Persistent Urogenital Sinus with Ambiguous Genitalia and Urethral Duplication in a Pubertal Female.

Urogenital sinus (UGS) and cloacal malformations are the spectra of disease affecting mainly females, resulting in an unusual confluence of the genital and urinary tract with or without the involvement of the gastrointestinal tract. Successful reconstruction of these anomalies depends on the accurate preoperative delineation of abnormal anatomy with the help of cross-sectional and other contrast studies like genitourogram along with cystourethroscopy wherever indicated. We hereby report a case of a 14-year-old female who presented with irregular cyclical hematuria and was diagnosed with persistent UGS with urethral duplication. After a thorough evaluation, the patient was successfully managed with surgical reconstruction, described in this study. Persistent UGS is a complex developmental anomaly. Complete characterization of anomaly requires a thorough evaluation such as hormonal assessment, endoscopy, cross-sectional, and radiological contrast study. Surgical reconstruction needs individualization and may need clitoroplasty, labioplasty, and urethral and vaginal mobilization. Morphological and functional outcome is satisfactory in a well-planned surgical reconstruction.

Ganglioneuroma presenting as an adrenal incidentaloma: Feasibility of adrenal-sparing surgery.

Adrenal ganglioneuromas (GNs) are very rare tumours that originate from neural crest cells. Most of the time, they are diagnosed incidentally as they are usually non-functional and remain asymptomatic. Nowadays, they are being detected more often due to better availability of imaging facilities such as computed tomography (CT)/magnetic resonance imaging (MRI). Minimally invasive adrenalectomy (laparoscopic or robotic) remains the standard of care for such lesions. Hereby, we report a case of a 15-year-old young girl with right adrenal incidentaloma which was diagnosed on CT with the features suggestive of GN. She underwent robot-assisted excision of the mass with adrenal-sparing surgery. Histopathology revealed benign GN and no adjuvant treatment was required. As GN is not known for recurrence or metastasis, minimal invasive adrenal-sparing surgery should be a preferred modality of choice.

Robot-assisted laparoscopic pyeloplasty: A retrospective case series review.

INTRODUCTION: Anderson-Hynes pyeloplasty has been gold standard in the management of pelviureteric junction obstruction (PUJO). It has evolved from open to laparoscopic and now robotic surgery. Open surgery has its drawback of long incision and scar mark, significant post-operative pain and long hospital stay. The main limitation of laparoscopic surgery had been the difficulty in endosuturing. Robotic surgery has incorporated the minimal access method of laparoscopy and endowrist movement of open surgery to overcome the challenge of intracorporeal suturing. Here, we present our initial experience of robotic pyeloplasty. PATIENTS AND METHODS: A total of 30 patients underwent robot-assisted laparoscopic pyeloplasty (RALP) over 19 months. Diagnosis of PUJO was made by computed tomography urography, diuretic renogram and retrograde pyelogram in selected patients. All patients underwent RALP by colon reflecting approach. Post-operative evaluation was done by DTPA scan at 3- and 6-month follow-up. Data were analysed after a mean follow-up of 11 months. RESULTS: The mean operative time was 148 min and the mean hospital stay was 3.5 days. While 93% of the patients showed objective improvement in their drainage pattern on DTPA renogram, 90% of the patients were symptom-free at the end of 6 months. CONCLUSIONS: Robotic pyeloplasty is a safe and easily conquerable technique with comparable outcomes in the hands of surgeons who are beginners in this technique.

Cytoprotective activated protein C averts Nlrp3 inflammasome-induced ischemia-reperfusion injury via mTORC1 inhibition.

