A 52-mg levonorgestrel-releasing intrauterine system vs bipolar radiofrequency nonresectoscopic endometrial ablation in women with heavy menstrual bleeding: long-term follow-up of a multicenter randomized controlled trial.

BACKGROUND: The symptom of heavy menstrual bleeding has a substantial impact on professional, physical, and social functioning. In 2021, results from a randomized controlled trial comparing a 52-mg levonorgestrel-releasing intrauterine system and radiofrequency nonresectoscopic endometrial ablation as treatments for women with heavy menstrual bleeding were published. Both treatment strategies were equally effective in treating heavy menstrual bleeding during 2-year follow-up. However, long-term results are also relevant for both patients and healthcare providers. OBJECTIVE: This study aimed to assess long-term differences in reintervention risk and menstrual blood loss in women with the symptom of heavy menstrual bleeding treated according to a strategy starting with a 52-mg levonorgestrel-releasing intrauterine system or radiofrequency nonresectoscopic endometrial ablation. STUDY DESIGN: This study was a long-term follow-up study of a multicenter randomized controlled trial (MIRA trial), in which women were allocated to either a 52-mg levonorgestrel-releasing intrauterine device (n=132) or radiofrequency nonresectoscopic endometrial ablation (n=138). Women from the original trial were contacted to fill out 6 questionnaires. The primary outcome was the reintervention rate after allocated treatment. Secondary outcomes included surgical reintervention rate, menstrual bleeding measured by the Pictorial Blood Loss Assessment Chart, (disease-specific) quality of life, sexual function, and patient satisfaction. RESULTS: From the 270 women who were randomized in the original trial, 196 (52-mg levonorgestrel-releasing intrauterine system group: n=94; radiofrequency nonresectoscopic endometrial ablation group: n=102) participated in this long-term follow-up study. Mean follow-up duration was 7.4 years (range, 6-9 years). The cumulative reintervention rate (including both medical and surgical reinterventions) was 40.0% (34/85) in the 52-mg levonorgestrel-releasing intrauterine system group and 28.7% (27/94) in the radiofrequency nonresectoscopic endometrial ablation group (relative risk, 1.39; 95% confidence interval, 0.92-2.10). The cumulative rate of surgical reinterventions only was significantly higher among patients with a treatment strategy starting with a 52-mg levonorgestrel-releasing intrauterine system compared with radiofrequency nonresectoscopic endometrial ablation (35.3% [30/85] vs 19.1% [18/94]; relative risk, 1.84; 95% confidence interval, 1.11-3.10). However, the hysterectomy rate was similar (11.8% [10/94] in the 52-mg levonorgestrel-releasing intrauterine system group and 18.1% [17/102] in the radiofrequency nonresectoscopic endometrial ablation group; relative risk, 0.65; 95% confidence interval, 0.32-1.34). Most reinterventions occurred during the first 24 months of follow-up. A total of 171 Pictorial Blood Loss Assessment Chart scores showed a median bleeding score of 0.0. No clinically relevant differences were found regarding quality of life, sexual function, and patient satisfaction. CONCLUSION: The overall risk of reintervention after long-term follow-up was not different between women treated according to a treatment strategy starting with a 52-mg levonorgestrel-releasing intrauterine system and those treated using a strategy starting with radiofrequency nonresectoscopic endometrial ablation. However, women allocated to a treatment strategy starting with a 52-mg levonorgestrel-releasing intrauterine system had a higher risk of surgical reintervention, which was driven by an increase in subsequent endometrial ablation. Both treatment strategies were effective in lowering menstrual blood loss over the long term. The results of this long-term follow-up study can support physicians in optimizing the counseling of women with heavy menstrual bleeding, thus promoting informed decision-making regarding choice of treatment.

Same-day discharge after laparoscopic hysterectomy for benign/premalignant disease: A multicentre randomised controlled trial.

