Sexually dimorphic renal expression of Klotho is directed by a kidney-specific distal enhancer responsive to HNF1b.

Transcription enhancers are genomic sequences regulating common and tissue-specific genes and their disruption can contribute to human disease development and progression. Klotho, a sexually dimorphic gene specifically expressed in kidney, is well-linked to kidney dysfunction and its deletion from the mouse genome leads to premature aging and death. However, the sexually dimorphic regulation of Klotho is not understood. Here, we characterize two candidate Klotho enhancers using H3K27ac epigenetic marks and transcription factor binding and investigate their functions, individually and combined, through CRISPR-Cas9 genome engineering. We discovered that only the distal (E1), but not the proximal (E2) candidate region constitutes a functional enhancer, with the double deletion not causing Klotho expression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism of Klotho as evidenced by qPCR and RNA-seq. Despite the sharp reduction of Klotho mRNA, unlike germline Klotho knockouts, mutant mice presented normal phenotype, including weight, lifespan, and serum biochemistry. Lastly, only males lacking E1 display more prominent acute, but not chronic kidney injury responses, indicating a remarkable range of potential adaptation to isolated Klotho loss, especially in female E1 knockouts, retaining renoprotection despite over 80% Klotho reduction.

Sexually dimorphic renal expression of Klotho is directed by a kidney-specific distal enhancer responsive to HNF1b.

Transcription enhancers are genomic sequences regulating common and tissue-specific genes and their disruption can contribute to human disease development and progression. Klotho, a sexually dimorphic gene specifically expressed in kidney, is well-linked to kidney dysfunction and its deletion from the mouse genome leads to premature aging and death. However, the sexually dimorphic regulation of Klotho is not understood. Here, we characterize two candidate Klotho enhancers using H3K27ac epigenetic marks and transcription factor binding and investigate their functions, individually and combined, through CRISPR-Cas9 genome engineering. We discovered that only the distal (E1), but not the proximal (E2) candidate region constitutes a functional enhancer, with the double deletion not causing Klotho expression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism of Klotho as evidenced by qPCR and RNA-seq. Despite the sharp reduction of Klotho mRNA, unlike germline Klotho knockouts, mutant mice presented normal phenotype, including weight, lifespan, and serum biochemistry. Lastly, only males lacking E1 display more prominent acute, but not chronic kidney injury responses, indicating a remarkable range of potential adaptation to isolated Klotho loss, especially in female E1 knockouts, retaining renoprotection despite over 80% Klotho reduction.

Indian Hedgehog release from TNF-activated renal epithelia drives local and remote organ fibrosis.

Progressive fibrosis is a feature of aging and chronic tissue injury in multiple organs, including the kidney and heart. Glioma-associated oncogene 1 expressing (Gli1+) cells are a major source of activated fibroblasts in multiple organs, but the links between injury, inflammation, and Gli1+ cell expansion and tissue fibrosis remain incompletely understood. We demonstrated that leukocyte-derived tumor necrosis factor (TNF) promoted Gli1+ cell proliferation and cardiorenal fibrosis through induction and release of Indian Hedgehog (IHH) from renal epithelial cells. Using single-cell-resolution transcriptomic analysis, we identified an "inflammatory" proximal tubular epithelial (iPT) population contributing to TNF- and nuclear factor κB (NF-κB)-induced IHH production in vivo. TNF-induced Ubiquitin D (Ubd) expression was observed in human proximal tubular cells in vitro and during murine and human renal disease and aging. Studies using pharmacological and conditional genetic ablation of TNF-induced IHH signaling revealed that IHH activated canonical Hedgehog signaling in Gli1+ cells, which led to their activation, proliferation, and fibrosis within the injured and aging kidney and heart. These changes were inhibited in mice by Ihh deletion in Pax8-expressing cells or by pharmacological blockade of TNF, NF-κB, or Gli1 signaling. Increased amounts of circulating IHH were associated with loss of renal function and higher rates of cardiovascular disease in patients with chronic kidney disease. Thus, IHH connects leukocyte activation to Gli1+ cell expansion and represents a potential target for therapies to inhibit inflammation-induced fibrosis.

JAK inhibitors dampen activation of interferon-activated transcriptomes and the SARS-CoV-2 receptor ACE2 in human renal proximal tubules.