Cytoprotection by activated protein C (aPC) after ischemia-reperfusion injury (IRI) is associated with apoptosis inhibition. However, IRI is hallmarked by inflammation, and hence, cell-death forms disjunct from immunologically silent apoptosis are, in theory, more likely to be relevant. Because pyroptosis (ie, cell death resulting from inflammasome activation) is typically observed in IRI, we speculated that aPC ameliorates IRI by inhibiting inflammasome activation. Here we analyzed the impact of aPC on inflammasome activity in myocardial and renal IRIs. aPC treatment before or after myocardial IRI reduced infarct size and Nlrp3 inflammasome activation in mice. Kinetic in vivo analyses revealed that Nlrp3 inflammasome activation preceded myocardial injury and apoptosis, corroborating a pathogenic role of the Nlrp3 inflammasome. The constitutively active Nlrp3A350V mutation abolished the protective effect of aPC, demonstrating that Nlrp3 suppression is required for aPC-mediated protection from IRI. In vitro aPC inhibited inflammasome activation in macrophages, cardiomyocytes, and cardiac fibroblasts via proteinase-activated receptor 1 (PAR-1) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Accordingly, inhibiting PAR-1 signaling, but not the anticoagulant properties of aPC, abolished the ability of aPC to restrict Nlrp3 inflammasome activity and tissue damage in myocardial IRI. Targeting biased PAR-1 signaling via parmodulin-2 restricted mTORC1 and Nlrp3 inflammasome activation and limited myocardial IRI as efficiently as aPC. The relevance of aPC-mediated Nlrp3 inflammasome suppression after IRI was corroborated in renal IRI, where the tissue protective effect of aPC was likewise dependent on Nlrp3 inflammasome suppression. These studies reveal that aPC protects from IRI by restricting mTORC1-dependent inflammasome activation and that mimicking biased aPC PAR-1 signaling using parmodulins may be a feasible therapeutic approach to combat IRI.

Homeostatic nuclear RAGE-ATM interaction is essential for efficient DNA repair.

The integrity of genome is a prerequisite for healthy life. Indeed, defects in DNA repair have been associated with several human diseases, including tissue-fibrosis, neurodegeneration and cancer. Despite decades of extensive research, the spatio-mechanical processes of double-strand break (DSB)-repair, especially the auxiliary factor(s) that can stimulate accurate and timely repair, have remained elusive. Here, we report an ATM-kinase dependent, unforeseen function of the nuclear isoform of the Receptor for Advanced Glycation End-products (nRAGE) in DSB-repair. RAGE is phosphorylated at Serine376 and Serine389 by the ATM kinase and is recruited to the site of DNA-DSBs via an early DNA damage response. nRAGE preferentially co-localized with the MRE11 nuclease subunit of the MRN complex and orchestrates its nucleolytic activity to the ATR kinase signaling. This promotes efficient RPA2S4-S8 and CHK1S345 phosphorylation and thereby prevents cellular senescence, IPF and carcinoma formation. Accordingly, loss of RAGE causatively linked to perpetual DSBs signaling, cellular senescence and fibrosis. Importantly, in a mouse model of idiopathic pulmonary fibrosis (RAGE-/-), reconstitution of RAGE efficiently restored DSB-repair and reversed pathological anomalies. Collectively, this study identifies nRAGE as a master regulator of DSB-repair, the absence of which orchestrates persistent DSB signaling to senescence, tissue-fibrosis and oncogenesis.

Maternal extracellular vesicles and platelets promote preeclampsia via inflammasome activation in trophoblasts.

Preeclampsia (PE) is a placenta-induced inflammatory disease associated with maternal and fetal morbidity and mortality. The mechanisms underlying PE remain enigmatic and delivery of the placenta is the only known remedy. PE is associated with coagulation and platelet activation and increased extracellular vesicle (EV) formation. However, thrombotic occlusion of the placental vascular bed is rarely observed and the mechanistic relevance of EV and platelet activation remains unknown. Here we show that EVs induce a thromboinflammatory response specifically in the placenta. Following EV injection, activated platelets accumulate particularly within the placental vascular bed. EVs cause adenosine triphosphate (ATP) release from platelets and inflammasome activation within trophoblast cells through purinergic signaling. Inflammasome activation in trophoblast cells triggers a PE-like phenotype, characterized by pregnancy failure, elevated blood pressure, increased plasma soluble fms-like tyrosine kinase 1, and renal dysfunction. Intriguingly, genetic inhibition of inflammasome activation specifically in the placenta, pharmacological inhibition of inflammasome or purinergic signaling, or genetic inhibition of maternal platelet activation abolishes the PE-like phenotype. Inflammasome activation in trophoblast cells of women with preeclampsia corroborates the translational relevance of these findings. These results strongly suggest that EVs cause placental sterile inflammation and PE through activation of maternal platelets and purinergic inflammasome activation in trophoblast cells, uncovering a novel thromboinflammatory mechanism at the maternal-embryonic interface.

