Sex differences in kidney metabolism may reflect sex-dependent outcomes in human diabetic kidney disease.

Diabetic kidney disease (DKD) is the main cause of chronic kidney disease (CKD) and progresses faster in males than in females. We identify sex-based differences in kidney metabolism and in the blood metabolome of male and female individuals with diabetes. Primary human proximal tubular epithelial cells (PTECs) from healthy males displayed increased mitochondrial respiration, oxidative stress, apoptosis, and greater injury when exposed to high glucose compared with PTECs from healthy females. Male human PTECs showed increased glucose and glutamine fluxes to the TCA cycle, whereas female human PTECs showed increased pyruvate content. The male human PTEC phenotype was enhanced by dihydrotestosterone and mediated by the transcription factor HNF4A and histone demethylase KDM6A. In mice where sex chromosomes either matched or did not match gonadal sex, male gonadal sex contributed to the kidney metabolism differences between males and females. A blood metabolomics analysis in a cohort of adolescents with or without diabetes showed increased TCA cycle metabolites in males. In a second cohort of adults with diabetes, females without DKD had higher serum pyruvate concentrations than did males with or without DKD. Serum pyruvate concentrations positively correlated with the estimated glomerular filtration rate, a measure of kidney function, and negatively correlated with all-cause mortality in this cohort. In a third cohort of adults with CKD, male sex and diabetes were associated with increased plasma TCA cycle metabolites, which correlated with all-cause mortality. These findings suggest that differences in male and female kidney metabolism may contribute to sex-dependent outcomes in DKD.

Humoral Responses in the Omicron Era Following 3-Dose SARS-CoV-2 Vaccine Series in Kidney Transplant Recipients.

Kidney transplant recipients (KTRs) have a diminished response to SARS-CoV-2 vaccination compared with immunocompetent individuals. Deeper understanding of antibody responses in KTRs following third-dose vaccination would enable identification of those who remain unprotected against Omicron. Methods: We profiled antibody responses in KTRs pre- and at 1 and 3 mo post-third-dose SARS-CoV-2 mRNA-based vaccine. Binding antibody levels were determined by ELISA. Neutralization against wild type, Beta, Delta, and Omicron (BA.1) variants was determined using a SARS-CoV-2 spike-pseudotyped lentivirus assay. Results: Forty-four KTRs were analyzed at 1 and 3 mo (n = 26) post-third dose. At 1 mo, the proportion of participants with a robust antibody response had increased significantly from baseline, but Omicron-specific neutralizing antibodies were detected in just 45% of KTRs. Median binding antibody levels declined at 3 mo, but the proportion of KTRs with a robust antibody response was unchanged; 38.5% KTRs maintained Omicron-specific neutralization at 3 mo. No clinical variables were significantly associated with Omicron-neutralizing antibodies, but antireceptor binding domain titers appeared to identify those with Omicron-specific neutralizing capacity. Conclusions: Over 50% of KTRs lack Omicron-specific neutralization capacity 1 mo post-third mRNA-vaccine dose. Antibody levels of responders were well preserved at 3 mo. Anti receptor binding domain antibody titers may identify patients with a detectable Omicron-neutralizing antibody response.

Single-cell profiling of healthy human kidney reveals features of sex-based transcriptional programs and tissue-specific immunity.

Knowledge of the transcriptional programs underpinning the functions of human kidney cell populations at homeostasis is limited. We present a single-cell perspective of healthy human kidney from 19 living donors, with equal contribution from males and females, profiling the transcriptome of 27677 cells to map human kidney at high resolution. Sex-based differences in gene expression within proximal tubular cells were observed, specifically, increased anti-oxidant metallothionein genes in females and aerobic metabolism-related genes in males. Functional differences in metabolism were confirmed in proximal tubular cells, with male cells exhibiting higher oxidative phosphorylation and higher levels of energy precursor metabolites. We identified kidney-specific lymphocyte populations with unique transcriptional profiles indicative of kidney-adapted functions. Significant heterogeneity in myeloid cells was observed, with a MRC1+LYVE1+FOLR2+C1QC+ population representing a predominant population in healthy kidney. This study provides a detailed cellular map of healthy human kidney, and explores the complexity of parenchymal and kidney-resident immune cells.

NUAK1 promotes organ fibrosis via YAP and TGF-ß/SMAD signaling.

