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Background: Long-term peritoneal dialysis (PD), especially with nonphysiological solutions, is afflicted with the severe complication of encapsulating peritoneal sclerosis (EPS). Physiologic PD solutions have been introduced to reduce pH trauma. Data on peritoneal biopsies in pediatrics with long-term PD using physiological solutions are scant. Case Report: We report an adolescent who had been on 10-h continuous hourly cycles using mostly 2.27% Physioneal™ for 5 years. There were two episodes of peritonitis in October 2017 (Klebsiella oxytoca) and May 2018 (Klebsiella pneumoniae), which were treated promptly. This adolescent, who lost two kidney transplants from recurrent focal and segmental glomerulosclerosis, underwent a peritoneal membrane biopsy at the time of a third PD catheter placement, 16 months after the second renal transplant. Laparoscopically, the peritoneum appeared grossly normal, but fibrosis and abundant hemosiderin deposition were noted on histology. The thickness of the peritoneum was 200–900 (mean 680) µm; normal for age of 14 years is 297 [IQR 229, 384] μm. The peritoneum biopsy did not show specific EPS findings, as the mesothelial cells were intact, and there was a lack of fibrin exudation, neo-membrane, fibroblast proliferation, infiltration, or calcification. Conclusions: While the biopsy was reassuring with respect to the absence of EPS, significant histopathological changes suggest that avoiding pH trauma may not ameliorate the effects of glucose exposure in long-term PD.

The increasing use of kidneys from donations after cardiac death (DCD) for renal transplantation is hindered by negative outcomes owing to organ injury after prolonged warm and cold ischemia-reperfusion. Recently, hydrogen sulfide (HWe tested a novel 30-day in vivo syngeneic murine model of DCD renal transplantation, in which the donor kidney was clamped for 30 minutes and stored for 18 hours in cold University of Wisconsin (UW) solution or UW with 150 μM sodium hydrogen sulfide (UW + NaHS) before transplantation. We also tested a 7-day in vivo porcine model of DCD renal autotransplantation, in which the left kidney was clamped for 60 minutes and preserved for 24 hours using hypothermic perfusion with UW or UW + 150 μM NaHS before autotransplantation. We collected blood and urine samples periodically, and collected kidney samples at the end point for histopathology and quantitative reverse transcription polymerase chain reaction.Rats that received HOur findings support the potential use of H

Inflammation posttransplant is directly linked to cell death programs including apoptosis and necrosis. Cell death leads to the release of cellular contents which can promote inflammation. Targeting of these pathways should be an effective strategy to prevent transplant rejection. Toll-like receptor 3 (TLR3) is emerging as a major endogenous sensor of inflammation. In this study, we assessed the role of TLR3 on cell death and transplant rejection. We showed that TLR3 is highly expressed on mouse microvascular endothelial cell (ECs) and the endothelium of cardiac grafts. We demonstrated that TLR3 interacting with dsRNA or self-RNA triggered apoptosis and necroptosis in ECs. Interestingly, TLR3-induced necroptosis led mitochondrial damage. Inhibition of the mitochondrial membrane permeability molecule Cyclophilin D prevented necroptosis in ECs. In vivo, endothelium damage and activities of caspase-3 and mixed lineage kinase domain-like protein were inhibited in TLR3-/- cardiac grafts compared with C57BL/6 grafts posttransplant (n = 5, p

Background and Purpose: Cisplatin-induced nephrotoxicity manifests as acute kidney injury (AKI) in approximately one third of patients receiving cisplatin therapy. Current measures of AKI are inadequate in detecting AKI prior to significant renal injury, and better biomarkers are needed for early diagnosis of cisplatin-induced AKI. Experimental Approach: C57BL/6 and FVB/N mice were treated with a single intraperitoneal injection of cisplatin (15 mg kg-1) or saline. Plasma, urine, and kidney samples were collected prior to cisplatin injection and 24-, 48-, 72-, and 96-hours following cisplatin injection. Untargeted metabolomics was employed using liquid chromatography-mass spectrometry to identify early diagnostic biomarkers for cisplatin-induced AKI. Key Results: There was clear metabolic discrimination between saline and cisplatin-treated mice at all timepoints (day 1 to day 4). In total, 26 plasma, urine, and kidney metabolites were identified as exhibiting early alterations following cisplatin treatment. Several of the metabolites showing early alterations were associated with mitochondrial function and energetics, including intermediates of the tricarboxylic acid cycle, regulators of mitochondrial function and indicators of fatty acid β-oxidation dysfunction. Furthermore, several metabolites were derived from the gut microbiome. Conclusion and Implications: Our results highlight the detrimental effects of cisplatin on mitochondrial function and demonstrate potential involvement of the gut microbiome in the pathophysiology of cisplatin-induced AKI. Here we provide a panel of metabolites to guide future clinical studies of cisplatin-induced AKI and provide insight into potential mechanisms behind cisplatin nephrotoxicity.

Pilomatrixoma is an uncommon, benign tumor with differentiation towards both the hair matrix and cells arising in the cortex, most frequently appearing in the first or second decade of life. In rare instances, pilomatrixomas can show malignant transformation. Pilomatrix carcinoma is extremely uncommon and has traditionally been considered a tumor of low malignant potential; however, a high local recurrence rate has been reported. There is a paucity of literature on these lesions, with only a few reports describing the spectrum of malignant changes seen in these lesions. In this case report, we present a case of pilomatrixoma in an adult patient showing atypical features. While the tumor is small, there are focal features that suggest progression to malignancy, but do not fulfill the criteria for pilomatrix carcinoma. These focal atypical features include a focal infiltrative pattern at the periphery, with a variable cytological atypia and an increased mitotic rate, up to five mitotic events/high-power field. Irregular foci of central necrosis (comedonecrosis) were present in several lobules. Some of the features identified were similar to a subset of pilomatrixoma, known as “proliferating pilomatrixoma.” However, our case did not have the diffuse changes or larger size that has been frequently reported in “proliferating pilomatrixoma.” In conclusion, given the lack of focality of the changes, the lesion in our case is best described as a pilomatricoma with atypical features. Furthermore, our case may highlight the need to ensure close clinical follow-up for these lesions with unexpected atypical features that raise concern of recurrence and malignant transformation.

