Evaluation of non-invasive biomarkers of kidney allograft rejection in a prospective multicenter unselected cohort study (EU-TRAIN).

Non-invasive biomarkers are promising tools for improving kidney allograft rejection monitoring, but their clinical adoption requires more evidence in specifically designed studies. To address this unmet need, we designed the EU-TRAIN study, a large prospective multicentric unselected cohort funded by the European Commission. Here, we included consecutive adult patients who received a kidney allograft in nine European transplant centers between November 2018 and June 2020. We prospectively assessed gene expression levels of 19 blood messenger RNAs, four antibodies targeting non-human leukocyte antigen (HLA) endothelial antigens, together with circulating anti-HLA donor-specific antibodies (DSA). The primary outcome was allograft rejection (antibody-mediated, T cell-mediated, or mixed) in the first year post-transplantation. Overall, 412 patients were included, with 812 biopsies paired with a blood sample. CD4 gene expression was significantly associated with rejection, while circulating anti-HLA DSA had a significant association with allograft rejection and a strong association with antibody-mediated rejection. All other tested biomarkers, including AKR1C3, CD3E, CD40, CD8A, CD9, CTLA4, ENTPD1, FOXP3, GZMB, ID3, IL7R, MS4A1, MZB1, POU2AF1, POU2F1, TCL1A, TLR4, and TRIB1, as well as antibodies against angiotensin II type 1 receptor, endothelin 1 type A receptor, C3a and C5a receptors, did not show significant associations with allograft rejection. The blood messenger RNAs and non-HLA antibodies did not show an additional value beyond standard of care monitoring parameters and circulating anti-HLA DSA to predict allograft rejection in the first year post-transplantation. Thus, our results open avenues for specifically designed studies to demonstrate the clinical relevance and implementation of other candidate non-invasive biomarkers in kidney transplantation practice.

Non-HLA antibodies targeting angiotensin II Type 1 receptor and endothelin-1 Type A receptors induce endothelial injury via ß2-arrestin link to mTOR pathway.

Functional non-HLA antibodies (antibodies to non-human leukocyte antigens) targeting the G protein-coupled receptors angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) are implicated in the pathogenesis of transplant vasculopathy. While ERK signaling (a regulator of cell growth) may represent a general cellular response to agonist stimulation, the molecular link between receptor stimulation and development of vascular obliteration has not been fully established. Here we hypothesize involvement of the versatile adaptor proteins, ß-arrestins, and the major regulator of cell growth, PI3K/mTOR signaling, in impaired endothelial repair. To test this, human microvascular endothelial cells were treated with AT1R/ETAR antibodies isolated from patients with kidney transplant vasculopathy. These antibodies activated both mTOR complexes via AT1R and ETAR in a PI3K-dependent and ERK-independent manner. The mTOR inhibitor, rapamycin, completely abolished activation of mTORC1 and mTORC2 after long-term treatment with receptor antibodies. Imaging studies revealed that ß2- but not ß1-arrestin was recruited to ETAR in response to ET-1 and patient antibodies but not with antibodies isolated from healthy individuals. Silencing of ß2-arrestin by siRNA transfection significantly reduced ERK1/2 and mTORC2 activation. Non-HLA antibodies impaired endothelial repair by AT1R- and ETAR-induced mTORC2 signaling. Thus, we provide evidence that functional AT1R/ETAR antibodies induce ERK1/2 and mTOR signaling involving ß2-arrestin in human microvascular endothelium. Hence, our data may provide a translational rationale for mTOR inhibitors in combination with receptor blockers in patients with non-HLA receptor recognizing antibodies.

Sex-specific molecular signature of mouse podocytes in homeostasis and in response to pharmacological challenge with rapamycin.

BACKGROUND: Sex differences exist in the prevalence and progression of major glomerular diseases. Podocytes are the essential cell-type in the kidney which maintain the physiological blood-urine barrier, and pathological changes in podocyte homeostasis are critical accelerators of impairment of kidney function. However, sex-specific molecular signatures of podocytes under physiological and stress conditions remain unknown. This work aimed at identifying sexual dimorphic molecular signatures of podocytes under physiological condition and pharmacologically challenged homeostasis with mechanistic target of rapamycin (mTOR) inhibition. mTOR is a crucial regulator involved in a variety of physiological and pathological stress responses in the kidney and inhibition of this pathway may therefore serve as a general stress challenger to get fundamental insights into sex differences in podocytes. METHODS: The genomic ROSAmT/mG-NPHS2 Cre mouse model was used which allows obtaining highly pure podocyte fractions for cell-specific molecular analyses, and vehicle or pharmacologic treatment with the mTOR inhibitor rapamycin was performed for 3 weeks. Subsequently, deep RNA sequencing and proteomics were performed of the isolated podocytes to identify intrinsic sex differences. Studies were supplemented with metabolomics from kidney cortex tissues. RESULTS: Although kidney function and morphology remained normal in all experimental groups, RNA sequencing, proteomics and metabolomics revealed strong intrinsic sex differences in the expression levels of mitochondrial, translation and structural transcripts, protein abundances and regulation of metabolic pathways. Interestingly, rapamycin abolished prominent sex-specific clustering of podocyte gene expression and induced major changes only in male transcriptome. Several sex-biased transcription factors could be identified as possible upstream regulators of these sexually dimorphic responses. Concordant to transcriptomics, metabolomic changes were more prominent in males. Remarkably, high number of previously reported kidney disease genes showed intrinsic sexual dimorphism and/or different response patterns towards mTOR inhibition. CONCLUSIONS: Our results highlight remarkable intrinsic sex-differences and sex-specific response patterns towards pharmacological challenged podocyte homeostasis which might fundamentally contribute to sex differences in kidney disease susceptibilities and progression. This work provides rationale and an in-depth database for novel targets to be tested in specific kidney disease models to advance with sex-specific treatment strategies.