SARS-CoV-2 infections initiate cytokine storms and activate genetic programs leading to progressive hyperinflammation in multiple organs of patients with COVID-19. While it is known that COVID-19 impacts kidney function, leading to increased mortality, cytokine response of renal epithelium has not been studied in detail. Here, we report on the genetic programs activated in human primary proximal tubule (HPPT) cells by interferons and their suppression by ruxolitinib, a Janus kinase (JAK) inhibitor used in COVID-19 treatment. Integration of our data with those from patients with acute kidney injury and COVID-19, as well as other tissues, permitted the identification of kidney-specific interferon responses. Additionally, we investigated the regulation of the recently discovered isoform (dACE2) of the angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. Using ChIP-seq, we identified candidate interferon-activated enhancers controlling the ACE2 locus, including the intronic dACE2 promoter. Taken together, our study provides an in-depth understanding of genetic programs activated in kidney cells.

Orphan nuclear receptor COUP-TFII enhances myofibroblast glycolysis leading to kidney fibrosis.

Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.

Interferon-regulated genetic programs and JAK/STAT pathway activate the intronic promoter of the short ACE2 isoform in renal proximal tubules.

Recently, a short, interferon-inducible isoform of Angiotensin-Converting Enzyme 2 (ACE2), dACE2 was identified. ACE2 is a SARS-Cov-2 receptor and changes in its renal expression have been linked to several human nephropathies. These changes were never analyzed in context of dACE2, as its expression was not investigated in the kidney. We used Human Primary Proximal Tubule (HPPT) cells to show genome-wide gene expression patterns after cytokine stimulation, with emphasis on the ACE2/dACE2 locus. Putative regulatory elements controlling dACE2 expression were identified using ChIP-seq and RNA-seq. qRT-PCR differentiating between ACE2 and dACE2 revealed 300- and 600-fold upregulation of dACE2 by IFNα and IFNß, respectively, while full length ACE2 expression was almost unchanged. JAK inhibitor ruxolitinib ablated STAT1 and dACE2 expression after interferon treatment. Finally, with RNA-seq, we identified a set of genes, largely immune-related, induced by cytokine treatment. These gene expression profiles provide new insights into cytokine response of proximal tubule cells.

Enhancer and super-enhancer dynamics in repair after ischemic acute kidney injury.

The endogenous repair process can result in recovery after acute kidney injury (AKI) with adaptive proliferation of tubular epithelial cells, but repair can also lead to fibrosis and progressive kidney disease. There is currently limited knowledge about transcriptional regulators regulating these repair programs. Herein we establish the enhancer and super-enhancer landscape after AKI by ChIP-seq in uninjured and repairing kidneys on day two after ischemia reperfusion injury (IRI). We identify key transcription factors including HNF4A, GR, STAT3 and STAT5, which show specific binding at enhancer and super-enhancer sites, revealing enhancer dynamics and transcriptional changes during kidney repair. Loss of bromodomain-containing protein 4 function before IRI leads to impaired recovery after AKI and increased mortality. Our comprehensive analysis of epigenetic changes after kidney injury in vivo has the potential to identify targets for therapeutic intervention. Importantly, our data also call attention to potential caveats involved in use of BET inhibitors in patients at risk for AKI.

Interleukin-1ß Activates a MYC-Dependent Metabolic Switch in Kidney Stromal Cells Necessary for Progressive Tubulointerstitial Fibrosis.

Background Kidney injury is characterized by persisting inflammation and fibrosis, yet mechanisms by which inflammatory signals drive fibrogenesis remain poorly defined.Methods RNA sequencing of fibrotic kidneys from patients with CKD identified a metabolic gene signature comprising loss of mitochondrial and oxidative phosphorylation gene expression with a concomitant increase in regulators and enzymes of glycolysis under the control of PGC1α and MYC transcription factors, respectively. We modeled this metabolic switch in vivo, in experimental murine models of kidney injury, and in vitro in human kidney stromal cells (SCs) and human kidney organoids.Results In mice, MYC and the target genes thereof became activated in resident SCs early after kidney injury, suggesting that acute innate immune signals regulate this transcriptional switch. In vitro, stimulation of purified human kidney SCs and human kidney organoids with IL-1ß recapitulated the molecular events observed in vivo, inducing functional metabolic derangement characterized by increased MYC-dependent glycolysis, the latter proving necessary to drive proliferation and matrix production. MYC interacted directly with sequestosome 1/p62, which is involved in proteasomal degradation, and modulation of p62 expression caused inverse effects on MYC expression. IL-1ß stimulated autophagy flux, causing degradation of p62 and accumulation of MYC. Inhibition of the IL-1R signal transducer kinase IRAK4 in vivo or inhibition of MYC in vivo as well as in human kidney organoids in vitro abrogated fibrosis and reduced tubular injury.Conclusions Our findings define a connection between IL-1ß and metabolic switch in fibrosis initiation and progression and highlight IL-1ß and MYC as potential therapeutic targets in tubulointerstitial diseases.