We have previously demonstrated benefits of kidney preservation utilizing an oxygenated subnormothermic ex vivo perfusion platform. Herein, we aim to compare pulsatile versus centrifugal (steady an...

Introduction: Cold ischemia-reperfusion injury (IRI) is an inevitable event that increases post-transplant complications. We have previously demonstrated that supplementation of University of Wisconsin (UW) solution with non-FDA-approved hydrogen sulfide (H2S) donor molecules minimizes cold IRI and improves renal graft function after transplantation. The present study investigates whether an FDA-approved H2S donor molecule, sodium thiosulfate (STS), will have the same or superior effect in a clinically relevant rat model of syngeneic orthotopic kidney transplantation. Method: Thirty Lewis rats underwent bilateral nephrectomy followed by syngeneic orthotopic transplantation of the left kidney after 24-hour preservation in either UW or UW+STS solution at 4 °C. Rats were monitored to post-transplant day 14 and sacrificed to assess renal function (urine output, serum creatinine and blood urea nitrogen). Kidney sections were stained with H&E, TUNEL, CD68, and myeloperoxidase (MPO) to detect acute tubular necrosis (ATN), apoptosis, macrophage infiltration, and neutrophil infiltration. Result: UW+STS grafts showed significantly improved graft function immediately after transplantation, with improved recipient survival compared to UW grafts (p

Introduction The current methods of preserving donor kidneys in nonoxygenated cold conditions minimally protect the kidney against ischemia-reperfusion injury (IRI), a major source of complications in clinical transplantation. However, preserving kidneys with oxygenated perfusion is not currently feasible due to the lack of an ideal perfusion mechanism that facilitates perfusion with blood at warm temperature. Here, we have designed an innovative renal pump circuit system that can perfuse blood or acellular oxygen carrier under flexible temperatures, pressures, and oxygenation. We have tested this apparatus to study optimal conditions of storage of our porcine model of donation after cardiac death (DCD) kidneys. Methods Porcine kidneys were retrieved after 30 minutes of cross-clamping renal pedicles in situ. Cessation of blood mimics postcardiac death in humans and simulates DCD warm ischemic injury. Procured kidneys were flushed and subjected to static cold storage (SCS) for 4 hours. For warm perfusion, kidneys were cannulated for pulsatile oxygenated perfusion with blood:PlasmaLyte for 4 hours at 15 °C, 22 °C, and 37 °C. To mimic posttransplant scenario, all kidneys were reperfused with blood for an additional 4 hours at 37 °C. Results Compared with all other groups, 22 °C perfusion resulted in significant reduction of acute tubular necrosis (ATN), apoptosis, kidney damage markers, Toll-like receptor signaling, and cytokine production. It was associated with maximal renal blood flow and urine output. Kidneys stored at 15 °C thrombosed within 2 hours under this condition. Martius Scarlet Blue staining confirmed that 22 °C was the optimal temperature to minimize hemorrhage and blood clots. Conclusion Our novel study shows that oxygenated perfusion at near-room-temperature provides optimal donor kidney storage conditions., Graphical abstract

Transplantation is invariably associated with programmed cell death including apoptosis and necrosis, resulting in delayed graft function and organ rejection. We have demonstrated the contribution of necroptosis to mouse microvascular endothelial cell (MVEC) death and transplant rejection. Organ injury results in the opening of mitochondrial permeability transition pores (mPTPs), which can trigger apoptotic molecules release that ultimately results in cell death. The effect of mPTPs in the necroptotic pathway remains controversial; importantly, their role in transplant rejection is not clear. In this study, tumor necrosis factor-α triggered MVECs to undergo receptor-interacting protein kinase family (RIPK1/3)-dependent necroptosis. Interestingly, inhibition of mPTP opening could also inhibit necroptotic cell death. Cyclophilin-D (Cyp-D) is a key regulator of the mPTPs. Both inhibition and deficiency of Cyp-D protected MVECs from necroptosis (n = 3, P < .00001). Additionally, inhibition of Cyp-D attenuated RIPK3-downstream mixed-lineage kinase domain-like protein phosphorylation. In vivo, Cyp-D-deficient cardiac grafts showed prolonged survival in allogeneic BALB/c mice posttransplant compared with wild-type grafts (n = 7, P < .0001). Our study results suggest that the mPTPs may be important mechanistic mediators of necroptosis in cardiac grafts. There is therapeutic potential in targeting cell death via inhibition of the mPTP-regulating molecule Cyp-D to prevent cardiac graft rejection.

Graft versus host disease is a rare but deadly complication of solid organ transplant. Clinical features of graft-versus-host-disease are non-specific, which may lead to delayed diagnosis as more common conditions including infections or drug reactions are considered. We describe a 54-year-old male patient who underwent liver transplantation for alcohol use disorder-related cirrhosis and developed acute graft-versus-host disease. Initial clinical presentation included dermatitis, bone marrow failure and enteritis. Results of skin biopsy and cytogenetic studies were consistent with liver transplant-associated acute graft-versus-host disease. The importance of this case is to highlight to transplant physicians and surgeons the challenges of diagnosing graft-versus-host-disease. In our case, pre-existing partnerships among the liver and hematopoietic stem cell transplant teams, transfusion medicine specialists, critical care specialists and facilitated timely communication relevant to confirming graft-versus-host disease. We propose an algorithm to assist in the workup of suspected graft-versus-host disease. Because this condition is characterized by high mortality, a high index of suspicion is imperative for prompt diagnosis and optimal management of the donor-recipient immune interaction when patients present with classic clinical features.

