Metabolic injury-induced NLRP3 inflammasome activation dampens phospholipid degradation.

The collateral effects of obesity/metabolic syndrome include inflammation and renal function decline. As renal disease in obesity can occur independently of hypertension and diabetes, other yet undefined causal pathological pathways must be present. Our study elucidate novel pathological pathways of metabolic renal injury through LDL-induced lipotoxicity and metainflammation. Our in vitro and in vivo analysis revealed a direct lipotoxic effect of metabolic overloading on tubular renal cells through a multifaceted mechanism that includes intralysosomal lipid amassing, lysosomal dysfunction, oxidative stress, and tubular dysfunction. The combination of these endogenous metabolic injuries culminated in the activation of the innate immune NLRP3 inflammasome complex. By inhibiting the sirtuin-1/LKB1/AMPK pathway, NLRP3 inflammasome dampened lipid breakdown, thereby worsening the LDL-induced intratubular phospholipid accumulation. Consequently, the presence of NLRP3 exacerbated tubular oxidative stress, mitochondrial damage and malabsorption during overnutrition. Altogether, our data demonstrate a causal link between LDL and tubular damage and the creation of a vicious cycle of excessive nutrients-NLRP3 activation-catabolism inhibition during metabolic kidney injury. Hence, this study strongly highlights the importance of renal epithelium in lipid handling and recognizes the role of NLRP3 as a central hub in metainflammation and immunometabolism in parenchymal non-immune cells.

Mucosal integrity and sensitivity to acid in the proximal esophagus in patients with gastroesophageal reflux disease.

Acid reflux episodes that extend to the proximal esophagus are more likely to be perceived. This suggests that the proximal esophagus is more sensitive to acid than the distal esophagus, which could be caused by impaired mucosal integrity in the proximal esophagus. Our aim was to explore sensitivity to acid and mucosal integrity in different segments of the esophagus. We used a prospective observational study, including 12 patients with gastroesophageal reflux disease (GERD). After stopping acid secretion-inhibiting medication, two procedures were performed: an acid perfusion test and an upper endoscopy with electrical tissue impedance spectroscopy and esophageal biopsies. Proximal and distal sensitivity to acid and tissue impedance were measured in vivo, and mucosal permeability and epithelial intercellular spaces at different esophageal levels were measured in vitro. Mean lag time to heartburn perception was much shorter after proximal acid perfusion (0.8 min) than after distal acid perfusion (3.9 min) (P = 0.02). Median in vivo tissue impedance was significantly lower in the distal esophagus (4,563 Ω·m) compared with the proximal esophagus (8,170 Ω·m) (P = 0.002). Transepithelial permeability, as measured by the median fluorescein flux was significantly higher in the distal (2,051 nmol·cm(-2)·h(-1)) than in the proximal segment (368 nmol·cm(-2)·h(-1)) (P = 0.033). Intercellular space ratio and maximum heartburn intensity were not significantly different between the proximal and distal esophagus. In GERD patients off acid secretion-inhibiting medication, acid exposure in the proximal segment of the esophagus provokes symptoms earlier than acid exposure in the distal esophagus, whereas mucosal integrity is impaired more in the distal esophagus. These findings indicate that the enhanced sensitivity to proximal reflux episodes is not explained by increased mucosal permeability.

Unique Renal Manifestation of Type I Cryoglobulinemia, With Massive Crystalloid Deposits in Glomerular Histiocytes, Podocytes, and Endothelial Cells.

OBJECTIVES: We describe a 62-year-old woman with a 15-year history of a plasma cell dyscrasia (monoclonal IgGκ), manifested by type I cryoglobulinemia and dermal vasculitis. METHODS: In addition to the clinical examinations, light microscopy with immunohistochemistry, sequential multicolor immunohistochemistry, and electron microscopy were used to characterize the crystalline deposits. RESULTS: At initial presentation and for a later flare, she was treated with cyclophosphamide and prednisolone with good clinical response. She had renal function decline, microscopic hematuria, and proteinuria. A renal biopsy specimen revealed the presence of glomerular macrophages and duplication of the capillary walls with cellular interposition. Glomerular cells contained abundant needle-shaped eosinophilic crystalline inclusions positive for κ light chain. Electron microscopy confirmed the presence of intracytoplasmatic crystalline structures in endothelial cells, podocytes, and macrophages but not in the tubular epithelium. Rituximab treatment was started. At follow-up (now up to 6 months), renal function remained stable. CONCLUSIONS: This patient displays a unique renal manifestation of type I cryoglobulinemia related to a plasma cell dyscrasia.

