Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion.

Podocyte function is tightly linked to the complex organization of its cytoskeleton and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. To identify novel genes that are important for podocyte function, we designed an in vitro genetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble Fms-like tyrosine kinase-1 (sFLT1)/Fc. A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against diphthamide biosynthesis protein 1-4 (DPH1-DPH4) were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for DPH1, DPH2, or DPH3, which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Finally, we showed that Drosophila nephrocyte-specific knockdown of Dph1, Dph2, and Dph4 resulted in altered nephrocyte function. In summary, we report here a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.

Surgical Approaches and Long-Term Outcomes in Adults with Complex Reoperative Hypospadias Repair.

PURPOSE: Patients with failed hypospadias repair are a challenging population for pediatric and reconstructive urologists. We describe our long-term outcomes and factors associated with complications of repeat hypospadias repair. MATERIALS AND METHODS: We retrospectively reviewed the records of 32 adult patients with a history of hypospadias repair who required subsequent urethroplasty between 2002 and 2012. Data on the presenting complaint, past medical and surgical history, demographic data, surgical approach, intraoperative findings and complications were collected and analyzed. RESULTS: Median patient age at urethroplasty was 32 years. Stricture of the penile urethra was the most common presentation. Urethroplasty was done in 30 patients as stricture treatment, 1 underwent perineal urethrostomy and 1 underwent diverticulectomy. Two-stage repair was performed in 90% of the men who underwent urethroplasty. The initial success rate was 83% in patients who underwent 1 or 2-stage urethroplasty. At a median followup of 9.5 years complications included 4 recurrent strictures and 1 fistula. Patient age, previous interventions, stricture length, hair present at the time of repair, the need to excise the urethral plate and the number of stages were not associated with complications or recurrence. If a graft was required, skin grafts were significantly associated with recurrence compared to buccal mucosa grafts. CONCLUSIONS: Excellent outcomes can be achieved using a 2-stage approach with replacement or augmentation of the urethral plate in adults with failed hypospadias repair. In our experience buccal mucosa appears to be associated with fewer complications and less stricture recurrence than skin grafts.

Inhibition of MTOR disrupts autophagic flux in podocytes.

Inhibitors of the mammalian target of rapamycin (MTOR) belong to a family of drugs with potent immunosuppressive, antiangiogenic, and antiproliferative properties. De novo or worsening proteinuria can occur during treatment with these agents, but the mechanism by which this occurs is unknown. We generated and characterized mice carrying a podocyte-selective knockout of the Mtor gene. Although Mtor was dispensable in developing podocytes, these mice developed proteinuria at 3 weeks and end stage renal failure by 5 weeks after birth. Podocytes from these mice exhibited an accumulation of the autophagosome marker LC3 (rat microtubule-associated protein 1 light chain 3), autophagosomes, autophagolysosomal vesicles, and damaged mitochondria. Similarly, human podocytes treated with the MTOR inhibitor rapamycin accumulated autophagosomes and autophagolysosomes. Taken together, these results suggest that disruption of the autophagic pathway may play a role in the pathogenesis of proteinuria in patients treated with MTOR inhibitors.

Clinical application of immune repertoire sequencing in solid organ transplant.

Background: Measurement of T cell receptor (TCR) or B cell receptor (BCR) gene utilization may be valuable in monitoring the dynamic changes in donor-reactive clonal populations following transplantation and enabling adjustment in therapy to avoid the consequences of excess immune suppression or to prevent rejection with contingent graft damage and to indicate the development of tolerance. Objective: We performed a review of current literature to examine research in immune repertoire sequencing in organ transplantation and to assess the feasibility of this technology for clinical application in immune monitoring. Methods: We searched MEDLINE and PubMed Central for English-language studies published between 2010 and 2021 that examined T cell/B cell repertoire dynamics upon immune activation. Manual filtering of the search results was performed based on relevancy and predefined inclusion criteria. Data were extracted based on study and methodology characteristics. Results: Our initial search yielded 1933 articles of which 37 met the inclusion criteria; 16 of these were kidney transplant studies (43%) and 21 were other or general transplantation studies (57%). The predominant method for repertoire characterization was sequencing the CDR3 region of the TCR ß chain. Repertoires of transplant recipients were found to have decreased diversity in both rejectors and non-rejectors when compared to healthy controls. Rejectors and those with opportunistic infections were more likely to have clonal expansion in T or B cell populations. Mixed lymphocyte culture followed by TCR sequencing was used in 6 studies to define an alloreactive repertoire and in specialized transplant settings to track tolerance. Conclusion: Methodological approaches to immune repertoire sequencing are becoming established and offer considerable potential as a novel clinical tool for pre- and post-transplant immune monitoring.