Reperfusion injury following cold and warm ischemia (IRI) is unavoidable during kidney transplantation and contributes to delayed graft function (DGF) and premature graft loss. Death of tubular epithelial cells (TECs) by necrosis during IRI releases pro-inflammatory mediators (e.g. HMGB1), propagating further inflammation (necroinflammation) and tissue damage. Kidney Injury Molecule-1 (KIM-1) is a phagocytic receptor upregulated on proximal TECs during acute kidney injury. We have previously shown that renal KIM-1 protects the graft against transplant associated IRI by enabling TECs to clear apoptotic and necrotic cells, and that recognition of necrotic cells by KIM-1 is augmented in the presence of the opsonin, apoptosis inhibitor of macrophages (AIM). Here, we tested whether recombinant AIM (rAIM) could be used to mitigate transplant associated IRI. We administered rAIM or vehicle control to nephrectomised B6 mice transplanted with a single B6 donor kidney. Compared to grafts in vehicle-treated recipients, grafts from rAIM-treated mice exhibited significantly less renal dysfunction, tubular cell death, tissue damage, tubular obstruction, as well as local and systemic inflammation. Both mouse and human rAIM enhanced the clearance of necrotic cells by murine and human TECs, respectively in vitro. These data support testing of rAIM as a potential therapeutic agent to reduce DGF following kidney transplantation.

Ischemia-reperfusion injury (IRI) is an inevitable consequence of organ transplant procedure and associated with acute and chronic organ rejection in transplantation. IRI leads to various forms of programmed cell death, which worsens tissue damage and accelerates transplant rejection. We recently demonstrated that necroptosis participates in murine cardiac microvascular endothelial cell (MVEC) death and murine cardiac transplant rejection. However, MVEC death under a more complex IRI model has not been studied. In this study, we found that simulating IRI conditions in vitro by hypoxia, reoxygenation and treatment with inflammatory cytokines induced necroptosis in MVECs. Interestingly, the apoptosis-inducing factor (AIF) translocated to the nucleus during MVEC necroptosis, which is regulated by the mitochondrial permeability molecule cyclophilin D (CypD). Furthermore, CypD deficiency in donor cardiac grafts inhibited AIF translocation and mitigated graft IRI and rejection (n = 7, p = 0.002). Our studies indicate that CypD and AIF play significant roles in MVEC necroptosis and cardiac transplant rejection following IRI. Targeting CypD and its downstream AIF may be a plausible approach to inhibit IRI-caused cardiac damage and improve transplant survival.

Introduction: We sought to design a partial nephrectomy (PN) with contralateral total nephrectomy porcine model and assess the underlying mechanisms of ischemia reperfusion injury (IRI) after PN using a novel, clinically approved resection device.Methods: Domestic male pigs (n=9) underwent left lower pole PN, allocated to either standard (Group 1) or no ischemia PN (Group 2), followed by contralateral nephrectomy. Biochemical studies were performed at baseline, Day 2, and Day 7; after sacrifice, kidneys were processed for histological analysis. Apoptotic markers were measured by Western blot analyses. Urinary biomarkers were measured to assess acute kidney injury.Results: At Day 2 following PN, there was a significant rise in serum creatinine in Group 1 compared to Group 2 (355 vs. 136 mmol/L; p=0.008). Intra-renal tissue oxygen saturation after PN was inversely correlated with postoperative creatinine (rs -0.75; p=0.012) and the grade of acute tubular necrosis (rs -0.70; p=0.036). We observed a rise in expression of pro-apoptotic markers and pro-inflammatory markers in Group 1 following PN compared to Group 2. Histological analysis revealed higher grade of apoptosis in Group 1.Conclusions: IRI associated with standard PN has a deleterious impact on acute renal function, markers of tissue injury, and histological parameters, compared to off-clamp PN using the ALTRUS device. We identified several intraoperative and postoperative markers that may be used as predictors for functional and histological injury following PN.

Background Despite advances in immunosuppressive therapies, the rate of chronic transplant loss remains substantial. Organ injury involves various forms of cell death including apoptosis and necrosis. We now recognize that early injury of cardiac transplants involves a newly described form of programmed necrotic cell death, termed necroptosis. Because this involves receptor-interacting protein (RIP) kinase 1/3, this study aimed to establish the role of RIP3 in chronic cardiac allograft rejection. Methods We used major histocompatibility complex class II mismatched C57BL/6N (H-2; B6) or B6.RIP3 (H-2; RIP3) mice to B6.C-H-2 (H2-Ab1; bm12) mouse cardiac transplantation. Microvascular endothelial cells (MVEC) were developed from B6 and RIP3 cardiac grafts. Result CD4 T cell-mediated cardiac graft rejection is inhibited using RIP3 deficient donor grafts, with reduced cellular infiltration and vasculopathy compared with wild type cardiac grafts. Alloreactive CD4 T cell-mediated MVEC death involves TNFα, Fas ligand (FasL) and granzyme B. Although necroptosis and release of danger molecule high-mobility group box 1 are eliminated by the absence of RIP3, CD4 T cells had attenuated MVEC death through granzyme B and FasL. Conclusions CD4 T cell-mediated MVEC death involves in TNFα, FasL and granzyme B. Necroptotic cell death and release of the danger molecule may promote inflammatory responses and transplant rejection. Although loss of RIP3 does not eliminate alloimmune responses, chronic graft injury is reduced. RIP3 is an important therapeutic target but additional granzyme and caspases inhibition is required for sufficiently improving long-term graft survival.

Summary: Thymine DNA glycosylase (TDG) is a nuclear receptor coactivator that plays an essential role in the maintenance of epigenetic stability in cells. Here, we demonstrate that the conditional deletion of TDG in adult mice results in a male-predominant onset of hepatocellular carcinoma (HCC). TDG loss leads to a prediabetic state, as well as bile acid (BA) accumulation in the liver and serum of male mice. Consistent with these data, TDG deletion led to dysregulation of the farnesoid X receptor (FXR) and small heterodimer partner (SHP) regulatory cascade in the liver. FXR and SHP are tumor suppressors of HCC and play an essential role in BA and glucose homeostasis. These results indicate that TDG functions as a tumor suppressor of HCC by regulating a transcriptional program that protects against the development of glucose intolerance and BA accumulation in the liver. : TDG is a base excision repair protein that is essential for embryonic development. Hassan et al. show that the conditional deletion of TDG in adult mice causes dysregulation of FXR signaling and a loss of glucose and bile acid homeostasis. This leads to a late-onset development of hepatocellular carcinoma. Keywords: thymine DNA glycosylase, active DNA demethylation, TET, hepatocellular carcinoma, FXR, bile acids, insulin resistance, conditional deletion, hepatoblastoma

