Evidence for Activation of the Unfolded Protein Response in Collagen IV Nephropathies

Thin-basement-membrane nephropathy (TBMN) and Alport syndrome (AS) are progressive collagen IV nephropathies caused by mutations in COL4A3/A4/A5 genes. These nephropathies invariably present with microscopic hematuria and frequently progress to proteinuria and CKD or ESRD during long-term follow-up. Nonetheless, the exact molecular mechanisms by which these mutations exert their deleterious effects on the glomerulus remain elusive. We hypothesized that defective trafficking of the COL4A3 chain causes a strong intracellular effect on the cell responsible for COL4A3 expression, the podocyte. To this end, we overexpressed normal and mutant COL4A3 chains (G1334E mutation) in human undifferentiated podocytes and tested their effects in various intracellular pathways using a microarray approach. COL4A3 overexpression in the podocyte caused chain retention in the endoplasmic reticulum (ER) that was associated with activation of unfolded protein response (UPR)–related markers of ER stress. Notably, the overexpression of normal or mutant COL4A3 chains differentially activated the UPR pathway. Similar results were observed in a novel knockin mouse carrying the Col4a3-G1332E mutation, which produced a phenotype consistent with AS, and in biopsy specimens from patients with TBMN carrying a heterozygous COL4A3-G1334E mutation. These results suggest that ER stress arising from defective localization of collagen IV chains in human podocytes contributes to the pathogenesis of TBMN and AS through activation of the UPR, a finding that may pave the way for novel therapeutic interventions for a variety of collagenopathies.Alport syndrome (AS) and thin-basement-membrane nephropathy (TBMN) are genetic diseases of the glomerular basement membrane (GBM), an important key component of the glomerular filtration barrier. They are caused by pathogenic changes in the genes encoding the α3/α4/α5 chains of type IV collagen, an abundant constituent of the GBM, and can lead to total or partial loss of its network.^1,2 Mutations have been identified in all three genes that encode the collagen IV chains in patients with AS and TBMN, while the number of these mutations exceeds 500 to date (Human Gene Mutation Database). AS is invariably associated with CKD and ESRD usually before 30 years of age, often accompanied by sensorineural deafness and/or ocular abnormalities. TBMN is characterized primarily by microscopic hematuria and was classically thought to be a benign disease with excellent prognosis. Still, our group and others have described particular COL4A3/COL4A4 mutations in patients with the dual diagnosis of TBMN and FSGS who developed CKD/ESRD, thus establishing TBMN as a far-from-benign condition, at least in a subset of patients.^3,4 Nonetheless, the exact molecular mechanisms by which these mutations exert their pathogenic effect remain unclear.Physiologically, the collagen IV α3, α4, and α5 chains assemble through recognition of their carboxy-terminal NC1 domains and form helical heterotrimers that undergo several enzymatic post-translational modifications in the cell’s endoplasmic reticulum (ER) before being secreted into the GBM. Once in the extracellular space, they form a multistructural network that has a dual role in providing strength to the membrane and participating in dynamic biological processes by interacting with many other proteins.^5,6 Previous studies showed that numerous COL4A3/A4/A5 mutations in the NC1 domains share destructive effects on heterotrimer formation and/or secretion of the heterotrimer from cells.^7–9 Therefore, mutant collagen IV heterotrimers could potentially cause deleterious extracellular effects due to their absence from the GBM, as well as putative intracellular effects due to accumulation of misfolded protein inside the producing cells. Importantly, it has been shown that the adult GBM collagen IV chains are solely produced by the podocyte and not by the endothelial or adjacent cells.¹⁰ This fact confers the podocyte a central role as the collagen IV producing and secreting cell. Of note, Heidet et al. demonstrated that in the AS podocyte, the COL4A3 protein, even when absent from the GBM, is detected within the cell, a finding that is distinct among patients with AS.¹¹Herein, we addressed the putative effect of overexpression of both normal and mutant COL4A3 chains on various intracellular pathways of the human podocyte. To target this, we profiled mRNA on human cultured podocytes that overexpressed the wild-type (WT) or mutant COL4A3-G1334E chain for panoramic assessment of gene expression. One of the pathways that emerged as highly deregulated in our analysis is the unfolded protein response pathway (UPR). Inside the cell, an imbalance between protein load and proper protein folding is called ER stress. As a defense mechanism against this imbalance, the cells have at hand a robust mechanism termed the UPR and activated by intracellular retention of unfolded protein.¹² UPR allows cells to recover from stress by temporarily halting protein translation and activating signaling pathways that lead to the production of molecular chaperones involved in protein folding. Once activated, this adaptive pathway may protect the cell from future insults.¹³ However, in cases of prolonged or deregulated ER stress owing to genetic or other factors, this pathway could become maladaptive and cytotoxic and lead to apoptosis.¹⁴We further examined this pathway by testing whether chaperone binding immunoglobulin protein (BiP), a central marker of UPR activation, is upregulated in renal biopsy specimens from patients with a confirmed COL4A3-G1334E mutation. Finally, we examined UPR activation using a relevant knockin mouse model we recently generated in our laboratory carrying the Col4a3-G1332E mutation (the equivalent of the COL4A3-G1334E mutation in patients with AS and TBMN). To our knowledge, this is the first mouse model carrying a missense glycine COL4A3 mutation, which causes AS and TBMN in humans.^4,15,16     