miR-182-5p Inhibition Ameliorates Ischemic Acute Kidney Injury.

Acute kidney injury (AKI) remains a major clinical event with high mortality rates. We previously identified renal miR-182 as the main driver of post-transplantation AKI. Therefore, we tested the causal inference of miR-182 by inhibiting its renal expression in vivo. In 45 rats AKI was induced by right nephrectomy and contralateral clamping of the renal pedicle for 40 minutes. Systemically administered antisense oligonucleotide (ASO) inhibited miR-182 in the kidneys up to 96 hours. The maximum creatinine elevation was on day 2 after injury (mg/dL; median and interquartile range): ASO 2.5mg/kg: 1.9 (1.3; 3.2), ASO 25mg/kg: 2.8 (0.7; 5.0), mismatch oligonucleotide (MM) 25mg/kg: 5.7 (5,0; 5.8), saline: 4.4 (3.5; 5.8) (P = 0.016, analysis of variance). Blinded semiquantitative histologic evaluation of renal biopsies showed better preserved morphology in both ASO groups than saline- and MM-treated kidneys (median and interquartile range of overall injury scores): ASO both concentrations 1 (1, 1), saline 3 (3, 3) and MM 3 (3, 3) (P< 0.001, analysis of variance). ASO facilitated cell proliferation, metabolism, and angiogenesis on a genome-wide level. ASO when applied in normothermic kidney machine perfusion reduced renal miR-182 expression by more than two magnitudes. In summary, we showed that in vivo inhibition of miR-182 by ASO improved kidney function and morphology after AKI. This technique may be applicable to reduce the high rate of AKI in the human renal transplantation setting.

Compound heterozygosity of two novel RHAG alleles leads to a considerable disruption of the Rh complex.

BACKGROUND: The Rhesus (Rh) complex consists of a core comprising the Rh proteins (RhD/RhCE) and the Rh-associated glycoprotein (RhAG) with accessory chains (GPB, LW, CD47). Molecular defects of the RHAG gene may cause a regulator Rhnull phenotype without Rh antigen expression or a Rhmod phenotype with decreased Rh antigen expression. STUDY DESIGN AND METHODS: Blood samples of a donor with strongly diminished Rh antigens and five family members were analyzed by serological phenotyping, flow cytometry, molecular testing, and gene expression analysis of Rh complex candidate genes. RESULTS: RHAG sequencing identified a missense mutation, c.241G>C (p.Gly81Arg) and a splice site mutation, c.640 + 3del14, among the cohort. Compound heterozygosity of these novel alleles identified in the propositus and two siblings gave rise to a strongly diminished expression of RhAG, Rh, and CD47 antigens on the RBC surface. CONCLUSION: The Rhmod phenotype was caused by a novel RHAG splice site mutation in association with a non-functional allele. The primary depression of RhAG is most likely due to posttranslational events that affect the interaction and processing of the RhAG glycoprotein and gave rise to a secondary depression of RhD, RhCE, and CD47, the major members of the Rh complex.

MicroRNAs in kidney transplantation.

The discovery of novel classes of non-coding RNAs (ncRNAs) has revolutionized medicine. Long thought to be a mere cellular housekeeper, surprising functions have recently been uncovered. MicroRNAs (miRNAs), are a representative of the class of short ncRNAs, play a fundamental role in the control of DNA and protein biosynthesis and activity as well as pathology. Currently, miRNAs are being investigated as diagnostic and prognostic markers and potential therapeutic targets in kidney transplantation for such indolent processes as ischaemia-reperfusion injury, humoral rejection or viral infections. It is realistic to believe that monitoring of renal allograft recipients in the future will include genome-wide miRNA profiling of biological fluids. Based on these individual profiles, an informed decision on therapeutic consequences will be possible. A first success with a specific suppression of miRNAs by antisense oligonucleotides was achieved in experimental studies of reperfusion injury and humoral rejection. Proof of this concept in men comes from studies in such indolent viral infections as Ebola and hepatitis C, where anti-miR therapy led to sustained viral clearance. In this review, we summarize the basis of the recent ncRNA revolution and its implication for kidney transplantation.