Background Ischemia-reperfusion (I/R) injury is the major cause of delayed renal graft function in kidney transplantation. To date, there are no effective therapeutic approaches for preventing I/R injury. We previously reported that treatment of animals with small interference RNA (siRNA) would prevent warm I/R injury in nontransplant models and cold I/R injury in heart transplantation. In the present study, we further explore the feasibility of protecting grafts from extended cold I/R injury as applied to kidney transplantation by downregulating I/R-associated genes using siRNA. Methods Donor kidneys were intra-arterially perfused with siRNA containing solution during donor excision and preserved in siRNA containing solution. The siRNA-treated donor organs were then implanted into syngeneic recipient mice, and the 2 original kidneys were removed from the recipient. The effect of siRNA solution on extended cold I/R injury was determined by assessing renal function, histopathological change, cell apoptosis, and inflammation. Results The perfused siRNA solution knocked down the expression of complement 3, RelB, and Fas in the kidney at the mRNA and protein levels. Administration of siRNA solution reduced the levels of blood urea nitrogen and serum creatinine as compared with control groups. The siRNA cocktail decreased cell apoptosis and histopathological changes in the kidney and prolonged graft survival. The siRNA cocktail also reduced the expression of proinflammatory cytokines, IL-6, and TNFα. Conclusions In conclusion, this is the first demonstration that perfusing donor organs with an siRNA cocktail solution can induce gene silencing in the kidney and prevent kidneys from extended cold I/R injury in kidney transplantation, highlighting the promise of the clinical application of siRNA-based therapies in the preservation of donor organs.

Introduction: Epstein-Barr Virus (EBV)-associated systemic T-cell lymphoproliferative disorder of childhood is a rare but severe manifestation of chronic EBV infection. Despite several case reports characterizing this rare hematological neoplasm, the literature describes extensive heterogeneity in the presentation of this disease. Case presentation: Here we present a complete autopsy of a 16-year-old girl who ultimately succumbed to EBV-associated systemic T-cell lymphoproliferative disorder of childhood. Her clinical presentation demonstrated a non-specific pharyngitis with positive mono spot test, evolving into fulminant multi-organ failure, disseminated intravascular coagulopathy, sepsis, and ultimately death. Conclusions: Post-mortem findings included extensive hemorrhage, and infiltration of the liver, spleen, lymph nodes and bone marrow with neoplastic T-cells. There was extensive hemophagocytic lymphohistiocytosis (HLH) within these organs, suggesting overlap between the EBV-associated systemic T-cell lymphoproliferative disorder of childhood and EBV-associated HLH. We hope these findings provide a more comprehensive overview of several possible manifestations of EBV-associated systemic T-cell lymphoproliferative disorder of childhood.

Background Ischaemia-reperfusion injury (IRI) is associated with programmed cell death that promotes inflammation and organ dysfunction. Necroptosis is mediated by members of receptor interacting protein kinases (RIPK1/3). Inhibition of RIPK1/3 provides a pro-survival benefit in kidney IRI. Caspase-8 initiates apoptosis and contributes to IRI. We studied whether inhibiting both RIPK3 and caspase-8 would provide an additional benefit in kidney IRI. Methods A clamp was applied to the left kidney pedicle for 45 min followed by right kidney nephrectomy. Kidney and serum from wild type, RIPK3-/- , and RIPK3-/- caspase-8-/- double knockout (DKO) mice were collected post-IRI for assessment of injury. Tubular epithelial cells (TEC) isolated from wild type, RIPK3-/- , and DKO mice were treated with interferons-γ and interleukin-1β to induce apoptotic death. Results Kidney IRI of DKO mice did not show improvement over RIPK3-/- mice. We have found that DKO triggered 'intrinsic' apoptosis in TEC in response to interleukin-1β and interferons-γ. Up-regulation of the B-cell lymphoma 2 (Bcl-2)-associated death promoter, the Bcl-2-homologous antagonist killer and Bcl-2-associated X protein and enhanced activation of caspase-3 and 9 were found in DKO TEC. TEC infected with Murine cytomegalovirus that encodes multiple cell death inhibitors resist to death. Conclusion We show that the deletion of both RIPK3 and caspase-8 does not provide additive benefit in IRI or TEC death and may enhance injury by up-regulation of intrinsic apoptosis. This suggests blocking multiple death pathways may be required for the prevention of kidney IRI clinically.

Background Carbon monoxide (CO) inhalation protects organ by reducing inflammation and cell death during transplantation processes in animal model. However, using CO in clinical transplantation is difficult due to its delivery in a controlled manner. A manganese-containing CO releasing molecules (CORM)-401 has recently been synthesized which can efficiently deliver 3 molar equivalents of CO. We report the ability of this anti-inflammatory CORM-401 to reduce ischemia reperfusion injury associated with prolonged cold storage of renal allografts obtained from donation after circulatory death in a porcine model of transplantation. Methods To stimulate donation after circulatory death condition, kidneys from large male Landrace pig were retrieved after 1 hour warm ischemia in situ by cross-clamping the renal pedicle. Procured kidneys, after a brief flushing with histidine-tryptophan-ketoglutarate solution were subjected to pulsatile perfusion at 4°C with University of Wisconsin solution for 4 hours and both kidneys were treated with either 200 μM CORM-401 or inactive CORM-401, respectively. Kidneys were then reperfused with normothermic isogeneic porcine blood through oxygenated pulsatile perfusion for 10 hours. Urine was collected, vascular flow was assessed during reperfusion and histopathology was assessed after 10 hours of reperfusion. Results We have found that CORM-401 administration reduced urinary protein excretion, attenuated kidney damage markers (kidney damage marker-1 and neutrophil gelatinase-associated lipocalin), and reduced ATN and dUTP nick end labeling staining in histopathologic sections. CORM-401 also prevented intrarenal hemorrhage and vascular clotting during reperfusion. Mechanistically, CORM-401 appeared to exert anti-inflammatory actions by suppressing Toll-like receptors 2, 4, and 6. Conclusions Carbon monoxide releasing molecules-401 provides renal protection after cold storage of kidneys and provides a novel clinically relevant ex vivo organ preservation strategy.