OBJECTIVE: To evaluate whether physical function and quality of life was influenced by discharge on the same-day after a total laparoscopic hysterectomy. DESIGN: Multicentre non-inferiority randomised controlled trial. SETTING: Five teaching hospitals and two university hospitals in the Netherlands. POPULATION: Patients undergoing laparoscopic hysterectomy for benign or premalignant disease. METHODS: Following informed consent, participants were allocated 1:1 either to same-day discharge (SDD) or next-day discharge (NDD). MAIN OUTCOME MEASURES: The primary outcome was physical function at 7 days after surgery measured by the Patient Reported Outcomes Measurement Information System (PROMIS) Physical Function short Form 10A. Secondary outcomes were physical function and quality of life at 1 and 3 days and 6 weeks after surgery measured with PROMIS short Form 10A and the EuroQol questionnaire (EQ-5D-5L). RESULTS: Two hundred and five patients were included of whom 105 were allocated to SDD and 100 to NDD. Physical function 7 days after surgery was 35.95 in the SDD group and 35.63 in the control group (mean difference 0.32; 95% CI [0.07-0.57]). As the upper limit of the 95% CI does not exceed the non-inferiority margin of 4 points, non-inferiority of SDD could be demonstrated. No difference in physical function nor quality of life on Days 1 and 3 and 6 weeks could be found. CONCLUSION: This research demonstrates same-day discharge after laparoscopic hysterectomy is non-inferior to next day discharge in physical function 7 days after surgery.

Do we more often opt for conservative management of ovarian tumors after changing the Dutch national guideline on enlarged ovaries? A nationwide cohort study.

INTRODUCTION: Increasing evidence shows that conservative management of ovarian tumors classified as benign, based on ultrasound assessment, is safe. Therefore, conservative management has been adopted as the preferred strategy for certain ovarian tumors assessed as benign in the Dutch national guideline on enlarged ovaries in 2013. The aim of this study was to examine whether implementation of this guideline has led to changes in the number of women/100 000 women undergoing surgery for an ovarian tumor in the Netherlands. MATERIAL AND METHODS: Histopathology reports were requested for all examinations of ovarian and fallopian tube specimens (including cyst enucleations) registered in Palga, the Dutch nationwide pathology databank, from 2011 (before guideline adaptation) and 2019 (after guideline adaptation). Reports on prophylactically removed adnexa, removal for other primary tumors (eg endometrial carcinoma), and for patients under 18 years of age, were excluded from the analysis. Interobserver agreement for the inclusion and classification of reports was assessed using Cohen's Kappa analysis. RESULTS: A total of 34 932 reports were retrieved, 13 917 of which were included in the analysis. In 2011 and 2019, respectively, 96.3/100 000 vs 68.8/100 000 women aged ≥18 underwent surgery for benign ovarian tumors, and 19.6/100 000 vs 18.3/100 000 for borderline and malignant tumors combined. The number of women/100 000 who had surgery for a benign ovarian tumor per 100 000 women declined by 28.5% (p < 0.001) between 2011 and 2019. The largest difference between 2011 and 2019 was observed in the number of women per 100 000 women who underwent surgery for a serous cystadenoma (-40.7%; 20.8/100 000 vs. 12.3/100 000), followed by endometrioma (-33.2%; 14.7/100 000 vs. 9.8/100 000), simple epithelial cyst (-57.3%; 8.4/100 000 vs. 3.6/100 000), and corpus luteum cyst (-57.0%; 4.0/100 000 vs. 1.7/100 000). Cohen's Kappa for the interobserver agreement was 0.96. CONCLUSIONS: The number of women/100 000 undergoing surgery for a benign ovarian tumor has substantially decreased in the Netherlands when comparing data before and after implementation of the national guideline in 2013, while the number of women/100 000 undergoing surgery for a malignant or borderline tumor remained the same. These findings suggest successful implementation of the updated guideline, and a measurable effect on increased adoption of conservative management for benign-looking ovarian tumors.

Procedural sedation and analgesia versus general anesthesia for hysteroscopic myomectomy (PROSECCO trial): A multicenter randomized controlled trial.