Effects of macroporous monofilament mesh on infection in a contaminated field

Background

The aim of this study was to evaluate whether the type of the mesh and proper surgical technique can influence the outcome of a tension-free hernia repair in a contaminated filed.

Materials and methods

This study was based on the model of bacterial peritonitis in rats induced with a mixture composed of Escherichia coli and Bacteroides fragilis. Two animals were used as a control group without induced peritonitis and 10 animals with mesh implanted inside of the peritoneal cavity. For the 20 animals in the studied group, bacterial fluid was applied into the abdominal cavity together with the mesh implantation. In 10 cases, the mesh was fixed flatly upon the surface of the peritoneum; in the other 10, the mesh was rolled and then fixed within the peritoneal cavity. After 5 weeks, the animals were operated on again, and the meshes, the peritoneal fluid and, if present, any granulomas were taken for bacterial cultivation.

Results

The results of the bacterial cultivation of the material from the control group (without mesh) and from the rats with flatly fixed mesh were almost completely negative (0/10 and 1/10, respectively). In 9 out of 10 rats that were exposed to the rolled mesh for 5 weeks, the colonisation of meshes with both B. fragilis and E. coli was found (p?Conclusions When properly fixed, flat mesh, even in a contaminated field, may allow for a proper mesh healing and does not influence the ability to cure bacterial peritonitis in an animal model. A bad surgical technique, such as inadequately positioned or rolled mesh, may cause persistent peritoneal bacteraemia.     

Axial plane analysis of Lenke 1A adolescent idiopathic scoliosis as an aid to identify curve characteristics

Background context

Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional (3D) deformity of the spine involving deviations in the frontal plane, modifications of the sagittal profile, and rotations in the transverse plane. Although Lenke classification system is based on 2D radiographs and includes sagittal thoracic and coronal lumbar modifiers, Lenke et al. suggested inclusion of axial thoracic and lumbar modifiers in the analysis.

Purpose

To analyze axial plane of Lenke 1A curves to identify curve characteristics.

Study design

Retrospective study.

Patient sample

Seventy patients (49 women, 21 men) with Lenke Type 1A idiopathic scoliosis were analyzed.

Outcome measures

Coronal, sagittal, and axial parameters were measured from plain radiographs that were obtained at initial medical examination of the patients.

Methods

Coronal and sagittal plane and whole spine segmental vertebra rotations from thoracic 1 to lumbar 5 were evaluated in 70 AIS patients with Lenke 1A curves by using Drerup method. Three different subgroups were identified according to magnitude and direction of lower end vertebra (LEV) rotation.

Results

In Group 1 (Lenke 1A1), the direction of LEV rotation was same with other vertebrae in the main curve and the magnitude of the LEV rotation was less than −0.5°. In Group 2 (Lenke 1A2), the rotation of LEV was between −0.5° and 0.5° and so was accepted as neutral. In Group 3 (Lenke 1A3), the rotation of LEV had opposite direction with vertebrae in the main curve and the magnitude of LEV rotation was more than 0.5°. The mean thoracic Cobb angle of patients with Lenke 1A idiopathic scoliosis was 51.1° (range 37°–80°), whereas the mean lumbar Cobb angle was 16.4° (range 0°–32°). The mean angle of trunk rotation of the patients was 5.7° (range 1°–16°). In terms of maximum thoracic vertebra rotation, the mean rotation angle of Lenke 1A idiopathic curves was −18.9° (range −(9.8°–44.7°)). The mean maximum lumbar vertebra rotation was 4.5° (range −7.2° to 15.1°).