Objectives: To assess the effects of slow-releasing H2S donor GYY4137 on post-obstructive renal function and injury following unilateral ureteral obstruction (UUO) by using the UUO and reimplantation (UUO-R) model in rats and to elucidate potential mechanisms by using an in vitro model of epithelial-mesenchymal transition (EMT). Methods: Male Lewis rats underwent UUO at the left ureterovesical junction. From post-operative day (POD) 1-13, rats received daily intraperitoneal (IP) injection of phosphate buffered saline (PBS, 1 mL) or GYY4137 (200 mu mol/kg/day in 1 mL PBS, IP). On POD 14, the ureter was reimplanted back into the bladder, followed by a right nephrectomy. Urine and serum samples were collected to monitor renal function. On POD 30, the left kidney was removed and tissue sections were stained with H&E, TUNEL, CD68, CD206, myeloperoxidase, and Masson's trichrome to determine cortical thickness, apoptosis, inflammation, and fibrosis. In our in vitro model of EMT, NRK52E cells were treated with 10 ng/mL TGF-beta 1, 10 mu M GYY4137 and/or 50 mu M GYY4137. Western blot analysis was performed to determine the expression of E-cadherin, vimentin, Smad7 and TGF-beta 1 receptor II (T beta RII). Results: GYY4137 led to a moderate decrease in post-obstructive serum creatinine, cystatin C and FENa. We also observed a trend towards a decrease in post-obstructive proteinuria following GYY4137 treatment. Histologically, we observed a significant decrease in apoptosis, inflammation, and fibrosis. Furthermore, our in vitro studies demonstrate that in the presence of TGF-beta 1, GYY4137 significantly decreases vimentin and T beta RII and significantly increases E-cadherin and Smad7. Conclusions: H2S may help to accelerate the recovery of renal function post-obstruction and attenuates renal injury associated with UUO. It is possible that H2S mitigates fibrosis by regulating the TGF-beta 1-mediated EMT pathway. Taken together, our data suggest that H2S may be a potential novel therapy for improving renal function and limiting renal injury associated with obstructive uropathy.

Ischemia-reperfusion injury during kidney transplantation predisposes to delayed graft function, rejection, and premature graft failure. Exacerbation of tissue damage and alloimmune responses may be explained by necroinflammation: an autoamplification loop of cell death and inflammation, which is mediated by the release of damage-associated molecular patterns (eg, high-mobility group box-1; HMGB1) from necrotic cells that activate both innate and adaptive immune pathways. Kidney injury molecule-1 (KIM-1) is a phosphatidylserine receptor that is upregulated on injured proximal tubular epithelial cells and enables them to clear apoptotic and necrotic cells. Here we show a pivotal role for clearance of dying cells in regulating necroinflammation in a syngeneic murine kidney transplant model. We found persistent KIM-1 expression in KIM-1+/+ kidney grafts posttransplantation. Compared to recipients of KIM-1+/+ kidneys, recipients of KIM-1-/- kidneys exhibited significantly more renal dysfunction, apoptosis and necrosis, tubular obstruction, and graft failure. KIM-1-/- grafts also had more inflammatory cytokines, infiltrating neutrophils, and macrophages compared to KIM-1+/+ grafts. Most significantly, passive release of HMGB1 from apoptotic and necrotic cells led to dramatically higher serum HMGB1 levels and increased proinflammatory macrophages in recipients of KIM-1-/- grafts. Our data identify an endogenous protective mechanism against necroinflammation in kidney grafts that may be of therapeutic relevance in transplantation.

Ischemic acute kidney injury is a serious untreatable condition. Activation of the G protein α12 (Gα12) subunit by reactive oxygen species is a major cause of tissue damage during renal ischemia-reperfusion injury. Kidney injury molecule-1 (KIM-1) is a transmembrane glycoprotein that is highly up-regulated during acute kidney injury, but the physiologic significance of this up-regulation is unclear. Here, we report for the first time that Kim-1 inhibits Gα12 activation and protects mice against renal ischemia-reperfusion injury. We reveal that Kim-1 physically interacts with and inhibits cellular Gα12 activation after inflammatory stimuli, including reactive oxygen species, by blocking GTP binding to Gα12. Compared with Kim-1 +/+ mice, Kim-1 −/− mice exhibited greater Gα12 and downstream Src activation both in primary tubular epithelial cells after in vitro stimulation with H 2 O 2 and in whole kidneys after unilateral renal artery clamping. Finally, we show that Kim-1–deficient mice had more severe kidney dysfunction and tissue damage after bilateral renal artery clamping, compared with wild-type mice. Our results suggest that KIM-1 is an endogenous protective mechanism against renal ischemia-reperfusion injury through inhibition of Gα12.

Cytokines and chemokines produced by tubular epithelial and infiltrating cells are critical to inflammation in renal ischemia-reperfusion injury. IL-37, a newly described IL-1 family member, inhibits IL-18-dependent pro-inflammatory cytokine production by its binding to IL-18 receptors and IL-18 binding protein. The potential role of IL-37 in renal ischemia-reperfusion injury is unknown. Here we found that exposure of tubular epithelial cells to exogenous IL-37 downregulated hypoxia and the IL-18-induced expression of TNFα, IL-6, and IL-1β. Importantly, human PT-2 tubular epithelial cells have inducible expression of IL-37. Moreover, pro-inflammatory cytokine expression was augmented in IL-37 mRNA-silenced tubular epithelial cells and inhibited by transfection with pCMV6-XL5-IL-37. In a mouse ischemic injury model, transgenic expression of human IL-37 inhibited kidney expression of TNFα, IL-6, and IL-1β and improved mononuclear cell infiltration, kidney injury, and function. Thus, human tubular epithelial cells express the IL-18 contra-regulatory protein IL-37 as an endogenous control mechanism to reduce inflammation. Augmenting kidney IL-37 may represent a novel strategy to suppress renal injury responses and promote kidney function after renal ischemic injury and transplantation.