BACKGROUND: Hysteroscopic resection is the first-choice treatment for symptomatic type 0 and 1 fibroids. Traditionally, this was performed under general anesthesia. Over the last decade, surgical procedures are increasingly being performed in an outpatient setting under procedural sedation and analgesia. However, studies evaluating safety and effectiveness of hysteroscopic myomectomy under procedural sedation are lacking. This study aims to investigate whether hysteroscopic myomectomy under procedural sedation and analgesia with propofol is noninferior to hysteroscopic myomectomy under general anesthesia. METHODS AND FINDINGS: This was a multicenter, randomized controlled noninferiority trial conducted in 14 university and teaching hospitals in the Netherlands between 2016 and 2021. Inclusion criteria were age ≥18 years, maximum number of 3 type 0 or 1 fibroids, maximum fibroid diameter 3.5 cm, American Society of Anesthesiologists class 1 or 2, and having sufficient knowledge of the Dutch or English language. Women with clotting disorders or with severe anemia (Hb < 5.0 mmol/L) were excluded. Women were randomized using block randomization with variable block sizes of 2, 4, and 6, between hysteroscopic myomectomy under procedural sedation and analgesia (PSA) with propofol or under general anesthesia (GA). Primary outcome was the percentage of complete resections, assessed on transvaginal ultrasonography 6 weeks postoperatively by a sonographer blinded for the treatment arm and surgical outcome. Secondary outcomes were the surgeon's judgment of completeness of procedure, menstrual blood loss, uterine fibroid related and general quality of life, pain, recovery, hospitalization, complications, and surgical reinterventions. Follow-up period was 1 year. The risk difference between both treatment arms was estimated, and a Farrington-Manning test was used to determine the p-value for noninferiority (noninferiority margin 7.5% of incomplete resections). Data were analyzed according to the intention-to-treat principle, including a per-protocol analysis for the primary outcome. A total of 209 women participated in the study and underwent hysteroscopic myomectomy with PSA (n = 106) or GA (n = 103). Mean age was 45.1 [SD 6.4] years in the PSA group versus 45.0 [7.7] years in the GA group. For 98/106 women in the PSA group and 89/103 women in the GA group, data were available for analysis of the primary outcome. Hysteroscopic resection was complete in 86/98 women (87.8%) in the PSA group and 79/89 women (88.8%) in the GA group (risk difference -1.01%; 95% confidence interval (CI) -10.36 to 8.34; noninferiority, P = 0.09). No serious anesthesiologic complications occurred, and conversion from PSA to GA was not required. During the follow-up period, 15 serious adverse events occurred (overnight admissions). All were unrelated to the intervention studied. Main limitations were the choice of primary outcome and the fact that our study proved to be underpowered. CONCLUSIONS: Noninferiority of PSA for completeness of resection was not shown, though there were no significant differences in clinical outcomes and quality of life. In this study, hysteroscopic myomectomy for type 0 and 1 fibroids with PSA compared to GA was safe and led to shorter hospitalization. These results can be used for counseling patients by gynecologists and anesthesiologists. Based on these findings, we suggest that hysteroscopic myomectomies can be performed under PSA in an outpatient setting. TRIAL REGISTRATION: The study was registered prospectively in the Dutch Trial Register (NTR 5357; registration date: 11 August 2015; Date of initial participant enrollment: 18 February 2016).

The ISTH-BAT score and outcomes after endometrial ablation in women with heavy menstrual bleeding.

BACKGROUND: The International Society on Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT), is used during the diagnostic workup of bleeding disorders. Data on ISTH-BAT scores in women with heavy menstrual bleeding (HMB) undergoing endometrial ablation (EA) could be essential in optimizing HMB counselling. OBJECTIVE: To investigate the postsurgical incidence of amenorrhea, dysmenorrhea, quality of life, re-intervention after EA, and ISTH-BAT score. METHODS: This study included women who have undergone EA because of HMB. During a follow-up of 2 to 5 years, ISTH-BAT, pictorial blood assessment chart (PBAC), and Short Form-36 survey (SF-36) were administered. At 10 years of follow-up surgical re-interventions were evaluated. RESULTS: Seventy-one women were included of whom 77% (n = 55) had an ISTH-BAT score < 6, versus 23% (n = 16) ISTH-BAT score ≥6 (mean age 46.3 versus 42.3, p = 0.004). In the ISTH-BAT ≥6 group versus < 6 group, amenorrhea occurred in 63% (10/16) versus 82% (45/55) (p = 0.111), dysmenorrhea in 38% (6/16) versus 18% (10/55) (p = 0.111), and surgical re-intervention in 19% (3/16) versus 25% (14/55) (p = 0.582). SF-36 item (Bodily) pain was lower in the ISTH-BAT ≥6 group versus < 6 (median score 58.7 vs. 80.0, p = 0.104). CONCLUSIONS: An ISTH-BAT score ≥6 may be related to a lower amenorrhea incidence and higher dysmenorrhea rate after EA.

The dysregulation of metabolic pathways and induction of the pentose phosphate pathway in renal ischaemia-reperfusion injury.