Molecular pathogenesis of post-transplant acute kidney injury: assessment of whole-genome mRNA and miRNA profiles.

Acute kidney injury (AKI) affects roughly 25% of all recipients of deceased donor organs. The prevention of post-transplant AKI is still an unmet clinical need. We prospectively collected zero-hour, indication as well as protocol kidney biopsies from 166 allografts between 2011 and 2013. In this cohort eight cases with AKI and ten matched allografts without pathology serving as control group were identified with a follow-up biopsy within the first twelve days after engraftment. For this set the zero-hour and follow-up biopsies were subjected to genome wide microRNA and mRNA profiling and analysis, followed by validation in independent expression profiles of 42 AKI and 21 protocol biopsies for strictly controlling the false discovery rate. Follow-up biopsies of AKI allografts compared to time-matched protocol biopsies, further baseline adjustment for zero-hour biopsy expression level and validation in independent datasets, revealed a molecular AKI signature holding 20 mRNAs and two miRNAs (miR-182-5p and miR-21-3p). Next to several established biomarkers such as lipocalin-2 also novel candidates of interest were identified in the signature. In further experimental evaluation the elevated transcript expression level of the secretory leukocyte peptidase inhibitor (SLPI) in AKI allografts was confirmed in plasma and urine on the protein level (p<0.001 and p = 0.003, respectively). miR-182-5p was identified as a molecular regulator of post-transplant AKI, strongly correlated with global gene expression changes during AKI. In summary, we identified an AKI-specific molecular signature providing the ground for novel biomarkers and target candidates such as SLPI and miR-182-5p in addressing AKI.

Left atrial diameter and survival among renal allograft recipients.

BACKGROUND AND OBJECTIVES: Sequential echocardiography is routinely performed in patients with ESRD listed for transplantation. The benefit of this labor- and time-intensive measure, however, remains unclear. Thus, this study elucidated the various obtained routine echocardiography parameters that best predicted mortality and graft survival after renal transplantation. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This study investigated 553 first renal transplant recipients listed in the Austrian Dialysis and Transplant Registry between 1992 and 2011 who had echocardiographic analysis at transplantation and survived at least 1 year. Cox proportional hazards models with the purposeful selection algorithms for covariables were used to identify predictors of mortality and graft loss. A Fine and Gray model was used to evaluate cause-specific death. RESULTS: During a median follow-up of 7.14 years, 81 patients died, and 59 patients experienced graft loss after the first year. The Kaplan-Meier analysis showed that 85% of patients with a left atrial diameter below the median of 53 mm were alive 10 years after transplantation, whereas only 70% of those patients with a left atrial diameter equal to or above the median had survived (P<0.001). In the multivariable model, left atrial diameter (per millimeter) independently predicted overall mortality (hazard ratio, 1.06; 95% confidence interval, 1.03 to 1.08; P<0.001) and cause-specific cardiac death (hazard ratio, 1.04; 95% confidence interval, 1.00 to 1.08; P=0.04). Functional graft loss was predicted by the right atrial diameter (hazard ratio, 1.04; 95% confidence interval, 1.02 to 1.07; P=0.001). CONCLUSION: The left atrial diameter determined at transplantation predicted overall and cardiac mortality. Patients with widely enlarged left atria exhibit a considerably reduced life expectancy. It remains to be determined, however, whether renal transplantation is futile in these patients.

miRNA profiling discriminates types of rejection and injury in human renal allografts.