Background and Aims EUS is a potentially useful modality to assess severity of inflammation in ulcerative colitis (UC). We assessed the reliability of existing EUS indices and correlated them with endoscopic and histologic scores. Methods Four blinded endosonographers assessed 58 endoscopic and EUS videos in triplicate, from patients with UC. Intrarater and interrater reliability of the hyperemia and Tsuga scores were estimated by using intra-class correlation coefficients (ICCs). Correlation with the Mayo endoscopy score, modified Baron score (MBS), Ulcerative Colitis Endoscopic Index of Severity (UCEIS), and Geboes histopathology score (GHS) were calculated by using bootstrapping methods. A RAND consensus process led to development of standardized definitions and a revised EUS-UC score. Results ICCs for intrarater reliability were 0.76 (95% confidence interval [CI], 0.71-0.80) for the hyperemia score and 0.85 (95% CI, 0.79-0.89) for the Tsuga score. Corresponding values for interrater reliability were 0.34 (95% CI, 0.25-0.42) and 0.36 (95% CI, 0.24-0.46). Correlation between hyperemia and Tsuga scores to Mayo scoring system, MBS, UCEIS, and the GHS were 0.39 (95% CI, 0.15-0.61) and 0.28 (95% CI, 0.04-0.51), 0.38 (95% CI, 0.16-0.57) and 0.25 (95% CI, -0.01-0.48), 0.41 (95% CI, 0.16-0.62) and 0.27 (95% CI, 0.01-0.50), 0.37 (95% CI, -0.01-0.48) and 0.24 (95% CI, 0.13-0.57), respectively. The revised EUS-UC score included bowel wall thickening, depth of inflammation, and hyperemia. Conclusions Although substantial to almost perfect intrarater agreement existed for EUS indices in UC, interrater agreement was fair. Standardization of item definitions with development of a revised evaluative instrument has potential application as an evaluative and prognostic tool for UC. (Clinical trial registration number: NCT01852760.)

BACKGROUND Protease inhibitor 9 (PI-9) is an intracellular serpin that specifically inhibits granzyme B, a cytotoxic serine protease found in the cytosolic granules of cytotoxic T lymphocytes and natural killer cells. Enhanced cortical expression of PI-9 has been observed in kidney allografts with subclinical rejection, suggesting that the tubular epithelial cell (TEC) expression of this protein may have a protective role and attenuate overt allograft rejection. METHODS AND RESULTS We demonstrate that TEC express SPI-6 protein, the murine homolog of PI-9, basally with a modest increase after cytokine exposure. Tubular epithelial cell expression of SPI-6 blocks granzyme B-mediated death because TEC from SPI-6 null kidneys have increased susceptibility to cytotoxic CD8+ cells in vitro. The role of SPI-6 was tested in a mouse kidney transplant model using SPI-6 null or wild type donor kidneys (H-2) into nephrectomized recipients (H-2). SPI-6 null kidney recipients demonstrated reduced renal function at day 8 after transplantation compared to controls (creatinine, 113±23 vs. 28±3 μmol/L; n=5; P

Cell death results in tissue damage and ultimately donor graft rejection and can occur as an active molecular process through apoptotic, necrotic and newly identified receptor interacting protein 1 and 3 kinase (RIPK1/3)-mediated necroptotic pathways. Necroptosis leads to the release of inflammatory molecules which can activate host immune cells. This pathway has yet to be studied in heart transplantation. We have found that necroptosis was induced in murine cardiac microvascular endothelial cell (MVEC) under anti-apoptotic condition following tumor necrosis factor alpha treatment. Necroptotic cell death and release of the danger molecule high mobility group box 1 (HMGB1) were inhibited by the RIPK1 inhibiting molecule necrostatin-1 and by genetic deletion of RIPK3. In addition, tissue necrosis, release of HMGB1 and graft cell infiltrate were attenuated in RIPK3 null heart allografts following transplantation. Finally, a brief sirolimus treatment markedly prolonged RIPK3 null cardiac allograft survival in allogeneic BALB/c recipients as compared to WT C57BL/6 donor grafts (95 ± 5.8 vs. 24 ± 2.6 days, p

Background: Renal ischemia reperfusion injury (IRI) leads to acute kidney injury (AKI) and the death of tubular epithelial cells (TEC). The release of high-mobility group box-1 (HMGB1) and other damage-associated molecular pattern moieties from dying cells may promote organ dysfunction and inflammation by effects on TEC. Glycyrrhizic acid (GZA) is a functional inhibitor of HMGB1, but its ability to attenuate the HMGB1-mediated injury of TEC has not been tested. Methods/Results: In vitro, hypoxia and cytokine treatment killed TEC and resulted in the progressive release of HMGB1 into the supernatant. GZA reduced the hypoxia-induced TEC death as measured by annexin-V and propidium iodide. Hypoxia increased the expression of MCP-1 and CXCL1 in TEC, which was reduced by GZA in a dose-dependent manner. Similarly, the HMGB1 activation of effector NK cells was inhibited by GZA. To test the effect of HMGB1 neutralization by GZA in vivo, mice were subjected to renal IRI. HMGB1 protein expression increased progressively in kidneys from 4 to 24 h after ischemia and was detected in tubular cells by 4 h using immunohistochemistry. GZA preserved renal function after IRI and reduced tubular necrosis and neutrophil infiltration by histological analyses and ethidium homodimer staining. Conclusions: Importantly, these data demonstrate for the first time that AKI following hypoxia and renal IRI may be promoted by HMGB1 release, which can reduce the survival of TEC and augment inflammation. Inhibition of the interaction of HMGB1 with TEC through GZA may represent a therapeutic strategy for the attenuation of renal injury following IRI and transplantation.

Ischemia-reperfusion injury is unavoidably caused by loss and subsequent restoration of blood flow during organ procurement, and prolonged ischemia-reperfusion injury IRI results in increased rates of delayed graft function and early graft loss. The endogenously produced gasotransmitter, hydrogen sulfide (H2 S), is a novel molecule that mitigates hypoxic tissue injury. The current study investigates the protective mitochondrial effects of H2 S during in vivo cold storage and subsequent renal transplantation (RTx) and in vitro cold hypoxic renal injury. Donor allografts from Brown Norway rats treated with University of Wisconsin (UW) solution + H2 S (150 μM NaSH) during prolonged (24-h) cold (4°C) storage exhibited significantly (p 1000-fold compared to similar levels of the nonspecific H2 S donor, GYY4137 and also improved syngraft function and survival following prolonged cold storage compared to UW solution. H2 S treatment mitigates cold IRI-associated renal injury via mitochondrial actions and could represent a novel therapeutic strategy to minimize the detrimental clinical outcomes of prolonged cold IRI during RTx.