Histological Response to Fluticasone Propionate in Patients With Eosinophilic Esophagitis Is Associated With Improved Functional Esophageal Mucosal Integrity.

OBJECTIVES: The esophageal mucosal integrity is impaired in patients with eosinophilic esophagitis (EoE). We aimed to evaluate the effect of fluticasone propionate on inflammation and functional and structural markers of esophageal mucosal barrier integrity in adult patients with EoE. METHODS: In this prospective study, we included 15 EoE patients (median age (IQR), 43 (30-45) years). Patients underwent upper endoscopy before and after an 8-week course of swallowed fluticasone propionate 500 µg BID. Several parameters of esophageal mucosal barrier integrity were evaluated: esophageal electrical tissue impedance in vivo during endoscopy, transepithelial electrical resistance (TER) and transepithelial molecule flux in Ussing chambers using esophageal biopsy specimens, and intercellular spaces as a structural marker of permeability using electron microscopy. Esophageal eosinophils and mast cells were counted, and expression of inflammatory cytokines and barrier integrity proteins was investigated using qPCR. Esophageal symptoms and signs were also assessed. RESULTS: Peak eosinophil and mast cell counts decreased significantly after fluticasone propionate treatment. The esophageal mucosal integrity increased substantially during treatment, as shown by increased extracellular impedance and TER (both P<0.01) and decreased transepithelial molecule flux in Ussing chambers (P<0.05). Whereas expression of genes encoding for inflammatory cytokines (IL5, IL13, eotaxin-3, periostin, TSLP) decreased after treatment, expression of genes encoding for barrier integrity proteins (filaggrin and desmoglein-1) increased. CONCLUSIONS: Fluticasone propionate treatment decreases eosinophilic inflammation and improves the esophageal mucosal barrier integrity in adult EoE patients. Improvement of the mucosal barrier integrity correlates with normalization of expression of desmoglein-1 and filaggrin marker genes.

Chronic kidney disease and an uncertain diagnosis of Fabry disease: approach to a correct diagnosis.

BACKGROUND AND OBJECTIVES: Screening for Fabry disease (FD), an X-linked lysosomal storage disorder, reveals a significant number of individuals with a genetic variant of unknown significance without classical FD manifestations; these variants in the α-galactosidase A gene often result in a high residual leukocyte α-galactosidase A and it is unclear whether these individuals suffer from FD. Therefore, a structured diagnostic approach is warranted. We present a diagnostic algorithm on how to approach adults with chronic kidney disease and an uncertain diagnosis of FD nephropathy. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: A modified Delphi procedure was conducted to reach consensus among 11 FD experts. A systematic review was performed to identify possible criteria that could confirm or exclude FD nephropathy. RESULTS: The gold standard for FD nephropathy was defined as characteristic storage on electron microscopy (EM) in a kidney biopsy in the absence of medication that may induce similar storage. The suggested criteria to confirm FD nephropathy are as follows: 'renal cysts', 'Maltese cross sign', 'immunohistochemical staining of Gb3 in urine' and 'high urinary Gb3'; and to exclude FD nephropathy: 'absence of renal cysts', 'small kidneys' and 'high protein excretion' were rejected because of low or uncertain specificity. Urinary Gb3 may be increased in other kidney diseases and there was no agreement on this criterion, although a third of the panel indicated that it is sufficient to diagnose FD nephropathy. The 'Maltese cross sign' and 'high urinary Gb3' were selected as red flags to suggest the possibility of FD nephropathy, but are not sufficient for a definite diagnosis of FD nephropathy. CONCLUSIONS: In adults with chronic kidney disease, an α-galactosidase A gene variant and an uncertain diagnosis of FD, a kidney biopsy with EM analysis should be performed to confirm or reject the diagnosis of FD nephropathy. Other criteria currently cannot substitute for a biopsy in these cases.