BACKGROUND: Increasing evidence accumulates on the central involvement of microRNAs (miRNAs) in disease pathophysiology. We identified distinctly deregulated miRNAs in human renal allograft biopsies from patients undergoing acute cellular rejection, antibody-mediated rejection (ABMR), and delayed graft function (DGF). METHODS: Sixty-five posttransplantation kidney biopsy samples covering 41 cases with acute rejection (15 vascular rejection, 15 interstitial rejection, and 11 ABMR), 14 DGF cases, and 10 protocol biopsies serving as controls were analyzed using the Affymetrix GeneChip miRNA Array. Differentially regulated miRNAs were identified by Student's t test and Bonferroni correction. Target genes for the set of miRNAs were retrieved from miRTarBase (experimentally verified targets) as well as by applying the target prediction routines DIANAmT, miRanda, and Targetscan. RESULTS: Patients with acute cellular rejection, ABMR, and DGF discriminate from the control group (protocol biopsies) in unsupervised clustering of miRNA profiles, clearly identifying deregulated miRNAs in rejection and DGF. Angiogenesis, apoptosis, and transforming growth factor-ß signaling were identified as relevant pathways in ischemic response following an integrative analysis of miRNA targets and mRNA expression profiles. Inflammation by chemokine and cytokine signaling, T-cell activation, and B-cell activation were identified as relevant in acute rejection accordingly. CONCLUSION: These data suggest that distinct miRNA signatures playing a role in specific biological pathways discriminate acute cellular and humoral rejection and DGF. This finding serves as valuable tool for a rational selection of diagnostic, prognostic, and potentially therapeutic molecular targets of posttransplantation events.

Sirolimus induced phosphaturia is not caused by inhibition of renal apical sodium phosphate cotransporters.

The vast majority of glomerular filtrated phosphate is reabsorbed in the proximal tubule. Posttransplant phosphaturia is common and aggravated by sirolimus immunosuppression. The cause of sirolimus induced phosphaturia however remains elusive. Male Wistar rats received sirolimus or vehicle for 2 or 7 days (1.5mg/kg). The urine phosphate/creatinine ratio was higher and serum phosphate was lower in sirolimus treated rats, fractional excretion of phosphate was elevated and renal tubular phosphate reabsorption was reduced suggesting a renal cause for hypophosphatemia. PTH was lower in sirolimus treated rats. FGF 23 levels were unchanged at day 2 but lower in sirolimus treated rats after 7 days. Brush border membrane vesicle phosphate uptake was not altered in sirolimus treated groups or by direct incubation with sirolimus. mRNA, protein abundance, and subcellular transporter distribution of NaPi-IIa, Pit-2 and NHE3 were not different between groups but NaPi-IIc mRNA expression was lower at day 7. Transcriptome analyses revealed candidate genes that could be involved in the phosphaturic response. Sirolimus caused a selective renal phosphate leakage, which was not mediated by NaPi-IIa or NaPi-IIc regulation or localization. We hypothesize that another mechanism such as a basolateral phosphate transporter may be responsible for the sirolimus induced phosphaturia.

Hemoglobin variability after renal transplantation is associated with mortality.

Anemia is a common problem after renal transplantation. Therefore, the patients are treated with erythropoietin stimulating agents (ESAs). The varying response to treatment contributes to hemoglobin variability, which might be associated with mortality. We conducted a retrospective cohort study of first kidney allograft recipients between 1990 and 2008 represented in the Austrian Transplant Registry. We included 1441 patients of whom 683 received ESAs at any time after transplantation. Cox regression with cubic splines and linear estimates and the purposeful selection algorithm of covariables were used. The measure of variability was the moving standard deviation computed at three monthly intervals for the entire graft life. The hazard ratio (HR) of mortality and graft loss in the spline models increased with hemoglobin variability. The linear HR for mortality was 2.35 (95% confidence interval 1.75-3.17, P<0.001) and functional graft loss 2.45 (1.76-3.40, P<0.001). In an adjusted Cox model (ESA use, hemoglobin, age, diabetes, days on dialysis, eGFR, biopsy confirmed acute rejection and year of transplantation), hemoglobin variability was associated with mortality (HR: 2.11; 1.51-2.94; P<0.001). No association with functional graft loss could be detected (HR: 1.34; 0.93-1.93; P=0.121). These findings suggest that hemoglobin variability is associated with mortality of renal allograft recipients.

The effect of steroid pretreatment of deceased organ donors on liver allograft function: a blinded randomized placebo-controlled trial.

