes. Rescues with inducible AGAL expression only partially normalized protein expression. A disturbed protein expression was confirmed in endothelial, epithelial and patient-specific cells, pointing toward fundamental pathway disturbances rather than to cell type-specific alterations in FD. We conclude that a loss of AGAL function results in profound changes of cellular pathways, which are ubiquitously in different cell types. Due to these profound alterations, current approved FD-specific therapies may not be sufficient to completely reverse all dysregulated pathways.

Data-Driven Kidney Transplant Phenotyping as a Histology-Independent Framework for Biomarker Discovery.

BACKGROUND: In transplant medicine, clinical decision making largely relies on histology of biopsy specimens. However, histology suffers from low specificity, sensitivity, and reproducibility, leading to suboptimal stratification of patients. We developed a histology-independent immune framework of kidney graft homeostasis and rejection. METHODS: We applied tailored RNA deconvolution for leukocyte enumeration and coregulated gene network analysis to published bulk human kidney transplant RNA transcriptomes as input for unsupervised, high-dimensional phenotype clustering. We used framework-based graft survival analysis to identify a biomarker that was subsequently characterized in independent transplant biopsy specimens. RESULTS: We found seven immune phenotypes that confirm known rejection types and uncovered novel signatures. The molecular phenotypes allow for improved graft survival analysis compared with histology, and identify a high-risk group in nonrejecting transplants. Two fibrosis-related phenotypes with distinct immune features emerged with reduced graft survival. We identified lysyl oxidase-like 2 (LOXL2)-expressing peritubular CD68+ macrophages as a framework-derived biomarker of impaired allograft function. These cells precede graft fibrosis, as demonstrated in longitudinal biopsy specimens, and may be clinically useful as a biomarker for early fibrogenesis. CONCLUSIONS: This study provides a comprehensive, data-driven atlas of human kidney transplant phenotypes and demonstrates its utility to identify novel clinical biomarkers.

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier.

BACKGROUND: Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified CRB2 mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS). METHODS: To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these Crb2podKO knockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein-tagged CRB2 wild type and mutants. RESULTS: Crb2podKO mice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conserved O-glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response. CONCLUSIONS: Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.

Deposition patterns of iatrogenic lanthanum and gadolinium in the human body depend on delivered chemical binding forms.

BACKGROUND: Recently, gadolinium from linear GBCAs has been reported to deposit in various regions of the body. Besides gadolinium, other lanthanides are used in medical care. In the current study, we investigated deposition of lanthanum in two patients who received lanthanum carbonate as a phosphate binder due to chronic kidney injury and compared it to additionally found Gd deposition. METHODS: Tissue specimens of two patients with long-term application of lanthanum carbonate as well as possible GBCA application were investigated. Spatial distribution of gadolinium and lanthanum was determined by quantitative laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging of tissue sections. The deposition of gadolinium and lanthanum in different organs was compared, and the ratio of Gd concentration to La concentration (Gd-to-La-ratio) was investigated on an individual pixel base within the images. RESULTS: Deposition of Gd and La was found in all investigated tissues of both patients. Gd and La exhibited high spatial correlation for all samples, with the main deposition being located in the middle coat (tunica media) of blood vessels. The Gd-to-La-ratio was similar in the tissues investigated (between 8 ±â€¯4 (mean ±â€¯standard deviation) and 10 ±â€¯2), except for the thyroid vasculature and surrounding tissue (90 ±â€¯17) as well as the cerebellum (270 ±â€¯18). Here, the ratio was significantly increased towards higher Gd concentration. CONCLUSION: The results of this study demonstrate long-term deposition of La and comparable localization of additionally found Gd in various tissues of the body. La deposition was relatively low, considering the total administered amount of lanthanum carbonate of up to 11.5 kg, indicating a low absorption and/or high excretion of lanthanum. However, the total amount of deposited La is significant and raises questions about possible adverse side effects. The ratio-approach allows for the usage of the additionally generated Gd data, without detailed knowledge about possible GBCA applications. The significantly decreased Gd-to-La-ratio in the brain might be explained by the lanthanum being released and taken up as free La3+ ion in the stomach that impedes a crossing of the blood-brain-barrier while the intravenously injected GBCAs might dechelate first when they have already crossed the blood-brain-barrier.

Extracellular ATP and Purinergic P2Y2 Receptor Signaling Promote Liver Tumorigenesis in Mice by Exacerbating DNA Damage.

