The Intersectoral Coordination Unit for the Sustainable Intensification of Peritoneal Dialysis in Schleswig-Holstein (SKIP-SH) cohort study.

BACKGROUND: Peritoneal dialysis (PD) remains underutilised in Germany, prompting the initiation of the Sustainable Intensification of Peritoneal Dialysis in Schleswig-Holstein (SKIP-SH) project. The SKIP-SH cohort study aims to demonstrate the presumed benefits of PD, including enhanced quality of life and reduced healthcare personnel requirements, and to generate data to strengthen the use of PD. METHODS: The prospective SKIP-SH cohort study recruits patients with advanced chronic kidney disease (CKD) and their caregivers. Comprehensive data, including demographic information, medical history, clinical course, laboratory data, and quality-of-life assessments, are collected. Additionally, biomaterials will be obtained. Primary study objectives are documenting the clinical course and complications, time on therapy for new dialysis patients, reasons influencing treatment modality choices, circumstances at the initiation of dialysis, and quality of life for patients with CKD and their caregivers. The collected biomaterials will serve as a basis for further translational research. Secondary objectives include identifying factors impacting disease-related quality of life, clinical complications, and therapy dropout, estimating ecological footprints, and evaluating healthcare costs and labour time for initiating and sustaining PD treatment. DISCUSSION: PD is notably underutilised in Germany. The current therapy approach for advanced CKD often lacks emphasis on patient-focused care and quality-of-life considerations. Furthermore, adequate explorative research programs to improve our knowledge of mechanisms leading to disease progression and therapy failure in PD patients are scarce. The overarching goal of the SKIP-SH cohort study is to address the notably low PD prevalence in Germany whilst advocating for a shift towards patient-focused care, quality-of-life considerations, and robust translational research. TRIAL REGISTRATION: This study was registered with the German trial registry (Deutsches Register klinischer Studien) on November 7, 2023, under trial number DRKS00032983.

Potential and Uncertainties of RejectClass in Acute Kidney Graft Dysfunction: An Independent Validation Study.

BACKGROUND: Kidney graft rejections are classified based on the Banff classification. The RejectClass algorithm, initially derived from a cohort comprising mostly protocol biopsies, identifies data-driven phenotypes of acute rejection and chronic pathology using Banff lesion scores. It also provides composite scores for inflammation activity and chronicity. This study independently evaluates the performance of RejectClass in a cohort consisting entirely of indication biopsies. METHODS: We retrospectively applied RejectClass to 441 patients from the German TRABIO (TRAnsplant BIOpsies) cohort who had received indication biopsies. The primary endpoint was death-censored graft failure during 2 y of follow-up. RESULTS: The application of RejectClass to our cohort demonstrated moderately comparable phenotypic features with the derivation cohort, and most clusters indicated an elevated risk of graft loss. However, the reproduction of all phenotypes and the associated risks of graft failure, as depicted in the original studies, was not fully accomplished. In contrast, adjusted Cox proportional hazards analyses substantiated that both the inflammation score and the chronicity score are independently associated with graft loss, exhibiting hazard ratios of 1.7 (95% confidence interval, 1.2-2.3; P = 0.002) and 2.2 (95% confidence interval, 1.8-2.6; P < 0.001), respectively, per 0.25-point increment (scale: 0.0-1.0). CONCLUSIONS: The composite inflammation and chronicity scores may already have direct utility in quantitatively assessing the disease stage. Further refinement and validation of RejectClass clusters are necessary to achieve more reliable and accurate phenotyping of rejection.

The German Transplantation Registry Reveals Deficiencies in the Listing Process for Kidney Transplantation.

