Sinonasal Lymphoepithelial Carcinoma With Aggressive Orbital Invasion.

Sinonasal lymphoepithelial carcinoma (LEC) is an extremely rare malignancy that shares some characteristics with nasopharyngeal carcinoma. In Asian populations, Epstein-Barr virus has been reported to be associated with LEC located outside of the nasopharynx. We report a rare case of sinonasal LEC with locoregional extension (brain and orbit). A 39-year-old Malay male initially presented with profound blurring of vision on the left eye (LE) and proptosis, followed by nasal symptoms of anosmia. Clinical examination revealed that the LE visual acuity was 6/36, with reduced optic nerve function with normal funduscopic findings, non-axial proptosis, and minimal limitation of extraocular movement. Subsequently, his vision worsened with perception of light in three days. Radioimaging studies showed soft tissue lesion at the ethmoid sinus with extensive local and intracranial extension. Microscopic analysis and immunohistochemistry confirmed the diagnosis of LEC. The patient was given induction chemotherapy followed by concurrent chemoradiotherapy with weekly intravenous cisplatin. Upon completing the fourth cycle of chemotherapy, the patient's ocular symptoms and general conditions worsened. Repeated imaging showed worsening intracranial extension with cerebral and cerebellar edema, and the patient succumbed to death. Sinonasal LEC is a rare malignant tumor with little mention in the literature. This case was reported to highlight the importance of a high index of suspicion for acute ocular symptoms with mass.

Comparative Analysis of Elderly Patients Undergoing Radical Cystectomy With Ureterocutaneostomy or Ileal Conduit With a Special Focus on Bowl Complications Requiring Surgical Revision.

Objectives: Ileal conduits (ICs) carry an additional perioperative complication risk due to the bowel procedure. This analysis compares surgical outcomes in patients ≥75 years of age with ureterocutaneostomy (UCN) and IC after cystectomy (Cx). Methods: Data of 527 patients included in a retrospective cystectomy database of two high volume centers (2008-2020) were queried to identify elderly patients (≥75 years) who underwent Cx either with IC or UCN. Patient characteristics of all patients [age, BMI, Charlson Comorbidity Index (CCI)], perioperative parameters (operation time, blood loss, transfusions, tumor stage), and postoperative complications (clavien >IIIA, intensive care unit (ICU) stay) were compared. As special focus, bowel complications requiring surgical revision (rBCs) were analyzed. In patients with IC, the rate of ureteral implantation stenosis (USt) was recorded. As a population of special interest, patients ≥80 years of age were analyzed separately. Categorical data were compared using Fisher exact test, and continuous data were compared using Mann-Whitney U test. Results: A total of 163 patients ≥75 years of age (125 IC, 38 UCN) were identified. Patients with UCN were older and presented with a higher CCI, though differences were not statistically different. Surgery with palliative intent was more frequent in patients with UCN (37 vs. 10%). Operation time in UCN was significantly shorter (233 vs. 305 min, p = 0.02), while blood loss and transfusion rate were comparable. Overall complication rate (Clavien-Dindo grade IIIA-IVB) was comparable (UCN 34% vs. IC 37%). However, rBC was a rare complication in UCN (3/38) as compared to patients with IC (15/125). Frequency of postoperative ICU stay (UCN 16% vs. IC 16%) and 90-day mortality did not differ (UCN 3/38 patients, IC 5/125 patients). Regarding long-term follow-up, USt requiring revision or permanent stenting was seen in 18/125 (14%) patients with IC. In patients >80 years of age, results were comparable to the main cohort. Low event rate regarding complications and bias inherent of a retrospective analysis (selection bias, unequal distribution in case numbers) precludes detection of statistical differences regarding patients' characteristics and overall complication rate. Conclusion: UCN is an alternative to IC in elderly and/or frail patients. Severe bowel complications are numerically less frequent and operation time is minimized.

Preoperative Parameters to Predict the Development of Symptomatic Lymphoceles after Radical Prostatectomy.