Fibrosis is a central pathway that drives progression of multiple chronic diseases, yet few safe and effective clinical antifibrotic therapies exist. In most fibrotic disorders, transforming growth factor-ß (TGF-ß)-driven scarring is an important pathologic feature and a key contributor to disease progression. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are two closely related transcription cofactors that are important for coordinating fibrogenesis after organ injury, but how they are activated in response to tissue injury has, so far, remained unclear. Here, we describe NUAK family kinase 1 (NUAK1) as a TGF-ß-inducible profibrotic kinase that is up-regulated in multiple fibrotic organs in mice and humans. Mechanistically, we show that TGF-ß induces a rapid increase in NUAK1 in fibroblasts. NUAK1, in turn, can promote profibrotic YAP and TGF-ß/SMAD signaling, ultimately leading to organ scarring. Moreover, activated YAP and TAZ can induce further NUAK1 expression, creating a profibrotic positive feedback loop that enables persistent fibrosis. Using mouse models of kidney, lung, and liver fibrosis, we demonstrate that this fibrogenic signaling loop can be interrupted via fibroblast-specific loss of NUAK1 expression, leading to marked attenuation of fibrosis. Pharmacologic NUAK1 inhibition also reduced scarring, either when initiated immediately after injury or when initiated after fibrosis was already established. Together, our data suggest that NUAK1 plays a critical, previously unrecognized role in fibrogenesis and represents an attractive target for strategies that aim to slow fibrotic disease progression.

Myofibroblast YAP/TAZ activation is a key step in organ fibrogenesis.

Fibrotic diseases account for nearly half of all deaths in the developed world. Despite its importance, the pathogenesis of fibrosis remains poorly understood. Recently, the two mechanosensitive transcription cofactors YAP and TAZ have emerged as important profibrotic regulators in multiple murine tissues. Despite this growing recognition, a number of important questions remain unanswered, including which cell types require YAP/TAZ activation for fibrosis to occur and the time course of this activation. Here, we present a detailed analysis of the role that myofibroblast YAP and TAZ play in organ fibrosis and the kinetics of their activation. Using analyses of cells, as well as multiple murine and human tissues, we demonstrated that myofibroblast YAP and TAZ were activated early after organ injury and that this activation was sustained. We further demonstrated the critical importance of myofibroblast YAP/TAZ in driving progressive scarring in the kidney, lung, and liver, using multiple transgenic models in which YAP and TAZ were either deleted or hyperactivated. Taken together, these data establish the importance of early injury-induced myofibroblast YAP and TAZ activation as a key event driving fibrosis in multiple organs. This information should help guide the development of new antifibrotic YAP/TAZ inhibition strategies.

Evaluation of a gene expression biomarker to identify operationally tolerant liver transplant recipients: the LITMUS trial.

LITMUS was a single-centre, Phase 2a study designed to investigate whether the gene biomarker FGL2/IFNG previously reported for the identification of tolerance in murine models could identify operationally tolerant liver transplant recipients. Multiplex RT-PCR was used to amplify eight immunoregulatory genes in peripheral blood mononuclear cells (PBMC) from 69 adult liver transplant recipients. Patients with PBMC FGL2/IFNG ≥ 1 and a normal liver biopsy underwent immunosuppression (IS) withdrawal. The primary end point was the development of operational tolerance. Secondary end points included correlation of tolerance with allograft gene expression and immune cell markers. Twenty-eight of 69 patients (38%) were positive for the PBMC tolerance biomarker and 23 proceeded to IS withdrawal. Nine of the 23 patients had abnormal baseline liver biopsies and were excluded. Of the 14 patients with normal biopsies, eight (57%) have achieved operational tolerance and are off IS (range 12-57 months). Additional studies revealed that all of the tolerant patients and only one non-tolerant patient had a liver gene ratio of FOXP3/IFNG ≥ 1 prior to IS withdrawal. Increased CD4+ T regulatory T cells were detected both in PBMC and livers of tolerant patients following IS withdrawal. Higher expression of SELE (gene for E-selectin) and lower expression of genes associated with inflammatory responses (GZMB, CIITA, UBD, LSP1, and CXCL9) were observed in the pre-withdrawal liver biopsies of tolerant patients by RNA sequencing. These results suggest that measurement of PBMC FGL2/IFNG may enrich for the identification of operationally tolerant liver transplant patients, especially when combined with intragraft measurement of FOXP3/IFNG. Clinical Trial Registration: ClinicalTrials.gov (LITMUS: NCT02541916).

Increased Autoantibodies Against Ro/SS-A, CENP-B, and La/SS-B in Patients With Kidney Allograft Antibody-mediated Rejection.