Renal tubular angiogenic dysregulation in anti-Thy1.1 glomerulonephritis.

Peritubular vascular changes and hypoxia after glomerular injury may explain subsequent tubulointerstitial injury and fibrosis. Several studies suggested that the expected tubulointerstitial angiogenic response is actively suppressed in this setting. The mechanism of this aberrant response has not been clearly identified. We used a common model of glomerular injury in rats to assess vascular changes and to identify potential factors associated with this aberrant response. Anti-Thy1.1 antibody administration (1 or 4 weekly doses) led to a dose-dependent renal damage characterized by elevated urea and tubulointerstitial fibrosis as assessed by Picro-Sirius Red staining. We quantified peritubular capillaries using CD31 and CD34 immunohistochemistry and showed that tubular angiogenic dysregulation was associated with peritubular capillary rarefaction. Using laser capture microdissection, we demonstrated an early induction of fibrogenic and angiogenic factors in the glomeruli and a subsequent dysregulated angiogenic response in the tubulointerstitial compartment. Proximal tubules of anti-Thy1.1-treated animals had increased pigment epithelial-derived factor (PEDF) expression by immunohistochemistry. Protein taken by laser capture microdissection also showed that PEDF was upregulated. Temporally associated with PEDF expression was a transient downregulation of tubular hypoxia-inducible factor (HIF)1α. In a human proximal tubular cell culture, we show that PEDF downregulates HIF1α protein and gene expression in cells exposed to 1% oxygen. In anti-Thy1.1 glomerulonephritis, there is aberrent tubular angiogenesis associated with glomerular injury and tubulointersititial fibrosis. We showed that PEDF may be involved by downregulating HIF1α. Further work is needed to elucidate the mechanism of PEDF upregulation and action in the tubules.

Comprehensive Immune Profiling of a Kidney Transplant Recipient With Peri-Operative SARS-CoV-2 Infection: A Case Report.

To date there is limited data on the immune profile and outcomes of solid organ transplant recipients who encounter COVID-19 infection early post-transplant. Here we present a unique case where the kidney recipient's transplant surgery coincided with a positive SARS-CoV-2 test and the patient subsequently developed symptomatic COVID-19 perioperatively. We performed comprehensive immunological monitoring of cellular, proteomic, and serological changes during the first 4 critical months post-infection. We showed that continuation of basiliximab induction and maintenance of triple immunosuppression did not significantly impair the host's ability to mount a robust immune response against symptomatic COVID-19 infection diagnosed within the first week post-transplant.

Cutaneous presentation of post-renal transplant lymphoproliferative disorder: a series of four cases.

We report detailed histological and molecular characteristics of four post transplant lymphoproliferative disorders (PTLD) presenting in the skin of renal transplant patients, and their clinical outcome. Three had B-cell lymphomas (cases 1-3), and one had a T-cell lymphoma (case 4). All B-cell lymphomas showed Epstein-Barr virus (EBV) by immunohistochemistry (IHC) or in situ hybridization (ISH). Cases 1 and 2 were large cell lymphomas, and case 3 a plasmacytoma. Case 1 showed light chain restriction and heavy chain gene rearrangement by polymerase chain reaction (PCR). The patient was then diagnosed with an abdominal lymphoma and died of sepsis. Case 2 had no recoverable DNA. Case 3 had a plasmacytoma that showed monoclonal light chain restriction on IHC and an oligoclonal heavy chain rearrangement by PCR. In cases 2 and 3, the lesions regressed following reduction of immunosuppression, and died 1.5 and 8 years later from unrelated medical causes. Case 4 was a CD 30+ anaplastic large T-cell lymphoma with no EBV detected by IHC, ISH and PCR, and died of heart failure 2 years later. Cutaneous manifestations of PTLD are rare, show wide array of clinical and pathological features, and generally have a favorable prognosis. EBV appears to be associated only with B-cell cutaneous lymphomas.