INTRODUCTION: Symptomatic lymphoceles (SLs) represent the most common complication after radical prostatectomy (RP) and pelvic lymph node dissection (PLND). To date, preoperative risk factors are missing. METHODS: Clinical and pathological data of 592 patients who underwent RP and PLND were evaluated. Included parameters were age, BMI, prostate-specific antigen (PSA), PSA ratio, PSA density, number of resected and/or positive lymph nodes, previous abdominal surgery/pelvic radiotherapy, anticoagulation, and surgical approach. RESULTS: Fifty-nine patients (10%) developed an SL, of which 57 underwent open retropubic radical prostatectomy (RRP) and 2 underwent robot-assisted radical prostatectomy (RARP). Multivariate logistic regression revealed the following parameters as statistically significant risk factors: PSA (odds ratio [OR] = 2.23; 95% CI [1.25; 5.04], p = 0.04), number of resected lymph nodes (OR = 1.47; 95% CI [1.10; 1.97], p < 0.01), previous abdominal surgery (OR = 2.58; 95% CI [1.38; 4.91], p < 0.01), and surgical approach (OR = 0.08; 95% CI [0.01; 0.27], p < 0.01). Previous oral anticoagulation showed almost statistically significant results (OR = 2.39 [0.92; 5.51], p = 0.05). CONCLUSION: The risk for SL might be predictable considering preoperative risk factors such as PSA, previous abdominal surgery and anticoagulation. To avoid SL, RARP should be the procedure of choice. If RRP is considered, patients at risk for SL may benefit from peritoneal fenestration during RP.

Novel Classification for Upper Tract Urothelial Carcinoma to Better Risk-stratify Patients Eligible for Kidney-sparing Strategies: An International Collaborative Study.

BACKGROUND: The European Association of Urology risk stratification dichotomizes patients with upper tract urothelial carcinoma (UTUC) into two risk categories. OBJECTIVE: To evaluate the predictive value of a new classification to better risk stratify patients eligible for kidney-sparing surgery (KSS). DESIGN, SETTING, AND PARTICIPANTS: This was a retrospective study including 1214 patients from 21 centers who underwent ureterorenoscopy (URS) with biopsy followed by radical nephroureterectomy (RNU) for nonmetastatic UTUC between 2000 and 2017. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: A multivariate logistic regression analysis identified predictors of muscle invasion (≥pT2) at RNU. The Youden index was used to identify cutoff points. RESULTS AND LIMITATIONS: A total of 811 patients (67%) were male and the median age was 71 yr (interquartile range 63-77). The presence of non-organ-confined disease on preoperative imaging (p < 0.0001), sessile tumor (p < 0.0001), hydronephrosis (p = 0.0003), high-grade cytology (p = 0.0043), or biopsy (p = 0.0174) and higher age at diagnosis (p = 0.029) were independently associated with ≥pT2 at RNU. Tumor size was significantly associated with ≥pT2 disease only in univariate analysis with a cutoff of 2 cm. Tumor size and all significant categorical variables defined the high-risk category. Tumor multifocality and a history of radical cystectomy help to dichotomize between low-risk and intermediate-risk categories. The odds ratio for muscle invasion were 5.5 (95% confidence interval [CI] 1.3-24.0; p = 0.023) for intermediate risk versus low risk, and 12.7 (95% CI 3.0-54.5; p = 0.0006) for high risk versus low risk. Limitations include the retrospective design and selection bias (all patients underwent RNU). CONCLUSIONS: Patients with low-risk UTUC represent ideal candidates for KSS, while some patients with intermediate-risk UTUC may also be considered. This classification needs further prospective validation and may help stratification in clinical trial design. PATIENT SUMMARY: We investigated factors predicting stage 2 or greater cancer of the upper urinary tract at the time of surgery for ureter and kidney removal and designed a new risk stratification. Patients with low or intermediate risk may be eligible for kidney-sparing surgery with close follow-up. Our classification scheme needs further validation based on cancer outcomes.

A 27-Year-Old Primigravida with a Right Renal Cell Carcinoma Removed at 30 Weeks of Gestation by Robot-Assisted Retroperitoneoscopic Partial Nephrectomy.