Cyclosporine versus everolimus: effects on the glomerulus.

Inhibitors of the mammalian target of rapamycin (mTOR) have been associated with proteinuria. We studied the development of proteinuria in renal transplant recipients (RTR) treated with the mTOR inhibitor everolimus in comparison with a calcineurin inhibitor. We related the presence of proteinuria to histopathological glomerular findings in two-yr protocol biopsies. In a single-center study, nested in a multicenter randomized controlled trial, we determined eGFR, proteinuria, and renal biopsy data (light- and electron microscopy) of RTR receiving prednisolone/everolimus (P/EVL) (n = 16) in comparison with patients treated with prednisolone/cyclosporine A (P/CsA) (n = 7). All patients had been on the above-described maintenance immunosuppression for 18 months. Renal function at two yr after transplantation did not differ between patients receiving P/EVL or P/CsA (eGFR 45.5 vs. 45.7 mL/min/1.73 m(2)). Proteinuria was slightly increased in P/EVL vs. P/CsA group (0.29 vs. 0.14 g/24 h, p = 0.06). There were no differences in light- or electron microscopic findings. We could not demonstrate increased podocyte effacement or changes in glomerular basement membrane (GBM) thickness in P/EVL-treated patients. In conclusion, long-term treatment with everolimus leaves the GBM and podocytes unaffected.

Hedgehog signalling stimulates precursor cell accumulation and impairs epithelial maturation in the murine oesophagus.

OBJECTIVE: In the intestine Hedgehog (Hh) signalling is directed from epithelium to mesenchyme and negatively regulates epithelial precursor cell fate. The role of Hh signalling in the oesophagus has not been studied in vivo. Here the authors examined the role of Hh signalling in epithelial homeostasis of oesophagus. DESIGN: The authors used transgenic mice in which the Hh receptor Patched1 (Ptch1) could be conditionally inactivated in a body-wide manner and mice in which Gli1 could be induced specifically in the epithelium of the skin and oesophagus. Effects on epithelial homeostasis of the oesophagus were examined using immunohistochemistry, in situ hybridisation, transmission electron microscopy and real-time PCR. Hh signalling was examined in patients with oesophageal squamous cell carcinoma (SCC) by quantitative real-time PCR. RESULTS: Sonic Hh is signalled in an autocrine manner in the basal layer of the oesophagus. Activation of Hh signalling resulted in an expansion of the epithelial precursor cell compartment and failure of epithelial maturation and migration. Levels of Hh targets GLI1, HHIP and PTCH1 were increased in SCC compared with normal tissue from the same patients. CONCLUSION: Here the authors find that Hh signalling positively regulates the precursor cell compartment in the oesophageal epithelium in an autocrine manner. Since Hh signalling targets precursor cells in the oesophageal epithelium and signalling is increased in SCCs, Hh signalling may be involved in oesophageal SCC formation.

Ultrastructural analysis of dermal fibroblasts in mucopolysaccharidosis type I: Effects of enzyme replacement therapy and hematopoietic cell transplantation.

In mucopolysaccharidosis type I (MPS I; alpha-L-iduronidase deficiency), glycosaminoglycans (GAGs) accumulate in different cell types, causing characteristic vacuolization. Hematopoietic cell transplantation (HCT) and enzyme replacement therapy (ERT) both aim to restore tissue morphology by delivering alpha-L-iduronidase to the deficient cells. The authors investigated the efficacy of both therapies on dermal fibroblast morphology in 12 patients by electron microscopy of repeated skin biopsies before and during 2 years of ERT as well as before and 6 months after HCT. Cell vacuolization was rated according to a semi-quantitative scoring system. At baseline all patients showed an increased vacuolization score as compared to controls. In addition the vacuolization score was significantly higher in patients with the severe phenotype of the disease (n = 7) compared to patients with attenuated phenotypes (n = 5) (p = .009). After initiation of ERT a significant decrease in cell vacuolization was observed (p = .012). However, the response rate varied among patients, as the vacuolization score remained high during the first year of ERT in 3 patients with the severe phenotype. In all patients who received a successful HCT (n = 3) only minimal disturbances in cell morphology were observed afterward. In conclusion, both ERT and HCT are capable of restoring, at least partially, dermal fibroblast morphology in MPS I.