BACKGROUND & AIMS: Brain death-associated inflammatory response contributes to increased risk of impaired early liver allograft function, which might be counterbalanced by steroid pretreatment of the organ donor. The aim of this randomized controlled trial was to elucidate whether steroid pretreatment of liver donors improves early liver allograft function, prevents rejection and prolongs survival. METHODS: A placebo-controlled blinded randomized clinical trial was performed in three different centers in Austria and Hungary between 2006 and 2008. Ninety deceased organ donors received either 1000 mg of methylprednisolone or placebo 6h before recovery of organs. The primary end point was the concentration slope of transaminases within the first week. The secondary end point included survival and biopsy-confirmed acute rejection (BCAR) within 3 years after transplantation. RESULTS: Of the 90 randomized donors, 83 recipients were eligible for study. The trajectories of ALT and AST were not different between treatments (p=0.40 and p=0.13, respectively). Eight subjects died in the steroid and 13 in the placebo group within 3 years after engraftment (RR=0.63 95% CI [0.29,1.36], p=0.31). Eleven recipients experienced biopsy-confirmed rejection (BCAR) in the steroid and 11 in the placebo group (RR=1.02 95% CI [0.50,2.10], p=1.00). No effect modification could be identified in the predefined strata of donor age, sex, cold ischemic time, and cause of donor death. CONCLUSIONS: Steroid pretreatment of organ donors did not improve outcomes after liver transplantation.

Effect of intraportal infusion of tacrolimus on ischaemic reperfusion injury in orthotopic liver transplantation: a randomized controlled trial.

The increased use of older and/or marginal donor organs in liver transplantation over the last decade calls for strategies to minimize ischaemic reperfusion (I/R) injury to prevent early graft failure. Tacrolimus, a very potent and effective calcineurin inhibitor, was selected because of its ability to ameliorate I/R injury. A randomized, blinded, controlled single-centre trial of 26 liver transplant recipients was performed between February 2008 and December 2009. Donor organs were randomized to be perfused intraportally during liver transplantation with 1.5 l 5% albumin infusion containing either 20 ng/ml tacrolimus or placebo. The primary end point was liver function as assessed by aspartate transaminase (AST) or alanine transaminase (ALT) levels 6 days after transplantation. Treatment effectiveness was tested by transcriptome-wide analysis of biopsies. There was no difference in the primary end point, i.e. AST (IU/l) and ALT (IU/l) at day 6 after transplantation between groups. Furthermore, choleastatic parameters as well as parameters of liver synthesis were not different between groups. However, tacrolimus treatment suppressed inflammation and immune response in the transplanted liver on a genome-wide basis. Intrahepatic administration of tacrolimus did not result in a reduction of AST and ALT within the first week after transplantation.

Computational analysis workflows for Omics data interpretation.

Progress in experimental procedures has led to rapid availability of Omics profiles. Various open-access as well as commercial tools have been developed for storage, analysis, and interpretation of transcriptomics, proteomics, and metabolomics data. Generally, major analysis steps include data storage, retrieval, preprocessing, and normalization, followed by identification of differentially expressed features, functional annotation on the level of biological processes and molecular pathways, as well as interpretation of gene lists in the context of protein-protein interaction networks. In this chapter, we discuss a sequential transcriptomics data analysis workflow utilizing open-source tools, specifically exemplified on a gene expression dataset on familial hypercholesterolemia.

Molecular biomarker candidates of acute kidney injury in zero-hour renal transplant needle biopsies.

The aim of this study was to assess gene expression levels of four biomarker candidates [lipocalin 2 (LCN2), the kidney injury molecule 1 (HAVCR1), netrin 1, and the cysteine-rich, angiogenic inducer, 61] in the tubulointerstitial and the glomerular compartment of zero-hour kidney biopsies in order to predict developing delayed graft function (DGF). Thirty-four needle kidney biopsy samples of deceased donors were manually microdissected. Relative gene expression levels were determined by real-time RT-PCR. For the validation of the biomarker candidates, we calculated a mixed model comparing kidneys with DGF, primary function and control samples from the healthy parts of tumor nephrectomies. Significant biomarker candidates were analyzed together with donor age in multivariable regression models to determine the prognostic value. Expression levels of LCN2 and HAVCR1 in the tubulointerstitium were significantly upregulated in the DGF group (LCN2: fold change = 3.78, P = 0.031 and HAVCR1: fold change = 3.44, P = 0.010). Odds ratios of both genes could not reach significance in the multivariable model together with donor age. The area under the curve of the receiver operating characteristic ranges between 0.75 and 0.83. LCN2 and HAVCR1 gene expression levels in zero-hour biopsies show potential to act as early biomarkers for DGF.