BACKGROUND Large renal tumors during pregnancy are rare findings (0.07-0.1%). Current guidelines recommend surgical removal. This surgery should be carefully planned in an interdisciplinary team and involves special risks for mother and fetus. This report describes a case of a 27-year-old primigravida woman with a right renal cell carcinoma involving the lower pole of the kidney, which was removed at 30 weeks of gestation by robot-assisted retroperitoneoscopic partial nephrectomy (RARPN). CASE REPORT The patient was referred by the treating obstetrician with a newly diagnosed right lower pole renal mass of 6×4 cm in greatest diameter extending deeply into the parenchyma. No metastasis or enlarged lymph nodes were described in subsequent magnetic resonance tomography. Clinical and laboratory examinations documented a healthy mother and fetus. A right-sided RARPN was advised and planned by an interdisciplinary team of treating physicians (gynecologists, oncologists, and urologists). The surgery was conducted under general anesthesia with an obstetrician on stand-by. Surgery was performed without any complications (operation time 95 min, renal-ischemia time 15 min, and negligible blood loss) and histopathology confirmed the diagnosis of a chromophobe renal cell carcinoma. Further follow-up consultations showed regular wound healing and normal progression of pregnancy, and the patient gave birth to a healthy child at term. Follow-up examinations of the patient were uneventful. CONCLUSIONS This case shows that RARPN can be a safe and effective surgical procedure for partial nephrectomy during pregnancy, where surgery is performed in a specialist center and by an interdisciplinary experienced surgical team. It seems to offer advantages and better risk profile over the laparoscopic approach.

Evaluation of Testicular Self Examination and Testicular Partner Examination in Medical versus Non-Medical Students.

OBJECTIVES: Although testicular cancer (TC) is the most common tumor in young men in Western countries, there is no official cancer detection/screening program for young men in Germany. The most important TC detection tool is self-examination of the testis. Hypothetically medical students may have a diagnosis lead time and detection superiority. This study was designed to analyze whether medical students have a possible knowledge advantage over students of other faculties concerning TC and to compare male and female cancer screening demeanor and mentality. METHODS: Male and female students of various faculties at the Goethe University Frankfurt/Main, Germany were invited to participate in this internet-based anonymous questionnaire with questions about TC awareness/knowledge, testicular (self) examination, and cancer screening behavior. RESULTS: In total 1,049 students (329 medical and 716 non-medical students) completed the questionnaire. In general, medical students had a significantly higher TC knowledge, especially in the more advanced stages of their medical studies (year 3-6). About 50% of medical students had knowledge of TC whereas only 21.3% of non-medical students knew about the disease (p < 0.01). In addition, medical students conducted scrotal examinations more frequently (34.7%) than non-medical students (18.8%). CONCLUSION: The knowledge about TC is low among students. In general, medical students are more aware of TC and perform more frequent testicular examinations compared to non-medical students. Female TC knowledge rises in the clinical part of studies to the same level as their male counterparts, with the result of more testicular partner examinations.

Maternal alloimmune IgG causes anti-glomerular basement membrane disease in perinatal transgenic mice that express human laminin α5.

Mammalian immune systems are not mature until well after birth. However, transfer of maternal IgG to the fetus and newborn usually provides immunoprotection from infectious diseases. IgG transfer occurs before birth in humans across the placenta and continues after birth across the intestine in many mammalian species, including rodents. Transfer, which is mediated by the neonatal IgG Fc receptor, occurs by transcytosis across placental syncytiotrophoblasts and intestinal epithelium. Although maternal IgG is generally beneficial, harmful maternal allo- and autoantibodies can also be transferred to the fetus/infant, resulting in serious disease. To test this we generated transgenic mice that widely express human laminin α5 in their basement membranes. When huLAMA5 transgenic males were crossed with wild-type females, there was a maternal anti-human laminin α5 immune response. Maternal IgG alloantibody crossed the yolk sac and post-natal intestine invivo and bound in bright, linear patterns to kidney glomerular basement membranes of transgenic fetuses/neonates but not those of wild-type siblings. By postnatal day 18, most transgenic mice were proteinuric, had glomerular C3 deposits and inflammatory cell infiltrates, thickened and split glomerular basement membranes, and podocyte foot process effacement. Thus, our novel model of perinatal anti-glomerular basement membrane disease may prove useful for studying pediatric glomerulopathies, formation of the fetomaternal interface, and maternal alloimmunization.

Low-radiation and high image quality coronary computed tomography angiography in "real-world" unselected patients.