Chronic obstructive uropathy can cause irreversible kidney injury, atrophy and inflammation, which can ultimately lead to fibrosis. Epithelial-mesenchymal transition is a key trigger of fibrosis that is caused by up-regulation of TGF-β1 (transforming growth factor-β1) and ANGII (angiotensin II). HFollowing unilateral ureteral obstruction male Lewis rats were given daily intraperitoneal administration of phosphate buffered saline vehicle (obstruction group) or phosphate buffered saline plus 200 μmol/kg GYY4137 (obstruction plus GYY4137 group) for 30 days. Urine and serum samples were collected to determine physiological parameters of renal function and injury. Kidneys were removed on postoperative day 30 to evaluate histopathology and protein expression. Epithelial-mesenchymal transition in LLC-PK1 pig kidney epithelial cells was induced with TGF-β1 and treated with GYY4137 to evaluate potential mechanisms via in vitro scratch wound assays.HTo our knowledge our findings demonstrate for the first time the protective effects of H

FT-IR to determine the biochemical structure and the matrix material content. The matrix material component was calculated as percentage to total biochemical components. The stone microanalysis for heavy metals and trace elements contents (22 elements/stone) was carried out by using ICP-OES. The correlation between matrix material content, biochemical structure and heavy metals was evaluated. RESULTS: FT-IR analyses revealed; 268 (48.6%) Ca oxalate, 166 (30.15) uric acid, 68 (12.3%) mixed and 19 (3.4) Ca phosphate, 13 (2.4%) struvite and 12 (2.2%) cystine stones. ICP-OES showed significant increase in the concentrationof 15 elements (Al, B, Ba,Ca,Cd,Cr, K, Mg, Na, P, Pb, S, Se, Sr and Zn) within the examined urinary stones. Phosphate-based stones (Ca phosphate and Struvite) had the highest concentration of Al, B, Ba, Cr, K, Mg, Na, P, Pb, Sr and Zinc. Calciumbased (Oxalate) stones had high levels of Cd,Fe, Mo and Se. Uric acid stones had high concentration of S. The mean percentage of matrix material was 6.4%. 195 (35.3%) stones had zero matrix content. 190 (34.4%) had matrix material < 6.4%. Zero matrix stones had high concentration of heavymetals (Al, As,Ba,Ca,Cd,Cr, Cu, Fe,Mn,Mo,Naand P). High matrix-containing stones had only higher Se than low matrix. CONCLUSIONS: The human urinary stones contained a significant concentration of heavy and trace elements. Phosphate-based stones had highest concentration of HMTE. Uric acid stones had the least contents of HMTE. Matrix-rich stones had less contents of HMTE. Poor Matrix-containing stones had highest contents of HMTE. These results indicate that matrix material could play an equally important role in stone formation beside other micro-components specially HMTE.

Ischemia-reperfusion injury is unavoidable during organ transplantation. Prolonged ischemia-reperfusion injury is detrimental to short-term and long-term graft function and survival. H2S is a recently characterized, endogenously produced gaseous molecule with important physiological roles that has been shown to be cytoprotective during tissue ischemia-reperfusion injury. The current study aimed to determine whether H2S could mitigate cold renal ischemia-reperfusion injury in the clinically relevant context of allogeneic renal transplantation.Following bilateral native nephrectomy Lewis rats underwent renal transplantation with kidneys from Brown Norway donor rats that were flushed with cold (4C) standard University of Wisconsin preservation solution (University of Wisconsin preservation solution group) or cold University of Wisconsin preservation solution plus 150 μM NaHS (H2S group) solution. Kidneys were stored for 6 hours at 4C in the same solution. Recipient animals were monitored for 14 days or until sacrifice using metabolic cages to assess various parameters of renal graft function.H2S treatment improved early allograft survival and function, and decreased early levels of necrosis, apoptosis and Kim-1 compared to University of Wisconsin preservation solution alone. H2S treatment did not affect allograft rejection. Rather, it modulated the early allograft transcriptome to decrease the expression of renal injury, coagulation and cellular stress response genes, and increase the expression of cellular proliferation and Ifn-γ induced genes compared to University of Wisconsin preservation solution alone.To our knowledge our findings are the first to show that H2S protects donor kidneys against cold ischemia-reperfusion injury in the context of allogeneic renal transplantation. This potentially represents a novel cost-effective therapeutic solution to mitigate ischemia-reperfusion injury and improve the clinical outcomes of renal transplantation.

Chronic allograft injury remains the leading cause of late kidney graft loss despite improvements in immunosuppressive drugs and a reduction in acute T cell-mediated rejection. We have recently demonstrated that natural killer (NK) cells are cytotoxic to tubular epithelial cells and contribute to acute kidney ischemia-reperfusion injury. The role of NK cells in kidney allograft rejection has not been studied.A "parent to F1" kidney transplant model was used to study NK cell-mediated transplant rejection.The C57BL/6 kidneys were transplanted into fully nephrectomized CB6F1 (C57BL/6 x BALB/c) mice. Serum creatinine levels increased from baseline (18.8 ± 5.0 μmol/L to 37.2 ± 5.9 μmol/L, P0.001) at 60 days after transplantation. B6Rag-to-CB6F1Rag (B6RagxBALB/cRag) recipients, which lack T and B cells but retain NK cells, showed similar levels of kidney dysfunction 65 days after transplantation (creatinine, 33.8 ± 7.9 μmol/L vs 17.5 ± 5.1 μmol/L in nontransplant Rag mice, P0.05). Importantly, depletion of NK cells in Rag1 recipients inhibited kidney injury (24.6 ± 5.5 μmol/L, P0.05). Osteopontin, which can activate NK cells to mediate tubular epithelial cell death in vitro, was highly expressed in 60 days kidney grafts. Osteopontin null kidney grafts had reduced injury after transplantation into CB6F1 mice (17.7 ± 3.1 μmol/L, P0.001).Collectively, these data demonstrate for the first time that independent of T and B cells, NK cells have a critical role in mediating long-term transplant kidney injury. Specific therapeutic strategies that target NK cells in addition to conventional immunosuppression may be required to attenuate chronic kidney transplant injury.