Release of ATP to the extracellular compartment and subsequent activation of purinergic receptors is a conserved mechanism mediating inflammatory responses and cell fate decisions in various organs including the liver. Previous findings suggest that extracellular ATP may promote liver tumorigenesis, however, the underlying mechanisms are poorly understood. Therefore, our aim was to dissect the functions of extracellular ATP and P2Y2 receptors (P2Y2R) during hepatocarcinogenesis. Liver tumors were induced in wild-type and P2y2r -/- knockout mice by intraperitoneal diethylnitrosamine (DEN) injection. Tumorigenesis was analyzed after 8 to 10 months and molecular analyses were performed at different stages of tumorigenesis in vivo, as well as in primary mouse hepatocytes in vitro. Liver tumor incidence and tumor numbers were strongly reduced in P2y2r -/- mice, whereas tumor size and morphology were comparable to wild-type controls, suggesting that P2Y2R contributes to tumor initiation. Mechanistically, hepatocyte proliferation in DEN-treated P2y2r -/- mice was reduced, which correlated with reduced c-JUN and CCND1 but increased p21 expression. Moreover, DNA damage as determined by hepatocellular expression of γH2A.X and of genes related to genotoxic stress, as well as STAT3 phosphorylation, was reduced in the absence of P2y2r. Administration of genotoxic agents to primary hepatocytes in vitro confirmed that DNA damage was indeed exacerbated by extracellular ATP, subsequent P2Y2R activation, and downstream intracellular calcium-dependent signal transduction. In conclusion, our data reveal that extracellular ATP and subsequent P2Y2R function stimulate DNA damage responses and hepatocyte proliferation, thereby promoting hepatocarcinogenesis. Targeting this pathway may be an attractive approach for chemoprevention of hepatocellular carcinoma. SIGNIFICANCE: Extracellular ATP and subsequent P2Y2 receptor function stimulate DNA damage responses and hepatocyte proliferation, thereby promoting hepatocarcinogenesis in mice. Targeting this pathway may be an attractive approach for chemoprevention of hepatocellular carcinoma. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/4/699/F1.large.jpg.

A Low Tacrolimus Concentration/Dose Ratio Increases the Risk for the Development of Acute Calcineurin Inhibitor-Induced Nephrotoxicity.

Fast tacrolimus metabolism is linked to inferior outcomes such as rejection and lower renal function after kidney transplantation. Renal calcineurin-inhibitor toxicity is a common adverse effect of tacrolimus therapy. The present contribution hypothesized that tacrolimus-induced nephrotoxicity is related to a low concentration/dose (C/D) ratio. We analyzed renal tubular epithelial cell cultures and 55 consecutive kidney transplant biopsy samples with tacrolimus-induced toxicity, the C/D ratio, C0, C2, and C4 Tac levels, pulse wave velocity analyses, and sublingual endothelial glycocalyx dimensions in the selected kidney transplant patients. A low C/D ratio (C/D ratio < 1.05 ng/mL×1/mg) was linked with higher C2 tacrolimus blood concentrations (19.2 ± 8.7 µg/L vs. 12.2 ± 5.2 µg/L respectively; p = 0.001) and higher degrees of nephrotoxicity despite comparable trough levels (6.3 ± 2.4 µg/L vs. 6.6 ± 2.2 µg/L respectively; p = 0.669). However, the tacrolimus metabolism rate did not affect the pulse wave velocity or glycocalyx in patients. In renal tubular epithelial cells exposed to tacrolimus according to a fast metabolism pharmacokinetic profile it led to reduced viability and increased Fn14 expression. We conclude from our data that the C/D ratio may be an appropriate tool for identifying patients at risk of developing calcineurin-inhibitor toxicity.

An integrative approach to cisplatin chronic toxicities in mice reveals importance of organic cation-transporter-dependent protein networks for renoprotection.

Cisplatin (CDDP) is one of the most important chemotherapeutic drugs in modern oncology. However, its use is limited by severe toxicities, which impair life quality after cancer. Here, we investigated the role of organic cation transporters (OCT) in mediating toxicities associated with chronic (twice the week for 4 weeks) low-dose (4 mg/kg body weight) CDDP treatment (resembling therapeutic protocols in patients) of wild-type (WT) mice and mice with OCT genetic deletion (OCT1/2-/-). Functional and molecular analysis showed that OCT1/2-/- mice are partially protected from CDDP-induced nephrotoxicity and peripheral neurotoxicity, whereas ototoxicity was not detectable. Surprisingly, proteomic analysis of the kidneys demonstrated that genetic deletion of OCT1/2 itself was associated with significant changes in expression of proinflammatory and profibrotic proteins which are part of an OCT-associated protein network. This signature directly regulated by OCT consisted of three classes of proteins, viz., profibrotic proteins, proinflammatory proteins, and nutrient sensing molecules. Consistent with functional protection, CDDP-induced proteome changes were more severe in WT mice than in OCT1/2-/- mice. Laser ablation-inductively coupled plasma-mass spectrometry analysis demonstrated that the presence of OCT was not associated with higher renal platinum concentrations. Taken together, these results redefine the role of OCT from passive membrane transporters to active modulators of cell signaling in the kidney.