Depletion of the colonic epithelial precursor cell compartment upon conditional activation of the hedgehog pathway.

BACKGROUND & AIMS: The intestinal epithelium is a homeostatic system in which differentiated cells are in dynamic equilibrium with rapidly cycling precursor cells. Wnt signaling regulates intestinal epithelial precursor cell fate and proliferation. Homeostatic systems exist by virtue of negative feedback loops, and we have previously identified the Hedgehog (Hh) pathway as a potential negative feedback signal in the colonic epithelium. Indian hedgehog (Ihh) is produced by the differentiated enterocytes and negatively regulates Wnt signaling in intestinal precursor cells. We studied the role of members of the Hh signaling family in the intestine using a conditional genetic approach. METHODS: We inactivated the Hh receptor Patched1 (Ptch1) in adult mice, resulting in constitutive activation of the Hh signaling pathway. Effects on colonic mucosal homeostasis were examined. Colon tissues were examined by immunohistochemistry, in situ hybridization, transmission electron microscopy, and real-time polymerase chain reaction. RESULTS: Ihh but not Sonic hedgehog (Shh) was expressed in colonic epithelium. Expression of Ptch1 and Gli1 was restricted to the mesenchyme. Constitutive activation of Hh signaling resulted in accumulation of myofibroblasts and colonic crypt hypoplasia. A reduction in the number of epithelial precursor cells was observed with premature development into the enterocyte lineage and inhibition of Wnt signaling. Activation of Hh signaling resulted in induction of the expression of bone morphogenetic proteins (Bmp) and increased Bmp signaling in the epithelium. CONCLUSIONS: Hh signaling acts in a negative feedback loop from differentiated cells via the mesenchyme to the colonic epithelial precursor cell compartment in the adult mouse.

Analysis of apoptosis in peripheral blood and synovial tissue very early after initiation of infliximab treatment in rheumatoid arthritis patients.

OBJECTIVE: Infliximab treatment results in a decrease in synovial cellularity as early as 48 hours after initiation of therapy in patients with rheumatoid arthritis (RA). This study was undertaken to investigate whether infliximab induces apoptosis within the first 24 hours after infusion. METHODS: The percentage of apoptotic cells was determined by flow cytometry in blood drawn from 21 patients directly before, 1 hour after, and 24 hours after infliximab infusion. Synovial tissue samples obtained before, 1 hour after (n = 5), or 24 hours after (n = 5) initiation of therapy were subjected to immunohistochemistry to detect active caspase 3 and to TUNEL assay and electron microscopy to detect apoptosis. In addition, plasma levels of nucleosomes (generated during apoptosis) and C4b/c (an indicator of complement activation) were measured. RESULTS: There were no signs of apoptosis induction in peripheral blood monocytes or lymphocytes after infliximab treatment. Circulating lymphocyte counts were increased within 1 hour after infusion (P < 0.05). There was no definite evidence of apoptosis induction in the synovium, except in 1 patient 24 hours after the infliximab infusion. Consistent with these results, there was no increase in nucleosome levels nor were there signs of complement activation. CONCLUSION: Our findings indicate that the rapid decrease in synovial cellularity observed after initiation of anti-tumor necrosis factor antibody therapy cannot be explained by apoptosis induction at the site of inflammation. It is tempting to speculate that the striking effects on synovial inflammation may be explained by other mechanisms, such as decreased migration toward the synovial compartment and reduced retention in the inflamed synovium.

Tubuloreticular structures in different types of myositis: implications for pathogenesis.

In dermatomyositis (DM) there is strong histopathological evidence of a microvascular pathogenesis, including endothelial microtubular inclusions. In nonspecific myositis, perimysial and perivascular infiltrates in the muscle biopsy similar to DM are found. Microtubular inclusions in endothelial cells were systematically searched for and found in 4 of the 20 muscle biopsies of nonspecific myositis patients (20%). Three had a CTD (SLE, scleroderma, and Sjogren syndrome). Ten patients with DM and 5 patients with sporadic inclusion body myositis served as positive and negative controls, respectively.