Introduction: The time from dialysis onset to enrollment on the kidney waiting list (listing time) is a crucial step on the path to receiving a kidney allograft; however, this process has received very little research attention in the Eurotransplant (ET) area. Methods: We retrospectively analyzed data from the German transplantation registry, including patients who were on the waiting list for a first kidney transplant in Germany between 2006 and 2016. Listing time was evaluated using a mixed linear model. The outcomes on the kidney waiting list were assessed using competing risk analyses. Results: We assessed a total of 43,955 patients. Listing occurred at a higher pace in patients receiving living donor transplantations (median 0.4 years from dialysis onset) than in deceased donor transplantations (Eurotransplant Kidney Allocation System [ETKAS] 1.1 years, European Senior Program [ESP] 1.4 years, Acceptable Mismatch program 1.3 years), with 28.5% of living donor transplantations performed preemptively. There was only modest variation in listing time between the transplant centers. Patients with a history of viral infection, high immunization; hemodialysis patients; and patients with a higher body mass index (BMI) had a delayed listing process. Two of 3 patients listed in the ETKAS, excluding those with potential bonus points (pediatric, other organ transplantations), were eventually transplanted. Older patients, male patients, patients with blood type O, and patients with diabetic nephropathy as the underlying renal disease had the highest risk not to proceed to transplantation. Conclusion: Although long waiting times remain the biggest hurdle for transplantation in Germany, there is ample room for improvement of the listing process.

The Tacrolimus Concentration/Dose Ratio Does Not Predict Early Complications After Kidney Transplantation.

Early-on post kidney transplantation, there is a high risk of graft rejection and opportunistic viral infections. A low tacrolimus concentration/dose (C/D) ratio as a surrogate marker of fast tacrolimus metabolism has been established for risk stratification 3 months post-transplantation (M3). However, many adverse events occurring earlier might be missed, and stratification at 1 month post-transplantation (M1) has not been investigated. We retrospectively analyzed case data from 589 patients who had undergone kidney transplantation between 2011 and 2021 at three German transplant centers. Tacrolimus metabolism was estimated by use of the C/D ratio at M1, M3, M6, and M12. C/D ratios increased substantially during the year, particularly between M1 and M3. Many viral infections and most graft rejections occurred before M3. Neither at M1 nor at M3 was a low C/D ratio associated with susceptibility to BKV viremia or BKV nephritis. A low C/D ratio at M1 could not predict acute graft rejections or impaired kidney function, whereas at M3 it was significantly associated with subsequent rejections and impairment of kidney function. In summary, most rejections occur before M3, but a low C/D ratio at M1 does not identify patients at risk, limiting the predictive utility of this stratification approach.

Epithelial X-Box Binding Protein 1 Coordinates Tumor Protein p53-Driven DNA Damage Responses and Suppression of Intestinal Carcinogenesis.

BACKGROUND & AIMS: Throughout life, the intestinal epithelium undergoes constant self-renewal from intestinal stem cells. Together with genotoxic stressors and failing DNA repair, this self-renewal causes susceptibility toward malignant transformation. X-box binding protein 1 (XBP1) is a stress sensor involved in the unfolded protein response (UPR). We hypothesized that XBP1 acts as a signaling hub to regulate epithelial DNA damage responses. METHODS: Data from The Cancer Genome Atlas were analyzed for association of XBP1 with colorectal cancer (CRC) survival and molecular interactions between XBP1 and p53 pathway activity. The role of XBP1 in orchestrating p53-driven DNA damage response was tested in vitro in mouse models of chronic intestinal epithelial cell (IEC) DNA damage (Xbp1/H2bfl/fl, Xbp1ΔIEC, H2bΔIEC, H2b/Xbp1ΔIEC) and via orthotopic tumor organoid transplantation. Transcriptome analysis of intestinal organoids was performed to identify molecular targets of Xbp1-mediated DNA damage response. RESULTS: In The Cancer Genome Atlas data set of CRC, low XBP1 expression was significantly associated with poor overall survival and reduced p53 pathway activity. In vivo, H2b/Xbp1ΔIEC mice developed spontaneous intestinal carcinomas. Orthotopic tumor organoid transplantation revealed a metastatic potential of H2b/Xbp1ΔIEC-derived tumors. RNA sequencing of intestinal organoids (H2b/Xbp1fl/fl, H2bΔIEC, H2b/Xbp1ΔIEC, and H2b/p53ΔIEC) identified a transcriptional program downstream of p53, in which XBP1 directs DNA-damage-inducible transcript 4-like (Ddit4l) expression. DDIT4L inhibits mechanistic target of rapamycin-mediated phosphorylation of 4E-binding protein 1. Pharmacologic mechanistic target of rapamycin inhibition suppressed epithelial hyperproliferation via 4E-binding protein 1. CONCLUSIONS: Our data suggest a crucial role for XBP1 in coordinating epithelial DNA damage responses and stem cell function via a p53-DDIT4L-dependent feedback mechanism.