Lipid accumulation is associated with various forms of acute renal injury; however, the causative factors and pathways underpinning this lipid accumulation have not been thoroughly investigated. In this study, we performed lipidomic profiling of renal tissue following ischaemia-reperfusion injury (IRI). We identified a significant accumulation of cholesterol and specific phospholipids and sphingolipids in kidneys 24 h after IRI. In light of these findings, we hypothesised that pathways involved in lipid metabolism may also be altered. Through the analysis of published microarray data, generated from sham and ischaemic kidneys, we identified nephron-specific metabolic pathways affected by IRI and validated these findings in ischaemic renal tissue. In silico analysis revealed the downregulation of several energy and lipid metabolism pathways, including mitochondrial fatty acid beta-oxidation (FAO), peroxisomal lipid metabolism, fatty acid (FA) metabolism, and glycolysis. The pentose phosphate pathway (PPP), which is fuelled by glycolysis, was the only metabolic pathway that was upregulated 24 h following IRI. In this study, we describe the effect of renal IRI on metabolic pathways and how this contributes to lipid accumulation. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Endometrial ablation plus levonorgestrel releasing intrauterine system versus endometrial ablation alone in women with heavy menstrual bleeding: study protocol of a multicentre randomised controlled trial; MIRA2 trial.

BACKGROUND: It is estimated that between 12 to 25% of women who undergo an endometrial ablation for heavy menstrual bleeding (HMB) are dissatisfied after two years because of recurrent menstrual bleeding and/or cyclical pelvic pain, with around 15% of these women ultimately having a hysterectomy. The insertion of a levonorgestrel-releasing intrauterine system (LNG-IUS) immediately after endometrial ablation may inactivate residual untreated endometrium and/or inhibit the regeneration of endometrial tissue. Furthermore, the LNG-IUS may prevent agglutination of the uterine walls preventing intrauterine adhesion formation associated with endometrial ablation. In these ways, insertion of an LNG-IUS immediately after endometrial ablation might prevent subsequent hysterectomies because of persisting uterine bleeding and cyclical pelvic pain or pain that arises de novo. Hence, we evaluate if the combination of endometrial ablation and an LNG-IUS is superior to endometrial ablation alone in terms of reducing subsequent rates of hysterectomy at two years following the initial ablative procedure. METHODS/DESIGN: We perform a multicentre randomised controlled trial in 35 hospitals in the Netherlands. Women with heavy menstrual bleeding, who opt for treatment with endometrial ablation and without contraindication for an LNG-IUS are eligible. After informed consent, participants are randomly allocated to either endometrial ablation plus LNG-IUS or endometrial ablation alone. The primary outcome is the hysterectomy rate at 24 months following endometrial ablation. Secondary outcomes include women's satisfaction, reinterventions, complications, side effects, menstrual bleeding patterns, quality of life, societal costs. DISCUSSION: The results of this study will help clinicians inform women with HMB who opt for treatment with endometrial ablation about whether concomitant use of the LNG-IUS is beneficial for reducing the need for hysterectomy due to ongoing bleeding and/or pain symptoms. Trial registration Dutch Trial registration: NL7817. Registered 20 June 2019, https://www.trialregister.nl/trial/7817 .

Urinary mitochondrial DNA associates with delayed graft function following renal transplantation.

BACKGROUND: Ischaemia-reperfusion (IR) injury is an important determinant of delayed graft function (DGF) affecting allograft function. Mitochondrial DNA (mtDNA) is released upon cell death and platelet activation into the extracellular environment and has been suggested to be a biomarker in several diseases. Whether extracellular mtDNA accumulates in plasma and/or urine upon renal IR and predisposes DGF is unknown. METHODS: C57BL/6J wild-type mice were subjected to renal IR. In addition, an observational case-control study was set up enrolling 43 patients who underwent kidney transplantation. One day post-IR in mice and a few days following renal transplantation in human, blood and urine were collected. Patients were stratified into DGF and non-DGF groups. RESULTS: mtDNA-encoded genes accumulate in urine and plasma in both mice subjected to renal IR injury and in humans following renal transplantation. In human renal transplant recipients, cold ischaemia time and renal function correlate with urinary mtDNA levels. Urinary mtDNA levels but not urinary nuclear DNA levels were significantly higher in the DGF group compared with the non-DGF group. Multiple receiver operating characteristic curves revealed significant diagnostic performance for mtDNA-encoded genes cytochrome c oxidase III (COXIII); nicotinamide adenine dinucleotide hydrogen subunit 1 (NADH-deh); mitochondrially encoded, mitochondrially encoded nicotinamide adenine dinucleotide dehydrogenase 2 (MT-ND2) with an area under the curve of, respectively, 0.71 [P = 0.03; 95% confidence interval (CI) 0.54-0.89], 0.75 (P = 0.01; 95% CI 0.58-0.91) and 0.74 (P = 0.02; 95% CI 0.58-0.89). CONCLUSIONS: These data suggest that renal ischaemia time determines the level of mtDNA accumulation in urine, which associates with renal allograft function and the diagnosis of DGF following renal transplantation.