Caspase-1, but Not Caspase-3, Promotes Diabetic Nephropathy.

Glomerular apoptosis may contribute to diabetic nephropathy (dNP), but the pathophysiologic relevance of this process remains obscure. Here, we administered two partially disjunct polycaspase inhibitors in 8-week-old diabetic (db/db) mice: M-920 (inhibiting caspase-1, -3, -4, -5, -6, -7, and -8) and CIX (inhibiting caspase-3, -6, -7, -8, and -10). Notably, despite reduction in glomerular cell death and caspase-3 activity by both inhibitors, only M-920 ameliorated dNP. Nephroprotection by M-920 was associated with reduced renal caspase-1 and inflammasome activity. Accordingly, analysis of gene expression data in the Nephromine database revealed persistently elevated glomerular expression of inflammasome markers (NLRP3, CASP1, PYCARD, IL-18, IL-1ß), but not of apoptosis markers (CASP3, CASP7, PARP1), in patients with and murine models of dNP. In vitro, increased levels of markers of inflammasome activation (Nlrp3, caspase-1 cleavage) preceded those of markers of apoptosis activation (caspase-3 and -7, PARP1 cleavage) in glucose-stressed podocytes. Finally, caspase-3 deficiency did not protect mice from dNP, whereas both homozygous and hemizygous caspase-1 deficiency did. Hence, these results suggest caspase-3-dependent cell death has a negligible effect, whereas caspase-1-dependent inflammasome activation has a crucial function in the establishment of dNP. Furthermore, small molecules targeting caspase-1 or inflammasome activation may be a feasible therapeutic approach in dNP.

Activated protein C ameliorates diabetic nephropathy by epigenetically inhibiting the redox enzyme p66Shc.

The coagulation protease activated protein C (aPC) confers cytoprotective effects in various in vitro and in vivo disease models, including diabetic nephropathy. The nephroprotective effect may be related to antioxidant effects of aPC. However, the mechanism through which aPC may convey these antioxidant effects and the functional relevance of these properties remain unknown. Here, we show that endogenous and exogenous aPC prevents glomerular accumulation of oxidative stress markers and of the redox-regulating protein p66(Shc) in experimental diabetic nephropathy. These effects were predominately observed in podocytes. In vitro, aPC inhibited glucose-induced expression of p66(Shc) mRNA and protein in podocytes (via PAR-1 and PAR-3) and various endothelial cell lines, but not in glomerular endothelial cells. Treatment with aPC reversed glucose-induced hypomethylation and hyperacetylation of the p66(Shc) promoter in podocytes. The hyperacetylating agent sodium butyrate abolished the suppressive effect of aPC on p66(Shc) expression both in vitro and in vivo. Moreover, sodium butyrate abolished the beneficial effects of aPC in experimental diabetic nephropathy. Inhibition of p66(Shc) expression and mitochondrial translocation by aPC normalized mitochondrial ROS production and the mitochondrial membrane potential in glucose-treated podocytes. Genetic ablation of p66(Shc) compensated for the loss of protein C activation in vivo, normalizing markers of diabetic nephropathy and oxidative stress. These studies identify a unique mechanism underlying the cytoprotective effect of aPC. Activated PC epigenetically controls expression of the redox-regulating protein p66(Shc), thus linking the extracellular protease aPC to mitochondrial function in diabetic nephropathy.

Activated Protein C Ameliorates Renal Ischemia-Reperfusion Injury by Restricting Y-Box Binding Protein-1 Ubiquitination.

Ischemia-reperfusion injury (IRI) is the leading cause of ARF. A pathophysiologic role of the coagulation system in renal IRI has been established, but the functional relevance of thrombomodulin (TM)-dependent activated protein C (aPC) generation and the intracellular targets of aPC remain undefined. Here, we investigated the role of TM-dependent aPC generation and therapeutic aPC application in a murine renal IRI model and in an in vitro hypoxia and reoxygenation (HR) model using proximal tubular cells. In renal IRI, endogenous aPC levels were reduced. Genetic or therapeutic reconstitution of aPC efficiently ameliorated renal IRI independently of its anticoagulant properties. In tubular cells, cytoprotective aPC signaling was mediated through protease activated receptor-1- and endothelial protein C receptor-dependent regulation of the cold-shock protein Y-box binding protein-1 (YB-1). The mature 50 kD form of YB-1 was required for the nephro- and cytoprotective effects of aPC in vivo and in vitro, respectively. Reduction of mature YB-1 and K48-linked ubiquitination of YB-1 was prevented by aPC after renal IRI or tubular HR injury. aPC preserved the interaction of YB-1 with the deubiquitinating enzyme otubain-1 and maintained expression of otubain-1, which was required to reduce K48-linked YB-1 ubiquitination and to stabilize the 50 kD form of YB-1 after renal IRI and tubular HR injury. These data link the cyto- and nephroprotective effects of aPC with the ubiquitin-proteasome system and identify YB-1 as a novel intracellular target of aPC. These insights may provide new impetus for translational efforts aiming to restrict renal IRI.