Activating Transcription Factor 6 Mediates Inflammatory Signals in Intestinal Epithelial Cells Upon Endoplasmic Reticulum Stress.

BACKGROUND & AIMS: Excess and unresolved endoplasmic reticulum (ER) stress in intestinal epithelial cells (IECs) promotes intestinal inflammation. Activating transcription factor 6 (ATF6) is one of the signaling mediators of ER stress. We studied the pathways that regulate ATF6 and its role for inflammation in IECs. METHODS: We performed an RNA interference screen, using 23,349 unique small interfering RNAs targeting 7783 genes and a luciferase reporter controlled by an ATF6-dependent ERSE (ER stress-response element) promoter, to identify proteins that activate or inhibit the ATF6 signaling pathway in HEK293 cells. To validate the screening results, intestinal epithelial cell lines (Caco-2 cells) were transfected with small interfering RNAs or with a plasmid overexpressing a constitutively active form of ATF6. Caco-2 cells with a CRISPR-mediated disruption of autophagy related 16 like 1 gene (ATG16L1) were used to study the effect of ATF6 on ER stress in autophagy-deficient cells. We also studied intestinal organoids derived from mice that overexpress constitutively active ATF6, from mice with deletion of the autophagy related 16 like 1 or X-Box binding protein 1 gene in IECs (Atg16l1ΔIEC or Xbp1ΔIEC, which both develop spontaneous ileitis), from patients with Crohn's disease (CD) and healthy individuals (controls). Cells and organoids were incubated with tunicamycin to induce ER stress and/or chemical inhibitors of newly identified activator proteins of ATF6 signaling, and analyzed by real-time polymerase chain reaction and immunoblots. Atg16l1ΔIEC and control (Atg16l1fl/fl) mice were given intraperitoneal injections of tunicamycin and were treated with chemical inhibitors of ATF6 activating proteins. RESULTS: We identified and validated 15 suppressors and 7 activators of the ATF6 signaling pathway; activators included the regulatory subunit of casein kinase 2 (CSNK2B) and acyl-CoA synthetase long chain family member 1 (ACSL1). Knockdown or chemical inhibition of CSNK2B and ACSL1 in Caco-2 cells reduced activity of the ATF6-dependent ERSE reporter gene, diminished transcription of the ATF6 target genes HSP90B1 and HSPA5 and reduced NF-κB reporter gene activation on tunicamycin stimulation. Atg16l1ΔIEC and or Xbp1ΔIEC organoids showed increased expression of ATF6 and its target genes. Inhibitors of ACSL1 or CSNK2B prevented activation of ATF6 and reduced CXCL1 and tumor necrosis factor (TNF) expression in these organoids on induction of ER stress with tunicamycin. Injection of mice with inhibitors of ACSL1 or CSNK2B significantly reduced tunicamycin-mediated intestinal inflammation and IEC death and expression of CXCL1 and TNF in Atg16l1ΔIEC mice. Purified ileal IECs from patients with CD had higher levels of ATF6, CSNK2B, and HSPA5 messenger RNAs than controls; early-passage organoids from patients with active CD show increased levels of activated ATF6 protein, incubation of these organoids with inhibitors of ACSL1 or CSNK2B reduced transcription of ATF6 target genes, including TNF. CONCLUSIONS: Ileal IECs from patients with CD have higher levels of activated ATF6, which is regulated by CSNK2B and HSPA5. ATF6 increases expression of TNF and other inflammatory cytokines in response to ER stress in these cells and in organoids from Atg16l1ΔIEC and Xbp1ΔIEC mice. Strategies to inhibit the ATF6 signaling pathway might be developed for treatment of inflammatory bowel diseases.

Epithelial RNase H2 Maintains Genome Integrity and Prevents Intestinal Tumorigenesis in Mice.

BACKGROUND & AIMS: RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS: We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS: The H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS: In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.