Excessive dietary lipid intake provokes an acquired form of lysosomal lipid storage disease in the kidney.

Obesity and dyslipidaemia are features of the metabolic syndrome and risk factors for chronic kidney disease. The cellular mechanisms connecting metabolic syndrome with chronic kidney disease onset and progression remain largely unclear. We show that proximal tubular epithelium is a target site for lipid deposition upon overnutrition with a cholesterol-rich Western-type diet. Affected proximal tubule epithelial cells displayed giant vacuoles of lysosomal or autophagosomal origin, harbouring oxidised lipoproteins and concentric membrane layer structures (multilamellar bodies), reminiscent of lysosomal storage diseases. Additionally, lipidomic analysis revealed renal deposition of cholesterol and phospholipids, including lysosomal phospholipids. Proteomic profiles of renal multilamellar bodies were distinct from those of epidermis or lung multilamellar bodies and of cytoplasmic lipid droplets. Tubular multilamellar bodies were observed in kidney biopsies of obese hypercholesterolaemic patients, and the concentration of the phospholipidosis marker di-docosahexaenoyl (22:6)-bis(monoacylglycerol) phosphate was doubled in urine from individuals with metabolic syndrome and chronic kidney disease. The enrichment of proximal tubule epithelial cells with phospholipids and multilamellar bodies was accompanied by enhanced inflammation, fibrosis, tubular damage markers, and higher urinary electrolyte content. Concomitantly to the intralysosomal lipid storage, a renal transcriptional response was initiated to enhance lysosomal degradation and lipid synthesis. In cultured proximal tubule epithelial cells, inhibition of cholesterol efflux transport or oxysterol treatment induced effects very similar to the in vivo situation, such as multilamellar body and phospholipid amassing, and induction of damage, inflammatory, fibrotic, and lipogenic molecules. The onset of phospholipidosis in proximal tubule epithelial cells is a novel pathological trait in metabolic syndrome-related chronic kidney disease, and emphasises the importance of healthy lysosomes and nutrition for kidney well-being. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Deletion of NLRX1 increases fatty acid metabolism and prevents diet-induced hepatic steatosis and metabolic syndrome.

NOD-like receptor (NLR)X1 (NLRX1) is an ubiquitously expressed inflammasome-independent NLR that is uniquely localized in mitochondria with as yet unknown effects on metabolic diseases. Here, we report that NLRX1 is essential in regulating cellular metabolism in non-immune parenchymal hepatocytes by decreasing mitochondrial fatty acid-dependent oxidative phosphorylation (OXPHOS) and promoting glycolysis. NLRX1 loss in mice has a profound impact on the prevention of diet-induced metabolic syndrome parameters, non-alcoholic fatty liver disease (NAFLD) progression, and renal dysfunction. Despite enhanced caloric intake, NLRX1 deletion in mice fed a western diet (WD) results in protection from liver steatosis, hepatic fibrosis, obesity, insulin resistance, glycosuria and kidney dysfunction parameters independent from inflammation. While mitochondrial content was equal, NLRX1 loss in hepatocytes leads to increased fatty acid oxidation and decreased steatosis. In contrast, glycolysis was decreased in NLRX1-deficient cells versus controls. Thus, although first implicated in immune regulation, we show that NLRX1 function extends to the control of hepatocyte energy metabolism via the restriction of mitochondrial fatty acid-dependent OXPHOS and enhancement of glycolysis. As such NLRX1 may be an attractive novel therapeutic target for NAFLD and metabolic syndrome.

Increased Circulating and Urinary Levels of Soluble TAM Receptors in Diabetic Nephropathy.