Conclusions

Addition of axial plane analysis to conventional coronal and sagittal evaluations in patients with Lenke 1A curves may reveal inherent structural differences that are not apparent in single planar radiographic assessments and may necessitate a different surgical strategy.     

Endplate deflection is a defining feature of vertebral fracture and is associated with properties of the underlying trabecular bone

Endplate deflection frequently occurs with vertebral failure, but the relationship between the two remains poorly defined. This study examined associations between endplate deflection under compressive loading and characteristics of the neighboring subchondral bone and intervertebral disc (IVD). Ten L1 vertebrae with adjacent IVDs were dissected, compressed axially in a stepwise manner to failure, and imaged with micro‐computed tomography before each loading step. From the images, deflection was measured across the surface of each endplate at each step. Trabecular microstructure and endplate volume fraction were evaluated in 5 mm regions just under the superior endplate. IVDs were assessed using computed tomography and histology. A marked increase in superior endplate deflection coincided with a drop in the load‐displacement curve. Endplate deflection was higher in regions with less robust bone microstructure (p < 0.009), though these associations tended to weaken as loading progressed. Immediately following the ultimate point, endplate deflection was higher in regions underlying the nucleus pulposus versus annulus fibrosus (p = 0.035), irrespective of disc grade (p = 0.346). These results indicate that a sudden increase in endplate deflection signals that the mechanical competence of the vertebra has been compromised. The mechanisms of endplate failure likely relate to anatomical features of the endplate, neighboring trabecular bone, and IVD. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:880–886, 2014.     

The influence of FDG PET-CT on the detection of extrahepatic disease in patients being considered for resection of colorectal liver metastasis

Introduction

The aim of this study was to evaluate the influence of flurodeoxyglucose positron emission tomography computed tomography (FDG PET-CT), as an adjunct to conventional CT staging, in the detection of extrahepatic disease in patients with potentially resectable colorectal liver metastasis.

Methods

Overall, 133 consecutive patients with colorectal liver metastases staged with CT and PET-CT referred to the East Lancashire regional hepatobiliary multidisciplinary team over a two-year period were included in this study. Abnormal findings on PET-CT were correlated with follow-up imaging and/or histology. All imaging was reviewed by specialist hepatobiliary radiologists for the presence/absence of extrahepatic disease. The influence of the PET-CT findings was categorised for each patient in relation to operability and other significant findings.

Results

PET-CT had a major impact on staging of extra hepatic disease in 20% of patients, in comparison with the initial CT. Six per cent of patients were upstaged from operable CT findings to inoperable findings on PET-CT because of the discovery of inoperable occult extrahepatic disease. Five per cent had operable local regional nodal disease detected on PET-CT. A further 3% had premalignant colorectal lesions detected on PET-CT. Six per cent of patients were downstaged from indeterminate or suspected inoperable CT findings to operable findings on PET-CT.

Conclusions

The use of PET-CT in this setting may prevent futile operations, guide the resection of local regional nodal disease and downstage a number of patients thought to have extrahepatic disease on conventional imaging. This study has shown similar results to other recent studies and supports the use of PET-CT as a necessary staging modality in patients with potentially resectable colorectal liver metastases.     

Which treatment option for paediatric femoral fractures in school-aged children: elastic nail or spica casting?

Objective

Titanium elastic nails and immediate spica casts are treatment options for femoral fractures in school-aged children (6–12 years). This study aimed to compare the results of elastic nail and immediate spica cast methods for treating femoral fractures in school-aged children.

Materials and methods

A retrospective evaluation was made of patients who underwent immediate spica cast (20 patients) or titanium elastic nail (22 patients) for femoral fracture. Groups were compared in terms of clinical and radiographic union, duration of hospitalisation, range of knee motion, walking independently and complications. The mean age was 9.8 ± 1.3 years for the elastic nail group and 6.4 ± 1 for the cast group. The mean follow-up period was 12.6 ± 5.2 months for the elastic nail group and 14.3 ± 6 months for the cast group.