The global burden of chronic kidney diseases is rapidly increasing and is projected to become the fifth most common cause of years of life lost worldwide by 2040. Sexual dimorphism in kidney diseases and transplantation is well known, yet sex-specific therapeutic strategies are still missing. One reason is the lack of knowledge due to the lack of inclusion of sex as a biological variable in study designs. This work aimed at identification of molecular signatures of male and female podocytes, gate-keepers of the glomerular filtration barrier. Like cardiomyocytes, podocytes are terminally differentiated cells which are highly susceptible towards pathological challenges. Podocytes are the decisive cell-type of the kidney to maintain the physiological blood-urine barrier, and disturbances of their homeostasis critically accelerate kidney function impairment. By help of a genomic mouse model, highly purified podocytes were obtained from male and female mice with and without pharmacological challenge of the mechanistic target of rapamycin (mTOR) signaling pathway which is known to be deregulated in major kidney diseases. Deep RNA sequencing, proteomics and metabolomics revealed strong intrinsic sex differences in the expression levels of mitochondrial, translation and structural transcripts, protein abundances and regulation of metabolic pathways which might fundamentally contribute to sex differences in kidney disease susceptibilities and progression. Remarkably, high number of previously reported kidney disease genes showed so far unknown intrinsic sexual dimorphism and/or different response patterns towards mTOR inhibition. Our work provides an in-depth database for novel targets to be tested in kidney disease models to advance with sex-specific treatment strategies.

Angiogenic Role of Mesothelium-Derived Chemokine CXCL1 During Unfavorable Peritoneal Tissue Remodeling in Patients Receiving Peritoneal Dialysis as Renal Replacement Therapy.

Peritoneal dialysis (PD) is a valuable 'home treatment' option, even more so during the ongoing Coronavirus pandemic. However, the long-term use of PD is limited by unfavourable tissue remodelling in the peritoneal membrane, which is associated with inflammation-induced angiogenesis. This appears to be driven primarily through vascular endothelial growth factor (VEGF), while the involvement of other angiogenic signaling pathways is still poorly understood. Here, we have identified the crucial contribution of mesothelial cell-derived angiogenic CXC chemokine ligand 1 (CXCL1) to peritoneal angiogenesis in PD. CXCL1 expression and peritoneal microvessel density were analysed in biopsies obtained by the International Peritoneal Biobank (NCT01893710 at www.clinicaltrials.gov), comparing 13 children with end-stage kidney disease before initiating PD to 43 children on chronic PD. The angiogenic potential of mesothelial cell-derived CXCL1 was assessed in vitro by measuring endothelial tube formation of human microvascular endothelial cells (HMECs) treated with conditioned medium from human peritoneal mesothelial cells (HPMCs) stimulated to release CXCL1 by treatment with either recombinant IL-17 or PD effluent. We found that the capillary density in the human peritoneum correlated with local CXCL1 expression. Both CXCL1 expression and microvessel density were higher in PD patients than in the age-matched patients prior to initiation of PD. Exposure of HMECs to recombinant CXCL1 or conditioned medium from IL-17-stimulated HPMCs resulted in increased endothelial tube formation, while selective inhibition of mesothelial CXCL1 production by specific antibodies or through silencing of relevant transcription factors abolished the proangiogenic effect of HPMC-conditioned medium. In conclusion, peritoneal mesothelium-derived CXCL1 promotes endothelial tube formation in vitro and associates with peritoneal microvessel density in uremic patients undergoing PD, thus providing novel targets for therapeutic intervention to prolong PD therapy.

Renal Ischemia/Reperfusion Injury in Soluble Epoxide Hydrolase-Deficient Mice.

AIM: 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP)-dependent eicosanoids that play opposite roles in the regulation of vascular tone, inflammation, and apoptosis. 20-HETE aggravates, whereas EETs ameliorate ischemia/reperfusion (I/R)-induced organ damage. EETs are rapidly metabolized to dihydroxyeicosatrienoic acids (DHETs) by the soluble epoxide hydrolase (sEH). We hypothesized that sEH gene (EPHX2) deletion would increase endogenous EET levels and thereby protect against I/R-induced acute kidney injury (AKI). METHODS: Kidney damage was evaluated in male wildtype (WT) and sEH-knockout (KO)-mice that underwent 22-min renal ischemia followed by two days of reperfusion. CYP-eicosanoids were analyzed by liquid chromatography tandem mass spectrometry. RESULTS: Contrary to our initial hypothesis, renal function declined more severely in sEH-KO mice as indicated by higher serum creatinine and urea levels. The sEH-KO-mice also featured stronger tubular lesion scores, tubular apoptosis, and inflammatory cell infiltration. Plasma and renal EET/DHET-ratios were higher in sEH-KO than WT mice, thus confirming the expected metabolic consequences of sEH deficiency. However, CYP-eicosanoid profiling also revealed that renal, but not plasma and hepatic, 20-HETE levels were significantly increased in sEH-KO compared to WT mice. In line with this finding, renal expression of Cyp4a12a, the murine 20-HETE-generating CYP-enzyme, was up-regulated both at the mRNA and protein level, and Cyp4a12a immunostaining was more intense in the renal arterioles of sEH-KO compared with WT mice. CONCLUSION: These results indicate that the potential beneficial effects of reducing EET degradation were obliterated by a thus far unknown mechanism leading to kidney-specific up-regulation of 20-HETE formation in sEH-KO-mice.