Kidney failure in mice lacking the tetraspanin CD151.

The tetraspanin CD151 is a cell-surface molecule known for its strong lateral interaction with the laminin-binding integrin alpha3beta1. Patients with a nonsense mutation in CD151 display end-stage kidney failure associated with regional skin blistering and sensorineural deafness, and mice lacking the integrin alpha3 subunit die neonatally because of severe abnormalities in the lung and kidney epithelia. We report the generation of Cd151-null mice that recapitulate the renal pathology of human patients, i.e., with age they develop massive proteinuria caused by focal glomerulosclerosis, disorganization of the glomerular basement membrane, and tubular cystic dilation. However, neither skin integrity nor hearing ability are impaired in the Cd151-null mice. Furthermore, we generated podocyte-specific conditional knockout mice for the integrin alpha3 subunit that show renal defects similar to those in the Cd151 knockout mice. Our results support the hypothesis that CD151 plays a key role in strengthening alpha3beta1-mediated adhesion in podocytes.

Podocyte foot process effacement is not correlated with the level of proteinuria in human glomerulopathies.

BACKGROUND: Nephrotic syndromes result from increased glomerular permeability to proteins and are structurally believed to be associated with podocyte foot process effacement. Despite increasing knowledge of the molecular composition of the glomerular filtration barrier, the relationship between proteinuria and foot process effacement is unclear. METHODS: We conducted a morphologic study on the relationship between podocyte foot process effacement and proteinuria. Electron microscope pictures of glomerular capillaries were randomly taken from 27 cases in various stages of minimal change nephrotic syndrome (MCNS), from six cases of IgA nephropathy (IgAN) with high proteinuria and from seven control kidneys. From each picture, the mean width of the foot processes (FPW) was quantitated. RESULTS: In normal kidney the mean FPW was 580 +/- 40 nm. In biopsies from patients with MCNS without treatment, foot processes were diffusely effaced, reflected by a FPW of 1600 +/- 440 nm. In biopsies from patients with MCNS relapsing under prednisolone treatment, foot processes were significantly less effaced than in untreated MCNS (FPW 920 +/- 200 nm). In biopsies displaying IgAN, effacement was significantly more segmental than in untreated MCNS (FPW 800 +/- 170 nm). Proteinuria did not differ significantly among the groups. Neither in MCNS nor in IgAN was the extent of foot process effacement correlated with the level of proteinuria. CONCLUSION: Podocyte foot process effacement is not correlated with proteinuria. The differences in podocyte effacement between MCNS, MCNS relapsing under prednisolone treatment, and IgAN may point to different mechanisms of podocyte injury in these diseases.

Timing and sequence of differentiation of embryonic rat hepatocytes along the biliary epithelial lineage.

To study the differentiation of hepatocytes along the biliary epithelial lineage in vivo, embryonic day 14 (E14) rat hepatocytes were isolated by differential centrifugation and transplanted as single-cell suspensions into the spleen of adult syngeneic rats. Hepatocytes and cholangiocytes were identified and their maturation characterized by the level of expression of alpha-fetoprotein (AFP), glutamate dehydrogenase (GDH), and carbamoyl phosphate synthetase I (CPS); annexin IV, annexin V, cytokeratin 19 (CK-19), and cystic fibrosis transmembrane conductance regulator (CFTR); and electron microscopy. By correlating morphologic changes with the timing in the expression of these markers, we show that the organization of the transplanted E14 hepatocytes into lobular structures is accompanied by the formation and maturation of bile ducts around these developing lobules. Morphologic differentiation of the emerging bile ducts was accompanied by a gradual loss of hepatocyte markers and a gradual acquisition of cholangiocyte markers, with markers identifying a large-cholangiocyte phenotype appearing latest. Once fully differentiated, the intrasplenic liver lobules developed cholestatic features. The accompanying proliferation of bile ducts was due to cholangiocyte proliferation, but ductular transformation of hepatocytes was also observed. In conclusion, (1) bile duct formation at the interface between hepatocytes and connective tissue is an inherent component of liver development and (2) the susceptibility of developing hepatocytes to bile duct-inducing signals is highest in the fetal liver but that (3) this capacity is not irreversibly lost in otherwise mature hepatocytes.