AIM: To determine the radiation dose and image quality in coronary computed tomography angiography (CCTA) using state-of-the-art dose reduction methods in unselected "real world" patients. METHODS: In this single-centre study, consecutive patients in sinus rhythm underwent CCTA for suspected coronary artery disease (CAD) using a 320-row detector CT scanner. All patients underwent the standard CT acquisition protocol at our institute (Morriston Hospital) a combination of dose saving advances including prospective electrocardiogram-gating, automated tube current modulation, tube voltage reduction, heart rate reduction, and the most recent novel adaptive iterative dose reconstruction 3D (AIDR3D) algorithm. The cohort comprised real-world patients for routine CCTA who were not selected on age, body mass index, or heart rate. Subjective image quality was graded on a 4-point scale (4 = excellent, 1 = non-diagnostic). RESULTS: A total of 543 patients were included in the study with a mean body weight of 81 ± 18 kg and a pre-scan mean heart rate of 70 ± 11 beats per minute (bpm). When indicated, patients received rate-limiting medication with an oral beta-blocker followed by additional intravenous beta-blocker to achieve a heart rate below 65 bpm. The median effective radiation dose was 0.88 mSv (IQR, 0.6-1.4 mSv) derived from a Dose Length Product of 61.45 mGy.cm (IQR, 42.86-100.00 mGy.cm). This also includes what we believe to be the lowest ever-reported radiation dose for a routine clinical CCTA (0.18 mSv). The mean image quality (± SD) was 3.65 ± 0.61, with a subjective image quality score of 3 ("good") or above for 93% of patient CCTAs. CONCLUSION: Combining a low-dose scan protocol and AIDR3D with a 320-detector row CT scanner can provide high quality images at exceptionally low radiation dose in unselected patients being investigated for CAD.

Neonatal Fc receptor promotes immune complex-mediated glomerular disease.

The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex-mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex-mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG.

Laminin and type IV collagen isoform substitutions occur in temporally and spatially distinct patterns in developing kidney glomerular basement membranes.

Kidney glomerular basement membranes (GBMs) undergo laminin and type IV collagen isoform substitutions during glomerular development, which are believed to be required for maturation of the filtration barrier. Specifically, GBMs of earliest glomeruli contain laminin α1ß1γ1 and collagen α1α2α1(IV), whereas mature glomeruli contain laminin α5ß2γ1 and collagen α3α4α5(IV). Here, we used confocal microscopy to simultaneously evaluate expression of different laminin and collagen IV isoforms in newborn mouse GBMs. Our results show loss of laminin α1 from GBMs in early capillary loop stages and continuous linear deposition of laminin bearing the α5 chain thereafter. In contrast, collagen α1α2α1(IV) persisted in linear patterns into late capillary loop stages, when collagen α3α4α5(IV) first appeared in discontinuous, non-linear patterns. This patchy pattern for collagen α3α4α5(IV) continued into maturing glomeruli where there were lengths of linear, laminin α5-positive GBM entirely lacking either isoform of collagen IV. Relative abundance of laminin and collagen IV mRNAs in newborn and 5-week-old mouse kidneys also differed, with those encoding laminin α1, α5, ß1, ß2, and γ1, and collagen α1(IV) and α2(IV) chains all significantly declining at 5 weeks, but α3(IV) and α4(IV) were significantly upregulated. We conclude that different biosynthetic mechanisms control laminin and type IV collagen expression in developing glomeruli.

Upregulated expression of integrin α1 in mesangial cells and integrin α3 and vimentin in podocytes of Col4a3-null (Alport) mice.

Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.

Mouse stem cells seeded into decellularized rat kidney scaffolds endothelialize and remodel basement membranes.