Antibody-mediated rejection (AMR) causes more than 50% of late kidney graft losses. In addition to anti-human leukocyte antigen (HLA) donor-specific antibodies, antibodies against non-HLA antigens are also linked to AMR. Identifying key non-HLA antibodies will improve our understanding of AMR. METHODS: We analyzed non-HLA antibodies in sera from 80 kidney transplant patients with AMR, mixed rejection, acute cellular rejection (ACR), or acute tubular necrosis. IgM and IgG antibodies against 134 non-HLA antigens were measured in serum samples collected pretransplant or at the time of diagnosis. RESULTS: Fifteen non-HLA antibodies were significantly increased (P < 0.05) in AMR and mixed rejection compared with ACR or acute tubular necrosis pretransplant, and 7 at diagnosis. AMR and mixed cases showed significantly increased pretransplant levels of IgG anti-Ro/Sjögren syndrome-antigen A (SS-A) and anti-major centromere autoantigen (CENP)-B, compared with ACR. Together with IgM anti-CENP-B and anti-La/SS-B, these antibodies were significantly increased in AMR/mixed rejection at diagnosis. Increased IgG anti-Ro/SS-A, IgG anti-CENP-B, and IgM anti-La/SS-B were associated with the presence of microvascular lesions and class-II donor-specific antibodies (P < 0.05). Significant increases in IgG anti-Ro/SS-A and IgM anti-CENP-B antibodies in AMR/mixed rejection compared with ACR were reproduced in an external cohort of 60 kidney transplant patients. CONCLUSIONS: This is the first study implicating autoantibodies anti-Ro/SS-A and anti-CENP-B in AMR. These antibodies may participate in the crosstalk between autoimmunity and alloimmunity in kidney AMR.

Transcriptome Analysis of Kidney Grafts Subjected to Normothermic Ex Vivo Perfusion Demonstrates an Enrichment of Mitochondrial Metabolism Genes.

Normothermic ex vivo kidney perfusion (NEVKP) has demonstrated superior outcomes for donation-after-cardiovascular death grafts compared with static cold storage (SCS). To determine the mechanisms responsible for this, we performed an unbiased genome-wide microarray analysis. METHODS: Kidneys from 30-kg Yorkshire pigs were subjected to 30 min of warm ischemia followed by 8 h of NEVKP or SCS, or no storage, before autotransplantation. mRNA expression was analyzed on renal biopsies on postoperative day 3. Gene set enrichment analysis was performed using hallmark gene sets, Gene Ontology, and pathway analysis. RESULTS: The gene expression profile of NEVKP-stored grafts closely resembled no storage kidneys. Gene set enrichment analysis demonstrated enrichment of fatty acid metabolism and oxidative phosphorylation following NEVKP, whereas SCS-enriched gene sets were related to mitosis, cell cycle checkpoint, and reactive oxygen species (q < 0.05). Pathway analysis demonstrated enrichment of lipid oxidation/metabolism, the Krebs cycle, and pyruvate metabolism in NEVKP compared with SCS (q < 0.05). Comparison of our findings with external data sets of renal ischemia-reperfusion injury revealed that SCS-stored grafts demonstrated similar gene expression profiles to ischemia-reperfusion injury, whereas the profile of NEVKP-stored grafts resembled recovered kidneys. CONCLUSIONS: Increased transcripts of key mitochondrial metabolic pathways following NEVKP storage may account for improved donation-after-cardiovascular death graft function, compared with SCS, which promoted expression of genes typically perturbed during IRI.

Normothermic Ex-vivo Kidney Perfusion in a Porcine Auto-Transplantation Model Preserves the Expression of Key Mitochondrial Proteins: An Unbiased Proteomics Analysis.

Normothermic ex-vivo kidney perfusion (NEVKP) results in significantly improved graft function in porcine auto-transplant models of donation after circulatory death injury compared with static cold storage (SCS); however, the molecular mechanisms underlying these beneficial effects remain unclear. We performed an unbiased proteomics analysis of 28 kidney biopsies obtained at three time points from pig kidneys subjected to 30 min of warm ischemia, followed by 8 h of NEVKP or SCS, and auto-transplantation. 70/6593 proteins quantified were differentially expressed between NEVKP and SCS groups (false discovery rate < 0.05). Proteins increased in NEVKP mediated key metabolic processes including fatty acid ß-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation. Comparison of our findings with external datasets of ischemia-reperfusion and other models of kidney injury confirmed that 47 of our proteins represent a common signature of kidney injury reversed or attenuated by NEVKP. We validated key metabolic proteins (electron transfer flavoprotein subunit beta and carnitine O-palmitoyltransferase 2, mitochondrial) by immunoblotting. Transcription factor databases identified members of the peroxisome proliferator-activated receptors (PPAR) family of transcription factors as the upstream regulators of our dataset, and we confirmed increased expression of PPARA, PPARD, and RXRA in NEVKP with reverse transcription polymerase chain reaction. The proteome-level changes observed in NEVKP mediate critical metabolic pathways. These effects may be coordinated by PPAR-family transcription factors and may represent novel therapeutic targets in ischemia-reperfusion injury.