Nlrp3-inflammasome activation in non-myeloid-derived cells aggravates diabetic nephropathy.

Diabetic nephropathy is a growing health concern with characteristic sterile inflammation. As the underlying mechanisms of this inflammation remain poorly defined, specific therapies targeting sterile inflammation in diabetic nephropathy are lacking. Intriguingly, an association of diabetic nephropathy with inflammasome activation has recently been shown, but the pathophysiological relevance of this finding remains unknown. Within glomeruli, inflammasome activation was detected in endothelial cells and podocytes in diabetic humans and mice and in glucose-stressed glomerular endothelial cells and podocytes in vitro. Abolishing Nlrp3 or caspase-1 expression in bone marrow-derived cells fails to protect mice against diabetic nephropathy. Conversely, Nlrp3-deficient mice are protected against diabetic nephropathy despite transplantation of wild-type bone marrow. Pharmacological IL-1R antagonism prevented or even reversed diabetic nephropathy in mice. Mitochondrial reactive oxygen species (ROS) activate the Nlrp3 inflammasome in glucose or advanced glycation end product stressed podocytes. Inhibition of mitochondrial ROS prevents glomerular inflammasome activation and nephropathy in diabetic mice. Thus, mitochondrial ROS and Nlrp3-inflammasome activation in non-myeloid-derived cells aggravate diabetic nephropathy. Targeting the inflammasome may be a potential therapeutic approach to diabetic nephropathy.

Nuclear factor erythroid-derived 2 (Nfe2) regulates JunD DNA-binding activity via acetylation: a novel mechanism regulating trophoblast differentiation.

We recently demonstrated that the bZip transcription factor nuclear factor erythroid-derived 2 (Nfe2) represses protein acetylation and expression of the transcription factor glial cell missing 1 (Gcm1) in trophoblast cells, preventing excess syncytiotrophoblast formation and permitting normal placental vascularization and embryonic growth. However, the Gcm1 promoter lacks a Nfe2-binding site and hence the mechanisms linking Nfe2 and Gcm1 expression remained unknown. Here we show that Nfe2 represses JunD DNA-binding activity to the Gcm1 promoter during syncytiotrophoblast differentiation. Interventional studies using knockdown and knockin approaches show that enhanced JunD DNA-binding activity is required for increased expression of Gcm1 and syncytiotrophoblast formation as well as impaired placental vascularization and reduced growth of Nfe2(-/-) embryos. Induction of Gcm1 expression requires binding of JunD to the -1441 site within the Gcm1 promoter, which is distinct from the -1314 site previously shown to induce Gcm1 expression by other bZip transcription factors. Nfe2 modulates JunD binding to the Gcm1 promoter via acetylation, as reducing JunD acetylation using the histone acetyltransferase inhibitor curcumin reverses the increased JunD DNA-binding activity observed in the absence of Nfe2. This identifies a novel mechanism through which bZip transcription factors interact. Within the placenta this interaction regulates Gcm1 expression, syncytiotrophoblast formation, placental vascularization, and embryonic growth.

Metabolic evaluation of first-time uncomplicated renal stone formers: A prospective study.