INTRODUCTION: To address transplant organ shortage, a promising strategy is to decellularize kidneys in a manner that the scaffold retains signals for seeded pluripotent precursor cells to differentiate and recapitulate native structures: matrix-to-cell signaling followed by cell-cell and cell-matrix interactions, thereby remodeling and replacing the original matrix. This would reduce scaffold antigenicity and enable xeno-allografts. RESULTS: DAPI-labeled cells in arterial vessels and glomeruli were positive for both endothelial lineage markers, BsLB4 and VEGFR2. Rat scaffold's basement membrane demonstrated immunolabeling with anti-mouse laminin ß1. Labeling intensified over time with 14 day incubations. CONCLUSION: We provide new evidence for matrix-to-cell signaling in acellular whole organ scaffolds that induces differentiation of pluripotent precursor cells to endothelial lineage. Production of mouse basement membrane supports remodeling of host (rat)-derived scaffolds and thereby warrants further investigation as a promising approach for xenotransplantation. METHODS: We previously showed that murine embryonic stem cells arterially seeded into acellular rat whole kidney scaffolds multiply and demonstrate morphologic, immunohistochemical and gene expression evidence for differentiation. Vascular cell endothelialization was now further tested by endothelial specific BsLB4 lectin and anti-VEGFR2 (Flk1) antibodies. Remodeling of the matrix basement membranes from rat to mouse ("murinization") was assessed by a monoclonal antibody specific for mouse laminin ß1 chain.

Transgenic expression of human LAMA5 suppresses murine Lama5 mRNA and laminin α5 protein deposition.

Laminin α5 is required for kidney glomerular basement membrane (GBM) assembly, and mice with targeted deletions of the Lama5 gene fail to form glomeruli. As a tool to begin to understand factors regulating the expression of the LAMA5 gene, we generated transgenic mice carrying the human LAMA5 locus in a bacterial artificial chromosome. These mice deposited human laminin α5 protein into basement membranes in heart, liver, spleen and kidney. Here, we characterized two lines of transgenics; Line 13 expressed ∼6 times more LAMA5 than Line 25. Mice from both lines were healthy, and kidney function and morphology were normal. Examination of developing glomeruli from fetal LAMA5 transgenics showed that the human transgene was expressed at the correct stage of glomerular development, and deposited into the nascent GBM simultaneously with mouse laminin α5. Expression of human LAMA5 did not affect the timing of the mouse laminin α1-α5 isoform switch, or that for mouse laminin ß1-ß2. Immunoelectron microscopy showed that human laminin α5 originated in both glomerular endothelial cells and podocytes, known to be origins for mouse laminin α5 normally. Notably, in neonatal transgenics expressing the highest levels of human LAMA5, there was a striking reduction of mouse laminin α5 protein in kidney basement membranes compared to wildtype, and significantly lower levels of mouse Lama5 mRNA. This suggests the presence in kidney of a laminin expression monitor, which may be important for regulating the overall production of basement membrane protein.

Deletion of von Hippel-Lindau in glomerular podocytes results in glomerular basement membrane thickening, ectopic subepithelial deposition of collagen {alpha}1{alpha}2{alpha}1(IV), expression of neuroglobin, and proteinuria.

Vascular endothelial growth factor, which is critical for blood vessel formation, is regulated by hypoxia inducible transcription factors (HIFs). A component of the E3 ubiquitin ligase complex, von Hippel-Lindau (VHL) facilitates oxygen-dependent polyubiquitination and proteasomal degradation of HIFalpha subunits. Hypothesizing that deletion of podocyte VHL would result in HIFalpha hyperstabilization, we crossed podocin promoter-Cre transgenic mice, which express Cre recombinase in podocytes beginning at the capillary loop stage of glomerular development, with floxed VHL mice. Vascular patterning and glomerular development appeared unaltered in progeny lacking podocyte VHL. However, urinalysis showed increased albumin excretion by 4 weeks when compared with wild-type littermates with several sever cases (>1000 microg/ml). Many glomerular ultrastructural changes were seen in mutants, including focal subendothelial delamination and widespread podocyte foot process broadening, and glomerular basement membranes (GBMs) were significantly thicker in 16-week-old mutants compared with controls. Moreover, immunoelectron microscopy showed ectopic deposition of collagen alpha1alpha2alpha1(IV) in GBM humps beneath podocytes. Significant increases in the number of Ki-67-positive mesangial cells were also found, but glomerular WT1 expression was significantly decreased, signifying podocyte death and/or de-differentiation. Indeed, expression profiling of mutant glomeruli suggested a negative regulatory feedback loop involving the HIFalpha prolyl hydroxylase, Egln3. In addition, the brain oxygen-binding protein, Neuroglobin, was induced in mutant podocytes. We conclude that podocyte VHL is required for normal maintenance of podocytes, GBM composition and ultrastructure, and glomerular barrier properties.

Cellular origins of type IV collagen networks in developing glomeruli.