Results

All fractures in both group were healed. Duration of hospitalisation was shorter (2.2/7.1) and range of knee motion was better (132°/129°) in the cast group. The duration for independent walking was shorter (49.2/79.8) in the nail group. These differences were significant (p < 0.001). Two superficial infections and two malalignment were detected in the nail group. Three superficial infections and four malalignment were detected in the cast group.

Conclusion

We detected that both treatment options were similar with regard to complications and results. Although the complications are similar in two treatment methods, complications of elastic nail are more challenging and may require new surgical procedure. If the elastic nail is selected, surgical complications should not be underestimated.     

Use of Deep Learning Networks and Statistical Modeling to Predict Changes in Mechanical Parameters of Contaminated Bone Cements

The purpose of the study was to test the usefulness of deep learning artificial neural networks and statistical modeling in predicting the strength of bone cements with defects. The defects are related to the introduction of admixtures, such as blood or saline, as contaminants into the cement at the preparation stage. Due to the wide range of applications of deep learning, among others in speech recognition, bioinformation processing, and medication design, the extent was checked to which it is possible to obtain information related to the prediction of the compressive strength of bone cements. Development and improvement of deep learning network (DLN) algorithms and statistical modeling in the analysis of changes in the mechanical parameters of the tested materials will enable determining an acceptable margin of error during surgery or cement preparation in relation to the expected strength of the material used to fill bone cavities. The use of the abovementioned computer methods may, therefore, play a significant role in the initial qualitative assessment of the effects of procedures and, thus, mitigation of errors resulting in failure to maintain the required mechanical parameters and patient dissatisfaction.     

Local atomic order of the amorphous TaOx thin films in relation to their chemical resistivity

The experimental and theoretical studies of the local atomic order and chemical binding in tantalum oxide amorphous films are presented. The experimental studies were performed on thin films deposited at the temperature of 100 °C by atomic layer deposition on silicon (100) and glass substrates. Thin films of amorphous tantalum oxide are known to exhibit an extremely large extent of oxygen nonstoichiometry. Performed X-ray absorption and photoelectron studies indicated the oxygen over-stoichiometric composition in the considered films. Surplus oxygen atoms have 1s electron level with binding energy about 1 eV higher than these in reference Ta₂O₅ oxide. The density functional theory was applied to find the possible location of additional oxygen atoms. Performed calculation indicated that additional atoms may form the dumbbell defects, which accumulate the dangling oxygen bonds in orthorhombic structure and lead to increase of oxygen 1s level binding energy. The presence of this kind of oxygen–oxygen bonding may be responsible for increase of amorphous film chemical resistivity which is very important in many applications.

The experimental and theoretical studies of the local atomic order and chemical binding in tantalum oxide amorphous films are presented.     

Atypical location of an osteoid osteoma with atypical anterior knee pain

INTRODUCTION

An osteoid osteoma is a painful tumor that most commonly affects the extra-articular parts of the long bones. An intra-articular location of an osteoid osteoma is rare. Various differential diagnoses may arise in connection with such an unusual location because it causes atypical clinical signs.

PRESENTATION OF CASE

A 24-year-old male developed pain in the central region of the right knee. Magnetic resonance imaging (MRI) showed no clear pathology in the knee joint. A technetium bone scan and computed tomography (CT) were then ordered and confirmed the presence of an osteoid osteoma in the knee joint. The patient was treated through an anteromedial approach to the knee, and the lesion was removed by excisional biopsy under fluoroscopy.

DISCUSSION

The diagnosis of intra-articular osteoid osteoma is challenging because the clinical presentation can be misleading. MRI is often requested as the first imaging method when dealing with knee symptoms, and radiologists are often unaware of the clinical presentation. Edema seen on MRI can be misleading with respect to the location of the nidus. CT is considered to be the best imaging method because it usually allows for clear visualization of the nidus. Different treatments have been proposed, ranging from open excision to arthroscopic resection.

CONCLUSION

Osteoid osteoma should be considered in young adult patients with chronic knee pain and no history of trauma.