Extracellular Matrix Injury of Kidney Allografts in Antibody-Mediated Rejection: A Proteomics Study.

BACKGROUND: Antibody-mediated rejection (AMR) accounts for >50% of kidney allograft loss. Donor-specific antibodies (DSA) against HLA and non-HLA antigens in the glomeruli and the tubulointerstitium cause AMR while inflammatory cytokines such as TNFα trigger graft injury. The mechanisms governing cell-specific injury in AMR remain unclear. METHODS: Unbiased proteomic analysis of laser-captured and microdissected glomeruli and tubulointerstitium was performed on 30 for-cause kidney biopsy specimens with early AMR, acute cellular rejection (ACR), or acute tubular necrosis (ATN). RESULTS: A total of 107 of 2026 glomerular and 112 of 2399 tubulointerstitial proteins was significantly differentially expressed in AMR versus ACR; 112 of 2026 glomerular and 181 of 2399 tubulointerstitial proteins were significantly dysregulated in AMR versus ATN (P<0.05). Basement membrane and extracellular matrix (ECM) proteins were significantly decreased in both AMR compartments. Glomerular and tubulointerstitial laminin subunit γ-1 (LAMC1) expression decreased in AMR, as did glomerular nephrin (NPHS1) and receptor-type tyrosine-phosphatase O (PTPRO). The proteomic analysis revealed upregulated galectin-1, which is an immunomodulatory protein linked to the ECM, in AMR glomeruli. Anti-HLA class I antibodies significantly increased cathepsin-V (CTSV) expression and galectin-1 expression and secretion in human glomerular endothelial cells. CTSV had been predicted to cleave ECM proteins in the AMR glomeruli. Glutathione S-transferase ω-1, an ECM-modifying enzyme, was significantly increased in the AMR tubulointerstitium and in TNFα-treated proximal tubular epithelial cells. CONCLUSIONS: Basement membranes are often remodeled in chronic AMR. Proteomic analysis performed on laser-captured and microdissected glomeruli and tubulointerstitium identified early ECM remodeling, which may represent a new therapeutic opportunity.

Spatial and Temporal Clustering of Anti-Glomerular Basement Membrane Disease.

BACKGROUND AND OBJECTIVES: An environmental trigger has been proposed as an inciting factor in the development of anti-GBM disease. This multicenter, observational study sought to define the national incidence of anti-GBM disease during an 11-year period (2003-2014) in Ireland, investigate clustering of cases in time and space, and assess the effect of spatial variability in incidence on outcome. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We ascertained cases by screening immunology laboratories for instances of positivity for anti-GBM antibody and the national renal histopathology registry for biopsy-proven cases. The population at risk was defined from national census data. We used a variable-window scan statistic to detect temporal clustering. A Bayesian spatial model was used to calculate standardized incidence ratios (SIRs) for each of the 26 counties. RESULTS: Seventy-nine cases were included. National incidence was 1.64 (95% confidence interval [95% CI], 0.82 to 3.35) per million population per year. A temporal cluster (n=10) was identified during a 3-month period; six cases were resident in four rural counties in the southeast. Spatial analysis revealed wide regional variation in SIRs and a cluster (n=7) in the northwest (SIR, 1.71; 95% CI, 1.02 to 3.06). There were 29 deaths and 57 cases of ESRD during a mean follow-up of 2.9 years. Greater distance from diagnosis site to treating center, stratified by median distance traveled, did not significantly affect patient (hazard ratio, 1.80; 95% CI, 0.87 to 3.77) or renal (hazard ratio, 0.76; 95% CI, 0.40 to 1.13) survival. CONCLUSIONS: To our knowledge, this is the first study to report national incidence rates of anti-GBM disease and formally investigate patterns of incidence. Clustering of cases in time and space supports the hypothesis of an environmental trigger for disease onset. The substantial variability in regional incidence highlights the need for comprehensive country-wide studies to improve our understanding of the etiology of anti-GBM disease.

Lipoxins attenuate renal fibrosis by inducing let-7c and suppressing TGFßR1.

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA4) attenuated TGF-ß1-induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-ß1 suppressed expression of let-7c. In cells pretreated with LXA4, upregulation of let-7c persisted despite subsequent stimulation with TGF-ß1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA4 analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-ß1 signaling pathway, including the TGF-ß receptor type 1. Consistent with this, LXA4-induced upregulation of let-7c inhibited both the expression of TGF-ß receptor type 1 and the response to TGF-ß1. Overexpression of let-7c mimicked the antifibrotic effects of LXA4 in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA4. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA4-mediated upregulation of let-7c suppresses TGF-ß1-induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.