Laminin and type IV collagen composition of the glomerular basement membrane changes during glomerular development and maturation. Although it is known that both glomerular endothelial cells and podocytes produce different laminin isoforms at the appropriate stages of development, the cellular origins for the different type IV collagen heterotrimers that appear during development are unknown. Here, immunoelectron microscopy demonstrated that endothelial cells, mesangial cells, and podocytes of immature glomeruli synthesize collagen alpha 1 alpha 2 alpha1(IV). However, intracellular labeling revealed that podocytes, but not endothelial or mesangial cells, contain collagen alpha 3 alpha 4 alpha 5(IV). To evaluate the origins of collagen IV further, we transplanted embryonic kidneys from Col4a3-null mutants (Alport mice) into kidneys of newborn, wildtype mice. Hybrid glomeruli within grafts containing numerous host-derived, wildtype endothelial cells never expressed collagen alpha 3 alpha 4 alpha 5(IV). Finally, confocal microscopy of glomeruli from infant Alport mice that had been dually labeled with anti-collagen alpha 5(IV) and the podocyte marker anti-GLEPP1 showed immunolabeling exclusively within podocytes. Together, these results indicate that collagen alpha 3 alpha 4 alpha 5(IV) originates solely from podocytes; therefore, glomerular Alport disease is a genetic defect that manifests specifically within this cell type.

LacZ transgenic mice and immunoelectron microscopy: an ultrastructural method for dual localization of beta-galactosidase and horseradish peroxidase.

Transgenic animals bearing the reporter gene, LacZ, encoding the histochemical enzyme, beta-galactosidase, are increasingly becoming available. Similarly, antibody conjugates consisting of specific IgGs coupled to horseradish peroxidase (HRP) are widely used for Western blotting, ELISA, and immunohistochemistry. Here we provide a detailed fixation and histochemical protocol for the simultaneous electron microscopic visualization and discrimination of beta-galactosidase and peroxidase reaction products within mouse kidney. After incubation of transgenic LacZ tissues with IgG-HRP conjugates, samples were lightly fixed with 2% paraformaldehyde and 0.4% glutaraldehyde and processed for peroxidase histochemistry. Tissues underwent beta-galactosidase histochemistry, were refixed with glutaraldehyde, osmicated, and embedded. In Flk1/LacZ mice, we immunolocalized anti-laminin beta1 chain IgG-HRP specifically to developing glomerular basement membranes, whereas Flk1/LacZ was expressed only by glomerular endothelial cells. In Epas1/LacZ mice, we immunolocalized anti-platelet endothelial cell adhesion molecule-1 specifically to glomerular endothelial plasma membranes, whereas Epas1/LacZ was expressed by both glomerular endothelial and mesangial cells. This dual ultrastructural localization technique should be broadly applicable for immunoelectron microscopic studies in LacZ transgenic animals, particularly those where LacZ expression and antibody-HRP binding are both relatively abundant.

Laminin compensation in collagen alpha3(IV) knockout (Alport) glomeruli contributes to permeability defects.

Alport disease is caused by mutations in genes encoding the alpha3, alpha4, or alpha5 chains of type IV collagen, which form the collagenous network of mature glomerular basement membrane (GBM). In the absence of alpha3, alpha4, alpha5 (IV) collagen, alpha1, alpha2 (IV) collagen persists, which ordinarily is found only in GBM of developing kidney. In addition to dysregulation of collagen IV, Alport GBM contains aberrant laminins, which may contribute to the progressive GBM thickening and splitting, proteinuria, and renal failure seen in this disorder. This study sought to characterize further the laminin dysregulation in collagen alpha3(IV) knockout mice, a model of Alport disease. With the use of confocal microscopy, laminin alpha1 and alpha5 abundance was quantified, and it was found that they co-distributed in significantly large amounts in areas of GBM thickening. In addition, labeling of entire glomeruli for laminin alpha5 was significantly greater in Alport mice than in wild-type siblings. Reverse transcriptase-PCR from isolated glomeruli demonstrated significantly more laminin alpha5 mRNA in Alport mice than in wild-type controls, indicating upregulated transcription of Lama5. For testing glomerular barrier function, ferritin was injected into 2-wk-old Alport and control mice, and GBM was examined by electron microscopy. Highest ferritin levels were seen in Alport GBM thickenings beneath effaced podocyte foot processes, but morphologically normal GBM was significantly permeable as well. We concluded that (1) ultrastructurally normal Alport GBM residing beneath differentiated podocyte foot processes is inherently and abnormally permeable, and (2) upregulation of Lama5 transcription and concentration of laminin alpha1 and alpha5 within Alport GBM thickenings contribute to abnormal permeabilities.