Background: Nephrolithiasis is a global health problem. The recurrence rate after the first stone clearance is approximately 50% at 5 years. Metabolic abnormalities are an important factor responsible for stone recurrence. Our prevalidated study aimed to evaluate metabolic abnormalities associated with first-time uncomplicated renal stone formers (FTURSF). Materials and methods: In this prospective, exploratory, time-bound, descriptive study, 30 first-time renal stone formers were evaluated for metabolic abnormalities. High-risk stone formers were excluded from the study. Data were collected in a predefined proforma, transferred to an Excel sheet, and analyzed using the Statistical Package for Social Sciences 20 and Epi Info 7. Fisher exact test, Mann-Whitney U test, paired t test, and Pearson correlation coefficient were used for statistical analyses. Results: The mean age of the participants was 35.57 ± 11.07 years, with a male-to-female ratio of 1.72. The most common abnormality was a 24-hour urine volume of <2.5 L in 73.33% of the participants. One or more metabolic abnormalities were detected in 76.67% of the participants. Other common metabolic abnormalities detected were hypocitraturia (60%), hypercalciuria (16.67%), hyperoxaluria (13.33%), and hyperuricosuria (3.33%). Parathyroid adenoma was detected in one participant (3.33%). Conclusions: Our study documented significant metabolic abnormalities in FTURSF. Therefore, a simplified metabolic evaluation protocol should be adopted while evaluating FTURSF. Detection of an underlying metabolic abnormality would enable the early institution of preventive measures to reduce stone recurrence and related complications.

Activated protein C modulates T-cell metabolism and epigenetic FOXP3 induction via α-ketoglutarate.

A direct regulation of adaptive immunity by the coagulation protease activated protein C (aPC) has recently been established. Preincubation of T cells with aPC for 1 hour before transplantation increases FOXP3+ regulatory T cells (Tregs) and reduces acute graft-versus-host disease (aGVHD) in mice, but the underlying mechanism remains unknown. Because cellular metabolism modulates epigenetic gene regulation and plasticity in T cells, we hypothesized that aPC promotes FOXP3+ expression by altering T-cell metabolism. To this end, T-cell differentiation was assessed in vitro using mixed lymphocyte reaction or plate-bound α-CD3/CD28 stimulation, and ex vivo using T cells isolated from mice with aGVHD without and with aPC preincubation, or analyses of mice with high plasma aPC levels. In stimulated CD4+CD25- cells, aPC induces FOXP3 expression while reducing expression of T helper type 1 cell markers. Increased FOXP3 expression is associated with altered epigenetic markers (reduced 5-methylcytosine and H3K27me3) and reduced Foxp3 promoter methylation and activity. These changes are linked to metabolic quiescence, decreased glucose and glutamine uptake, decreased mitochondrial metabolism (reduced tricarboxylic acid metabolites and mitochondrial membrane potential), and decreased intracellular glutamine and α-ketoglutarate levels. In mice with high aPC plasma levels, T-cell subpopulations in the thymus are not altered, reflecting normal T-cell development, whereas FOXP3 expression in splenic T cells is reduced. Glutamine and α-ketoglutarate substitution reverse aPC-mediated FOXP3+ induction and abolish aPC-mediated suppression of allogeneic T-cell stimulation. These findings show that aPC modulates cellular metabolism in T cells, reducing glutamine and α-ketoglutarate levels, which results in altered epigenetic markers, Foxp3 promoter demethylation and induction of FOXP3 expression, thus favoring a Treg-like phenotype.

Penile Fracture with Urethral Rupture: The Feasibility of Repair through Penoscrotal Approach.

Penile fracture with an associated urethral injury is a rare urological emergency resulting from trauma to the erect penis during vigorous sexual intercourse. The patient often presents with swelling of the penis, discoloration of the penile skin, localized pain, and hematuria with a typical history of sudden detumescence during intercourse. Subcoronal penile degloving incision has been conventionally described and is frequently used by many clinicians for the management of penile fracture-urethral injury. Here, we describe a case of complex penile fracture managed through the vertical penoscrotal incision. The penoscrotal approach confers excellent exposure to both the ruptured corpus cavernosum and urethra. This approach ensures successful outcomes in such an emergency procedure without having disadvantages of the degloving incision.

When to Avoid a Restaging Procedure for Non-muscle Invasive Bladder Cancer? Inferences from a Tertiary Care Center.