L-selectin shedding affects bacterial clearance in the lung: a new regulatory pathway for integrin outside-in signaling.

Pneumonia induced by Gram-negative bacteria is a common and serious disease associated with high morbidity and mortality. Elimination of bacterial pathogens relies on the recruitment and functions of neutrophils. The adhesion molecule L-selectin has recently been implicated in integrin activation in neutrophils (inside-out signaling). However, the molecular mechanism by which L-selectin participates in host defense against Klebsiella pneumoniae-induced pulmonary inflammation is unknown. We demonstrate that L-selectin-deficient mice are prone to pulmonary infection compared with wild-type controls. Mechanistically, L-selectin cleavage from the neutrophil surface triggered by integrin engagement is involved in neutrophil recruitment into the lung and bacterial clearance. Downstream of integrin ligation, the metalloproteinase A disintegrin and metalloproteinase 17 (ADAM17) sheds L-selectin from the neutrophil surface in an IRhom2-dependent manner. L-selectin cleavage enhances integrin-mediated outside-in signaling, resulting in increased neutrophil effector functions. Thus, we identify a novel regulatory mechanism in neutrophils required for an adequate immune response triggered by integrin engagement during K pneumoniae-induced pulmonary inflammation.

Nephrin Signaling Results in Integrin ß1 Activation.

BACKGROUND: Patients with certain mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and display severe proteinuria. Many adult-onset glomerulopathies also feature alterations in Nephrin expression and function. Nephrin signals from the podocyte slit diaphragm to the Actin cytoskeleton by recruiting proteins that can interact with C3G, a guanine nucleotide exchange factor of the small GTPase Rap1. Because Rap activity affects formation of focal adhesions, we hypothesized that Nephrin transmits signals to the Integrin receptor complex, which mediates podocyte adhesion to the extracellular matrix. METHODS: To investigate Nephrin's role in transmitting signals to the Integrin receptor complex, we conducted genetic studies in Drosophila nephrocytes and validated findings from Drosophila in a cultured human podocyte model. RESULTS: Drosophila nephrocytes form a slit diaphragm-like filtration barrier and express the Nephrin ortholog Sticks and stones (Sns). A genetic screen identified c3g as necessary for nephrocyte function. In vivo, nephrocyte-specific gene silencing of sns or c3g compromised nephrocyte filtration and caused nephrocyte diaphragm defects. Nephrocytes with impaired Sns or C3G expression displayed an altered localization of Integrin and the Integrin-associated protein Talin. Furthermore, gene silencing of c3g partly rescued nephrocyte diaphragm defects of an sns overexpression phenotype, pointing to genetic interaction of sns and c3g in nephrocytes. We also found that activated Nephrin recruited phosphorylated C3G and resulted in activation of Integrin ß1 in cultured podocytes. CONCLUSIONS: Our findings suggest that Nephrin can mediate a signaling pathway that results in activation of Integrin ß1 at focal adhesions, which may affect podocyte attachment to the extracellular matrix.

Increased soluble fms-like tyrosine kinase 1 after ischemia reperfusion contributes to adverse clinical outcomes following kidney transplantation.

Renal ischemia reperfusion injury (IRI) adversely affects clinical outcomes following kidney transplantation. Understanding the cellular mechanisms and the changes in gene/protein expression following IRI may help to improve these outcomes. Serum soluble fms-like tyrosine kinase 1 (sFlt-1), a circulating antiangiogenic protein, is increased in the first week following kidney transplantation. We evaluated the casual relationship of elevated sFlt-1 levels with renal microvascular dysfunction following IRI in a longitudinal study of 93 kidney transplant recipients and in several animal models. Transplant recipients with higher sFlt-1 levels had higher odds of delayed graft function, graft rejection, impaired graft function, and death. In a subgroup of 25 participants who underwent kidney biopsy within 4 months of kidney transplantation, peritubular capillary area was lower in those with elevated serum sFtl-1 levels. The administration of recombinant sFlt-1 into rodents resulted in significant structural and functional changes of the renal microvasculature, including reduced peritubular capillary density and intracapillary blood volume, and lead to increased expression of inflammatory genes and increased fibrosis. In a murine model of IRI, the kidney was a site of sFlt-1 production, and systemic neutralization of sFlt-1 preserved peritubular capillary density and alleviated renal fibrosis. Our data indicate that high sFlt-1 levels after IRI play an important role in the pathogenesis of microvascular dysfunction, thereby contributing to adverse clinical outcomes following kidney transplantation.

Nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant results in albuminuria and mesangial expansion.

Intracellular trafficking processes play a key role for the establishment and maintenance of membrane surfaces in renal epithelia. Therefore, dysfunctions of these trafficking processes could be key events and important determinants in the onset and progression of diseases. The presence of cellular vacuoles-observed in many histologic analyses of renal diseases-is a macroscopic hint for disturbed intracellular trafficking processes. However, how vacuoles develop and which intracellular pathways are directly affected remain largely unknown. Previous studies showed that in some cases, vacuolization is linked to malfunction of the Vac14 complex. This complex, including the scaffold protein Vac14, the lipid kinase PIKfyve, and its counteracting lipid phosphatase Fig4, regulates intracellular phosphatidylinositol phosphate levels, which in turn, control the maturation of early-into-late endosomes, as well as the processing of autophagosomes into autophagolysosomes. Here, we analyzed the role of Vac14 in mice and observed that the nephron-specific knockin of the PIKfyve-binding-deficient Vac14L156R mutant led to albuminuria, accompanied by mesangial expansion, increased glomerular size, and an elevated expression of several kidney injury markers. Overexpression of this Vac14 variant in podocytes did not reveal a strong in vivo phenotype, indicating that Vac14-dependent trafficking processes are more important for tubular than for glomerular processes in the kidney. In vitro overexpression of Vac14L156R in Madin-Darby canine kidney cells had no impact on apico-basal polarity defects but resulted in a faster reassembly of junctional structures after Ca2+ depletion and delayed endo- and transcytosis rates. Taken together, our data suggest that increased albuminuria of Vac14L156R-overexpressing mice is a consequence of a lowered endo- and transcytosis of albumin in renal tubules.

The ITIM Domain-Containing NK Receptor Ly49Q Impacts Pulmonary Infection by Mediating Neutrophil Functions.

Pulmonary infection is a frequent pathology associated with excessive neutrophil infiltration. Ly49Q, an ITIM domain-bearing receptor expressed on different leukocytes, has been recently reported to impact neutrophil migration and polarization. Utilizing a murine model of Klebsiella pneumoniae-induced pulmonary infection in combination with additional in vivo and in vitro assays, we show that Ly49Q is critically involved in different steps of the leukocyte adhesion cascade. Ly49Q deficiency is associated with a reduced rolling velocity, impaired crawling capacity, and diminished transmigration. We show that overactivation of the neutrophil ß2 integrins Mac-1 and LFA-1 is responsible for increased adhesion and reduced neutrophil transmigration, resulting in a strongly impaired immune defense against pulmonary infection. Structure function analysis in vitro and in vivo demonstrated that different domains of Ly49Q are important for its function. In summary, Ly49Q regulates integrin activation and neutrophil recruitment and is required for an adequate immune response in pulmonary infection.

WW and C2 domain-containing proteins regulate hepatic cell differentiation and tumorigenesis through the hippo signaling pathway.

The Hippo pathway regulates cell differentiation, proliferation, and apoptosis. Upon activation, it inhibits the import of the transcriptional coactivator yes-associated protein (YAP) into the nucleus, thus suppressing transcription of pro-proliferative genes. Hence, dynamic and precise control of the Hippo pathway is crucial for organ size control and the prevention of tumor formation. Hippo signaling is controlled by a growing number of upstream regulators, including WW and C2 domain-containing (WWC) proteins, which trigger a serine/threonine kinase pathway. One component of this is the large tumor suppressor (LATS) kinase, which phosphorylates YAP, trapping it in the cytoplasm. WWC proteins have been shown to interact with LATS in vitro and stimulate its kinase activity, thus directly promoting cytoplasmic accumulation of phosphorylated YAP. However, the function of the WWC proteins in the regulation of cell proliferation, organ size control, and tumor prevention in vivo has not yet been determined. Here, we show that loss of hepatic WWC expression in mice leads to tissue overgrowth, inflammation, fibrosis, and formation of liver carcinoma. WWC-deficient mouse livers display reduced LATS activity, increased YAP-mediated gene transcription, and enhanced proliferation of hepatic progenitor cells. In addition, loss of WWC expression in the liver accelerates the turnover of angiomotin proteins, which act as negative regulators of YAP activity. CONCLUSION: Our data define an essential in vivo function for WWC proteins as regulators of canonical and noncanonical Hippo signaling in hepatic cell growth and liver tumorigenesis. Thus, expression of WWC proteins may serve as novel prognostic factors in human liver carcinoma. (Hepatology 2018;67:1546-1559).