Partial rescue of glomerular laminin alpha5 mutations by wild-type endothelia produce hybrid glomeruli.

Both endothelial cells and podocytes are sources for laminin alpha1 at the inception of glomerulogenesis and then for laminin alpha5 during glomerular maturation. Why glomerular basement membranes (GBM) undergo laminin transitions is unknown, but this may dictate glomerular morphogenesis. In mice that genetically lack laminin alpha5, laminin alpha5beta2gamma1 is not assembled, vascularized glomeruli fail to form, and animals die at midgestation with neural tube closure and placental deficits. It was previously shown that renal cortices of newborn mice contain endothelial progenitors (angioblasts) and that when embryonic day 12 kidneys are transplanted into newborn kidney, hybrid glomeruli (host-derived endothelium and donor-derived podocytes) result. Reasoning that host endothelium may correct the glomerular phenotype that is seen in laminin alpha5 mutants, alpha5 null embryonic day 12 metanephroi were grafted into wild-type newborn kidney. Hybrid glomeruli were identified in grafts by expression of a host-specific LacZ lineage marker. Labeling of glomerular hybrid GBM with chain-specific antibodies showed a markedly stratified distribution of laminins: alpha5 was found only on the inner endothelial half of GBM, whereas alpha1 located to outer layers beneath mutant podocytes. For measurement of the contribution of host endothelium to hybrid GBM, immunofluorescent signals for laminin alpha5 were quantified: Hybrid GBM contained approximately 50% the normal alpha5 complement as wild-type GBM. Electron microscopy of glomerular hybrids showed vascularization, but podocyte foot processes were absent. It was concluded that (1) endothelial and podocyte-derived laminins remain tethered to their cellular origin, (2) developing endothelial cells contribute large amounts of GBM laminins, and (3) podocyte foot process differentiation may require direct exposure to laminin alpha5.

Kidney development and gene expression in the HIF2alpha knockout mouse.

The hypoxia-inducible transcription factor-2 (HIF2), a heterodimer composed of HIF2alpha and HIF1beta subunits, drives expression of genes essential for vascularization, including vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2, Flk-1). Here, we used a HIF2alpha/LacZ transgenic mouse to define patterns of HIF2alpha transcription during kidney development and maturation. Our results from embryonic heterozygotes showed HIF2alpha/LacZ expression by apparently all renal endothelial cells. At 4 weeks of age, glomerular mesangial and vascular smooth muscle cells were also positive together with endothelial cells. These expression patterns were confirmed by electron microscopy using Bluo-gal as a beta-galactosidase substrate. Small numbers of glomerular and tubular epithelial cells were also positive at all stages examined. Light and electron microscopic examination of kidneys from HIF2alpha null embryos showed no defects in renal vascular development or nephrogenesis. Similarly, the same amounts of Flk-1 protein were seen on Western blots of kidney extracts from homozygous and heterozygous HIF2alpha mutants. To examine responsiveness of HIF2alpha null kidneys to hypoxia, embryonic day 13.5 metanephroi were cultured in room air or in mild (5% O(2)) hypoxia. For both heterozygous and null samples, VEGF mRNA levels doubled when metanephroi were cultured in mild hypoxia. Anterior chamber grafts of embryonic HIF2alpha knockouts were morphologically indistinguishable from heterozygous grafts. Endothelial markers, platelet endothelial cell adhesion molecule and BsLB4, as well as glomerular epithelial markers, GLEPP1 and WT-1, were all expressed appropriately. Finally, we undertook quantitative real-time polymerase chain reaction of kidneys from HIF2alpha null embryos and wild-type siblings and found no compensatory up-regulation of HIF1alpha or -3alpha. Our results show that, although HIF2alpha was widely transcribed by kidney endothelium and vascular smooth muscle, knockouts displayed no detectable deficits in vessel development or VEGF or Flk-1 expression.