TAM receptors (Tyro3, Axl, and Mer) have been implicated in innate immunity. Circulating TAM receptor soluble forms (sTyro3, sAxl, sMer) are related to autoimmune disorders. We investigated TAM and their ligand protein S in patients with diabetes. Urinary and plasma levels of protein S, sTyro3, sAxl, and sMer were determined in 126 patients with diabetes assigned to a normoalbuminuric or macroalbuminuric (urinary albumin excretion <30 mg/24 hours and >300 mg/24 hours, respectively) study group and 18 healthy volunteers. TAM and protein S immunostaining was performed on kidney biopsy specimens from patients with diabetic nephropathy (n = 9) and controls (n = 6). TAM expression and shedding by tubular epithelial cells were investigated by PCR and enzyme-linked immunosorbent assay in an in vitro diabetes model. Patients with macroalbuminuria diabetes had higher circulating levels of sMer and more urinary sTyro3 and sMer than normoalbuminuric diabetics. Increased clearance of sTyro3 and sMer was associated with loss of tubular Tyro3 and Mer expression in diabetic nephropathy tissue and glomerular depositions of protein S. During in vitro diabetes, human kidney cells had down-regulation of Tyro3 and Mer mRNA and increased shedding of sTyro3 and sMer. Renal injury in diabetes is associated with elevated systemic and urine levels of sMer and sTyro3. This is the first study reporting excretion of sTAM receptors in urine, identifying the kidney as a source of sTAM.

No difference in renal injury and fibrosis between wild-type and NOD1/NOD2 double knockout mice with chronic kidney disease induced by ureteral obstruction.

BACKGROUND: Chronic kidney disease (CKD) is characterized by sustained tissue damage and ongoing tubulo-interstitial inflammation and fibrosis. Pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) and NOD-like receptors (NLRs) can sense endogenous ligands released upon tissue damage, leading to sterile inflammation and eventually irreversible kidney disease. It is known that NOD1 and NOD2 contribute to the pathogenesis of various inflammatory diseases, including acute kidney injury. However their role in chronic kidney disease is largely unknown. The aim of this study was therefore to investigate the contribution of NOD1 and NOD2 in renal interstitial fibrosis and obstructive nephropathy. METHODS: To do so, we performed unilateral ureteral obstruction (UUO) in wild type (WT) and NOD1/NOD2 double deficient (DKO) mice and analysed renal damage, fibrosis and inflammation. Data were analysed using the non-parametric Mann-Whitney U-test. RESULTS: Minor changes in inflammatory response were observed in NOD1/2 DKO mice, while no effects were observed on renal injury and the development of fibrosis. CONCLUSION: No difference in renal injury and fibrosis between WT and NOD1/NOD2 DKO mice following obstructive nephropathy induced by ureteral obstruction.

Depletion of Gut Microbiota Protects against Renal Ischemia-Reperfusion Injury.

An accumulating body of evidence shows that gut microbiota fulfill an important role in health and disease by modulating local and systemic immunity. The importance of the microbiome in the development of kidney disease, however, is largely unknown. To study this concept, we depleted gut microbiota with broad-spectrum antibiotics and performed renal ischemia-reperfusion (I/R) injury in mice. Depletion of the microbiota significantly attenuated renal damage, dysfunction, and remote organ injury and maintained tubular integrity after renal I/R injury. Gut flora-depleted mice expressed lower levels of F4/80 and chemokine receptors CX3CR1 and CCR2 in the F4/80+ renal resident macrophage population and bone marrow (BM) monocytes than did control mice. Additionally, compared with control BM monocytes, BM monocytes from gut flora-depleted mice had decreased migratory capacity toward CX3CL1 and CCL2 ligands. To study whether these effects were driven by depletion of the microbiota, we performed fecal transplants in antibiotic-treated mice and found that transplant of fecal material from an untreated mouse abolished the protective effect of microbiota depletion upon renal I/R injury. In conclusion, we show that depletion of gut microbiota profoundly protects against renal I/R injury by reducing maturation status of F4/80+ renal resident macrophages and BM monocytes. Therefore, dampening the inflammatory response by targeting microbiota-derived mediators might be a promising therapy against I/R injury.

High glucose induces HGF-independent activation of Met receptor in human renal tubular epithelium.