The increasing incidence of urinary bladder carcinoma is alarming. Approximately seventy percent of these patients are non-muscle invasive bladder cancer (NMIBC). Restage transurethral resection of bladder tumor (TURBT) is the current recommendation for any T1 and or high-grade non muscle invasive bladder cancers (NMIBC) to accurately stage the malignancy. The question whether a second surgery is always required as a restage procedure is still unanswered. The patient's concern about completeness, morbidity, and financial considerations of a major surgery cannot be overlooked. Moreover, it also puts a strain on the already overburdened healthcare system. To answer this question, whether it is oncologically sound to omit a second resection, the current study evaluated the outcomes of patients undergoing restage TURBT, and analyzed the preoperative factors predicting a change in the staging of this malignancy. The study design was a prospective observational including NMIBC patients from September 2018 to February 2020. A total of 72 patients underwent restage TURBT. Their demographic data, imaging and cystoscopic findings, and histopathological data were recorded. The objective was to study the clinico-pathological correlations and factors predicting recurrence and upstaging of tumor in NMIBC patients undergoing restage TURBT. A total of 101 patients were found eligible for restage TURBT. Eventually, 72 underwent restage TURBT. Twelve (16.7%) patient had recurrence at restage while 3(4.16%) were upstaged to T2. Presence of lower urinary tract symptoms (LUTS) was independently associated with the risk of recurrence of same stage compared to no recurrence (p-0.025, OR-8.793, 95% CI-1.316-98.773). Chemical exposure (p-0.042) was also significantly associated with the same. Presence of lymphadenopathy on CT was independently associated with the risk of upstaging compared to no recurrence (p-0.032, OR-18.25, 95% CI-1.292-257.85). The study concluded that in the presence of a well-performed and adequate initial TURBT, restage TURBT could be skipped for further management. However, in small subgroup of patients with lymphadenopathy on preoperative imaging having a higher risk of tumor recurrence and upstaging, and patients with a history of chemical exposure and previous lower urinary tract symptoms having a high risk of recurrence alone, restage TURBT should still be performed to accurately stage the disease. Further studies with large patient cohort are needed to confirm and reinforce the facts proposed. Supplementary Information: The online version contains supplementary material available at 10.1007/s13193-022-01516-8.

A naturally occurring variant in human TLR9, P99L, is associated with loss of CpG oligonucleotide responsiveness.

The innate immune system employs Toll-like receptors (TLRs) for the detection of invading microorganisms based on distinct molecular patterns. For example, TLR9 is activated by microbial DNA and also by short therapeutic CpG-containing oligonucleotides (CpG-ODN). TLR9 activation leads to the production of interferons and the priming of humoral adaptive immune responses. Unfortunately, the principles of ligand recognition by TLR9 are poorly understood, and genetic variants of TLR9, which may affect its function, have not been characterized systematically on the molecular level. We therefore sought to functionally characterize reported single nucleotide polymorphisms of TLR9 in the HEK293 model system. We discovered that two variants, P99L and M400I, are associated with altered receptor function regarding NF-κB activation and cytokine induction. Our investigations show that for the most functionally impaired variant, P99L, the ability to respond to physiological and therapeutic TLR9 ligands is severely compromised. However, CpG-ODN binding is normal. CpG-ODN recognition by TLR9 thus appears to involve two separate events, CpG-ODN binding and sensing. Our studies highlight Pro-99 as a residue important for the latter process. In genotyping studies, we confirmed that both M400I (rs41308230) and P99L (rs5743844) are relatively rare variants of TLR9. Our data add rs41308230 and rs5743844 to the list of functionally important TLR variants and warrant further research into their relevance for infectious disease susceptibility or responsiveness to CpG-ODN-based therapies.

Papillary Renal Cell Carcinoma with Sarcomatoid Differentiation in a Native Kidney of Transplant Recipient: A Case Report and Review of Literature.

Renal cell carcinoma (RCC) developing in a transplant recipient is about 5-20 times higher than the general population. It is more common in native kidneys than graft kidney, and incidence varies between 0.3% and 4.8%. Clear cell and papillary types are more frequently reported. Most RCC of allograft recipient is usually low-grade with favorable prognosis. We present a case of papillary RCC with sarcomatoid differentiation (SD) in a native kidney of renal transplant (RT) recipient. The coexistence of sarcomatoid variant with papillary RCC, as in our case, makes it a high grade (WHO/ISUP grade 4) and portends a poor prognosis. Relative aggressiveness and rarity of this variant histology in transplant recipients prompted us to report this case and carry out an extensive search of the available literature.