Identification of gene signature for treatment response to guide precision oncology in clear-cell renal cell carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is a common therapy resistant disease with aberrant angiogenic and immunosuppressive features. Patients with metastatic disease are treated with targeted therapies based on clinical features: low-risk patients are usually treated with anti-angiogenic drugs and intermediate/high-risk patients with immune therapy. However, there are no biomarkers available to guide treatment choice for these patients. A recently published phase II clinical trial observed a correlation between ccRCC patients' clustering and their response to targeted therapy. However, the clustering of these groups was not distinct. Here, we analyzed the gene expression profile of 469 ccRCC patients, using featured selection technique, and have developed a refined 66-gene signature for improved sub-classification of patients. Moreover, we have identified a novel comprehensive expression profile to distinguish between migratory stromal and immune cells. Furthermore, the proposed 66-gene signature was validated using a different cohort of 64 ccRCC patients. These findings are foundational for the development of reliable biomarkers that may guide treatment decision-making and improve therapy response in ccRCC patients.

Peritoneal morphological and functional changes associated with platelet-derived growth factor B.

BACKGROUND: Morphological changes associated with long-term peritoneal dialysis (PD) include increased vascular surface area due to angiogenesis, submesothelial fibrosis and epithelial mesenchymal transition. Platelet-derived growth factor (PDGF) has been associated with all of these phenomena, and is a prototypical 'response to injury' growth factor. METHODS: Rats received an intraperitoneal injection of adenoviral vector expressing PDGF-B. At sacrifice, we analysed the structure and function of the peritoneal membrane. Gene expression in the peritoneal tissue was assessed for changes suggestive of epithelial mesenchymal transition. RESULTS: Over-expression of PDGF in the rat peritoneum led to significant angiogenesis, cellular proliferation and submesothelial thickening. Although PDGF induced expression of transforming growth factor beta, there was a lack of activation of this growth factor, and we believe that this explains the lack of significant collagen accumulation observed by a hydroxyproline assay. Despite evidence of angiogenesis and subsequent increased solute transport, we observed only a transient, non-significant impact on ultrafiltration function. This suggests that increased vascular surface area is necessary, but not sufficient, to produce ultrafiltration dysfunction. There was no evidence of epithelial mesenchymal transition observed either in regulation of associated genes such as Snail or E-Cadherin or in the lack of dual-labelled epithelial and mesenchymal cells on immunofluorescence. Mesothelial cells exposed to PDGF-B demonstrated increased collagen gene expression. CONCLUSIONS: PDGF-B induced angiogenesis without fibrosis in the peritoneum. The lack of significant ultrafiltration dysfunction and epithelial mesenchymal transition, as observed in patients on PD, suggests that PDGF-B may play a role, but is not the integral component, in response to peritoneal injury.

Management of Recurrent Pelvic Fluid Collections in Adult Male Bladder Exstrophy Patients With Maintenance of Erectile Function.

OBJECTIVE: "To describe management options for pelvic fluid collections in adult patients with classic bladder exstrophy." METHODS: A single institution retrospective chart review was performed of patients who presented between 1998 and 2016 with a history of bladder exstrophy and pelvic fluid collections and 3 patients were identified. Patients had been followed for a mean of 9.0 years (1-23). RESULTS: All 3 patients required urinary diversions at various intervals following their exstrophy repair as newborns. All initially presented with symptomatic fluid collections located inferior to the bladder visualized by cross-sectional imaging. Mean age at presentation was 32.3 years (26-38 years). Two patients underwent drainage and sclerosing of cystic fluid collections with durable symptomatic relief for 1 patient. The other had recurrence of the fluid collections so he underwent marsupialization of the fluid collection which failed to sufficiently alleviate his symptoms. Ultimately, he along with the last patient, underwent open excision of the presumed hypoplastic prostate leading to resolution of pain symptoms, though the last patient did have some persistence of the fluid collection. All patients maintained their erectile function subsequent to these interventions. CONCLUSION: Adult patients with bladder exstrophy can present with painful cystic fluid collections potentially due to secretions from presumed hypoplastic prostate tissue. Sclerosing of the cyst can be successful in a subset of these patients, though some may require removal of the presumed prostatic tissue, which is curative and can be achieved with preservation of erectile function.

MTOR regulates autophagic flux in the glomerulus.

Sirolimus (rapamycin), an inhibitor of the mechanistic target of rapamycin (MTOR), was originally proposed as an immunosuppressant to prevent rejection of solid organ transplants. There were expectations that MTOR inhibitors would replace nephrotoxic calcineurin inhibitors (CNIs). Despite its potential advantages, evidence that sirolimus causes de novo or worsening proteinuria is unequivocal. Given the well-recognized proteinuric effect of MTOR inhibitors, we were interested in understanding its role in maintaining the glomerular filtration barrier. To investigate this in vivo, we developed a mouse model with a podocyte selective deletion of the Mtor gene (Mtor pod-KO).