CONTEXT: The role of hepatocyte growth factor (HGF) in diabetic kidney damage remains controversial. OBJECTIVE: To test the hypothesis that high glucose levels activate pathways related to HGF and its receptor Met and that this could participate in glucose-induced renal cell damage. MATERIALS AND METHODS: HK2 cells, a human proximal tubule epithelial cell line, were stimulated with high glucose for 48 hours. Levels of pMet/Met, pEGFR/EGFR, pSTAT3/STAT3, pAkt/Akt and pERK1/2/ERK1/2 were studied by immunoblotting. Absence of HGF was verified by qRT-PCR and ELISA. RESULTS: High glucose level activated Met and its downstream pathways STAT3, Akt and ERK independently of HGF. High glucose induced an integrin ligand fibronectin. HGF-independent Met phosphorylation was prevented by inhibition of integrin α5ß1, Met inhibitor crizotinib, Src inhibitors PP2 and SU5565, but not by EGFR inhibitor AG1478. High glucose increased the expression of TGFß-1, CTGF and the tubular damage marker KIM-1 and increased apoptosis of HK2 cells, effects inhibited by crizotinib. CONCLUSION: High glucose activated Met receptor in HK2 cells independently of HGF, via induction of integrin α5ß1 and downstream signaling. This mode of Met activation was associated with tubular cell damage and apoptosis and it may represent a novel pathogenic mechanism and a treatment target in diabetic nephropathy.

The calcium-binding protein complex S100A8/A9 has a crucial role in controlling macrophage-mediated renal repair following ischemia/reperfusion.

Upon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by Toll-like receptor-4 and its activation is known to deleteriously contribute to renal I/R-induced injury. To further test this, wild-type and S100A9 knockout mice (deficient for S100A8/A9 complex) were subjected to renal I/R. The expression of S100A8/A9 was significantly increased 1 day after I/R and was co-localized with Ly6G (mouse neutrophil marker)-positive cells. These knockout mice displayed similar renal dysfunction and damage and neutrophil influx compared with wild-type mice at this early time point. Interestingly, S100A9 knockout mice displayed altered tissue repair 5 and 10 days post I/R, as reflected by increased renal damage, sustained inflammation, induction of fibrosis, and increased expression of collagens. This coincided with enhanced expression of alternatively activated macrophage (M2) markers, while the expression of classically activated macrophage (M1) markers was comparable. Similarly, S100A9 deficiency affected M2, but not M1 macrophage polarization in vitro. During the repair phase following acute kidney injury, S100A9 deficiency affects M2 macrophages in mice leading to renal fibrosis and damage. Thus, S100A8/A9 plays a crucial part in controlling macrophage-mediated renal repair following I/R.

A tissue-specific role for Nlrp3 in tubular epithelial repair after renal ischemia/reperfusion.

Ischemia/reperfusion injury is a major cause of acute kidney injury. Improving renal repair would represent a therapeutic strategy to prevent renal dysfunction. The innate immune receptor Nlrp3 is involved in tissue injury, inflammation, and fibrosis; however, its role in repair after ischemia/reperfusion is unknown. We address the role of Nlrp3 in the repair phase of renal ischemia/reperfusion and investigate the relative contribution of leukocyte- versus renal-associated Nlrp3 by studying bone marrow chimeric mice. We found that Nlrp3 expression was most profound during the repair phase. Although Nlrp3 expression was primarily expressed by leukocytes, both leukocyte- and renal-associated Nlrp3 was detrimental to renal function after ischemia/reperfusion. The Nlrp3-dependent cytokine IL-1ß remained unchanged in kidneys of all mice. Leukocyte-associated Nlrp3 negatively affected tubular apoptosis in mice that lacked Nlrp3 expression on leukocytes, which correlated with reduced macrophage influx. Nlrp3-deficient (Nlrp3KO) mice with wild-type bone marrow showed an improved repair response, as seen by a profound increase in proliferating tubular epithelium, which coincided with increased hepatocyte growth factor expression. In addition, Nlrp3KO tubular epithelial cells had an increased repair response in vitro, as seen by an increased ability of an epithelial monolayer to restore its structural integrity. In conclusion, Nlrp3 shows a tissue-specific role in which leukocyte-associated Nlrp3 is associated with tubular apoptosis, whereas renal-associated Nlrp3 impaired wound healing.

Sensing damage by the NLRP3 inflammasome.

The NLRP3 inflammasome is activated in response to a variety of signals that are indicative of damage to the host including tissue damage, metabolic stress, and infection. Upon activation, the NLRP3 inflammasome serves as a platform for activation of the cysteine protease caspase-1, which leads to the processing and secretion of the proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-18. Dysregulated NLRP3 inflammasome activation is associated with both heritable and acquired inflammatory diseases. Here, we review new insights into the mechanism of NLRP3 inflammasome activation and its role in disease pathogenesis.

CCAAT-enhancer binding protein delta (C/EBPδ) attenuates tubular injury and tubulointerstitial fibrogenesis during chronic obstructive nephropathy.