Polyoma BK virus nephropathy is a common complication after renal transplantation and is rarely seen in non-renal transplant recipients. There are only a couple of case reports of BK virus nephropathy in native kidneys in non-transplant patients, including a recent report of a 73-year-old patient with chronic lymphatic leukemia. A variety of treatment options, including leflunomide and cidofovir, were reported in these patients.Here we report the case of a 10-year-old boy with acute lymphatic leukemia who presented with non-oliguric hypertensive acute kidney injury at the 12th maintenance cycle of his chemotherapy. The workup supported the clear diagnosis of BK virus nephropathy with tubulointerstitial changes, and the patient responded favorably to intravenous immunoglobulin therapy.Pediatric nephrologists need to consider BK virus nephropathy as a differential diagnosis of acute kidney injury in immunocompromised non-transplant patients.

Background While cold storage of organs prior to transplantation is the clinical standard of care, it has been shown to exacerbate renal ischemia-reperfusion injury (IRI). Adding hydrogen sulfide (H2S) to cold University of Wisconsin (UW) solution has been shown to ameliorate graft function and injury. However, normothermic preservation alternatives are also of interest. In this study, we investigate the effect of normothermic preservation with UW solution containing a novel, mitochondria-targeted H2S donor (AP39) using a clinically relevant ex vivo porcine model of renal transplantation. Methods Warm ischemia was induced via 30m of renal pedicle clamping. The left kidney was flushed with and stored in cold UW solution for 4h. The right kidney was flushed with UW+200nM AP39 and then perfused ex vivo with non-stressed blood at 21°C for 4h. Both kidneys were then perfused with stressed blood at 37°C for 4h to simulate renal transplantation. Hourly urine and blood samples were analyzed. Tissue injury was evaluated using histopathology. Results Kidneys treated with UW+AP39 had greater mean total urine output compared to UW preserved kidneys. UW+AP39 kidneys also showed lower mean urine protein excretion compared to the UW only kidneys, suggestive of diminished glomerular injury. Histopathological data supported these findings. Conclusion We demonstrate, for the first time, that normothermic preservation with AP39-supplemented UW solution leads to superior graft function and reduced tissue injury compared to the current standard of clinical care.

Kidney transplant injury occurs with ischemia and alloimmunity. Members of the receptor interacting protein kinase family (RIPK1,3) are key regulators of "necroptosis," a newly recognized, regulated form of necrosis. Necroptosis and apoptosis death appear to be counterbalanced as caspase-8 inhibition can divert death from apoptosis to necrosis. Inhibition of necroptosis in donor organs to limit injury has not been studied in transplant models. In this study, necroptosis was triggered in caspase inhibited tubular epithelial cells (TEC) exposed to tumor necrosis factor alpha in vitro, while RIPK1 inhibition with necrostatin-1 or use of RIPK3(-/-) TEC, prevented necroptosis. In vivo, short hairpin RNA silencing of caspase-8 in donor B6 mouse kidneys increased necroptosis, enhanced high-mobility group box 1 release, reduced renal function and accelerated rejection when transplanted into BALB/c recipients. Using ethidium homodimer perfusion to assess necrosis in vivo, necrosis was abrogated in RIPK3(-/-) kidneys postischemia. Following transplantation, recipients receiving RIPK3(-/-) kidneys had longer survival (p = 0.002) and improved renal function (p = 0.03) when compared to controls. In summary, we show for the first time that RIPK3-mediated necroptosis in donor kidneys can promote inflammatory injury, and has a major impact on renal ischemia-reperfusion injury and transplant survival. We suggest inhibition of necroptosis in donor organs may similarly provide a major clinical benefit.

Introduction: The incidence of renal cell carcinoma (RCC) continues to rise concurrently with the increased prevalence of end-stage renal disease worldwide. Treatment for small renal masses continues to be partial nephrectomy mostly involving the clamping of renal blood vessels. Although necessary, this technique results in warm renal ischemia and reperfusion injury (IRI) to the afflicted kidney. We have recently demonstrated that hydrogen sulfide (H2S), a novel endogenous gaseous molecule, protects against prolonged cold and short-term warm renal IRI. In the current study, we examined whether exogenous H2S has long-term protective effects against warm renal IRI associated with renal surgical procedures.Methods: Uni-nephrectomized Lewis rats underwent 1 hour of warm ischemia induced by clamping of the renal pelvis. Animals underwent either intraperitoneal treatment with phosphate buffered saline (PBS; IRI group) or PBS supplemented with 150 μM NaHS (H2S group), and were compared against Sham-operated rats.Results: H2S treatment improved long-term renal function as serum creatinine at day 7 was significantly decreased in the H2S group compared to IRI animals (p < 0.05). H2S treatment decreased the expression of pro-inflammatory markers TLR-4, TNF-α, IFNγ, IL-2and ICAM-1, increased the expression of pro-survival molecule Bcl-2 and decreased the expression of pro-apoptotic marker BID at postoperative day 1. H2S-treated kidneys also showed a significant decrease (p < 0.05) in infiltration of macrophages at day 7 post-IRI compared to no treatment.Conclusion: H2S treatment improved long-term renal function and decreased long-term inflammation associated with warm IRI, and may offer a novel therapeutic approach to preventing warm IRI-induced renal injury associated with renal surgical procedures.

Ganglioneuromas are rare benign tumors originating from the sympathetic nervous system and neural crest cells. A 4-year-old girl presented with numerous urinary tract infections. Ultrasound and computed tomography revealed a large mass within the right kidney. A right nephrectomy and sampling of surrounding lymph nodes were performed. Pathology confirmed that the mass was a mature ganglioneuroma. The patient remains disease-free, more than 2 years after surgery. We present this rare case of renal ganglioneuroma as well as a review of the literature.