CCAAT-enhancer-binding protein delta (C/EBPδ) is a transcription factor mainly known for its role in inflammation and apoptosis/proliferation. Considering that these are key processes in renal fibrosis, we hypothesized that C/EBPδ would potentiate renal fibrosis. In line with this hypothesis, C/EBPδ has recently been suggested to regulate the fibrotic response during glomerulonephritis. Here we determined the importance of C/EBPδ in the development of renal tubulointerstitial fibrosis by subjecting 8- to 12-week-old C/EBPδ-deficient mice and age- and sex-matched wild-type controls to the unilateral ureteral obstruction model. Mice were killed at 1, 3, or 7 days post surgery, and renal tissues were obtained for RNA, protein, and immunohistochemical analysis. We show that C/EBPδ deficiency resulted in a more profound fibrotic response as evident from enhanced tubular injury, collagen deposition in the interstitial area, and higher expression of transforming growth factor-ß. Moreover, we show that the increase in renal fibrosis in C/EBPδ-deficient mice does not depend on an altered proliferation/apoptosis balance or on a differential inflammatory response in the obstructed kidney. In conclusion, our study provides direct evidence that C/EBPδ is a novel mediator of renal fibrosis. Modulating C/EBPδ expression could consequently be a potential antifibrotic strategy in patients with chronic kidney disease.

Nlrp3 is a key modulator of diet-induced nephropathy and renal cholesterol accumulation.

Metabolic syndrome (MetSyn) is a major health concern and associates with the development of kidney disease. The mechanisms linking MetSyn to renal disease have not been fully elucidated but are known to involve hyperuricemia, inflammation, and fibrosis. Since the innate immune receptor Nlrp3 is an important mediator of obesity and inflammation, we sought to determine whether Nlrp3 is involved in the development of MetSyn-associated nephropathy by giving wild-type or Nlrp3-knockout mice a Western-style compared to a normal diet or water without or with fructose. A plausible driver of pathology, the Nlrp3-dependent cytokine IL-1ß was not increased in the kidney. Interestingly, Nlrp3-dependent renal cholesterol accumulation, another well-known driver of renal pathology, was enhanced during MetSyn. We also determined the role of Nlrp3 and fructose-fortified water on the development of MetSyn and kidney function since fructose is an important driver of obesity and kidney disease. Surprisingly, fructose did not induce MetSyn but, irrespective of this, did induce Nlrp3-dependent renal inflammation. The presence of Nlrp3 was crucial for the development of Western-style diet-induced renal pathology as reflected by the prevention of renal inflammation, fibrosis, steatosis, microalbuminuria, and hyperuricemia in the Nlrp3-knockout mice. Thus, Nlrp3 may mediate renal pathology in the context of diet-induced MetSyn.

Opposite role of CD44-standard and CD44-variant-3 in tubular injury and development of renal fibrosis during chronic obstructive nephropathy.

Chronic kidney diseases (CKDs) are characterized by tubular atrophy and interstitial fibrosis. We previously showed that in obstructive nephropathy de novo CD44 renal expression contributes to renal fibrosis but attenuates tubular damage/apoptosis. As CD44-standard (CD44s) has been linked to TGF-ß1-mediated actions and CD44-variant-3 (CD44v3) favors HGF-c-Met binding, we compared the functional properties of these CD44 isoforms in the progression of obstructive nephropathy, using specific CD44-variant knockout/knockin mice. The presence of CD44v3 diminished tubular damage during obstructive nephropathy, decreased apoptosis, and increased proliferation of tubular epithelial cells, and prevented renal fibrosis development. In contrast, expression of CD44s led to increased tubular damage and tubular epithelial cell apoptosis, and more renal fibrosis. A relative increase in renal ß-catenin expression, HGF production, and HGF/c-Met signaling, together with a relative inhibition of TGF-ß1 downstream signaling and TGF-ß type I receptor expression, was found in CD44v3 mice compared with CD44s littermates. In line with this, Wnt3a/HGF treatment of tubular cells resulted in higher ß-catenin/p-AKT levels in CD44v3(+) tubular epithelial cells, whereas TGF-ß1 induced a mild collagen I upregulation in CD44v3(+) mouse embryonic fibroblasts as compared with CD44s(+) cells. Thus, CD44s and CD44v3 exert opposite roles in the progression of obstructive nephropathy, with CD44v3-v10 being the protective isoform that delays evolution of the renal pathology.