Assessment and Risk Prediction of Chronic Kidney Disease and Kidney Fibrosis Using Non-Invasive Biomarkers.

Effective management of chronic kidney disease (CKD), a major health problem worldwide, requires accurate and timely diagnosis, prognosis of progression, assessment of therapeutic efficacy, and, ideally, prediction of drug response. Multiple biomarkers and algorithms for evaluating specific aspects of CKD have been proposed in the literature, many of which are based on a small number of samples. Based on the evidence presented in relevant studies, a comprehensive overview of the different biomarkers applicable for clinical implementation is lacking. This review aims to compile information on the non-invasive diagnostic, prognostic, and predictive biomarkers currently available for the management of CKD and provide guidance on the application of these biomarkers. We specifically focus on biomarkers that have demonstrated added value in prospective studies or those based on prospectively collected samples including at least 100 subjects. Published data demonstrate that several valid non-invasive biomarkers of potential value in the management of CKD are currently available.

Non-Invasive Biomarkers for Diagnosis, Risk Prediction, and Therapy Guidance of Glomerular Kidney Diseases: A Comprehensive Review.

Effective management of glomerular kidney disease, one of the main categories of chronic kidney disease (CKD), requires accurate diagnosis, prognosis of progression, assessment of therapeutic efficacy, and, ideally, prediction of drug response. Multiple biomarkers and algorithms for the assessment of specific aspects of glomerular diseases have been reported in the literature. Though, the vast majority of these have not been implemented in clinical practice or are not available on a global scale due to limited access, missing medical infrastructure, or economical as well as political reasons. The aim of this review is to compile all currently available information on the diagnostic, prognostic, and predictive biomarkers currently available for the management of glomerular diseases, and provide guidance on the application of these biomarkers. As a result of the compiled evidence for the different biomarkers available, we present a decision tree for a non-invasive, biomarker-guided diagnostic path. The data currently available demonstrate that for the large majority of patients with glomerular diseases, valid biomarkers are available. However, despite the obvious disadvantages of kidney biopsy, being invasive and not applicable for monitoring, especially in the context of rare CKD etiologies, kidney biopsy still cannot be replaced by non-invasive strategies.

Deficiency of Complement C3a and C5a receptors Does Not Prevent Angiotensin II-Induced Hypertension and Hypertensive End-Organ Damage.

BACKGROUND: Complement may drive the pathology of hypertension through effects on innate and adaptive immune responses. Recently an injurious role for the anaphylatoxin receptors C3aR (complement component 3a receptor) and C5aR1 (complement component 5a receptor) in the development of hypertension was shown through downregulation of Foxp3+ (forkhead box protein 3) regulatory T cells. Here, we deepen our understanding of the therapeutic potential of targeting both receptors in hypertension. METHODS: Data from the European Renal cDNA Bank, single cell sequencing and immunohistochemistry were examined in hypertensive patients. The effect of C3aR or C3aR/C5aR1 double deficiency was assessed in two models of Ang II (angiotensin II)-induced hypertension in knockout mice. RESULTS: We found increased expression of C3aR, C5aR1 and Foxp3 cells in kidney biopsies of patients with hypertensive nephropathy. Expression of both receptors was mainly found in myeloid cells. No differences in blood pressure, renal injury (albuminuria, glomerular filtration rate, glomerular and tubulointerstitial injury, inflammation) or cardiac injury (cardiac fibrosis, heart weight, gene expression) between control and mutant mice was discerned in C3aR-/- as well as C3aR/C5aR1-/- double knockout mice. The number of renal Tregs was not decreased in Ang II as well as in DOCA salt induced hypertension. CONCLUSIONS: Hypertensive nephropathy in mice and men is characterized by an increase of renal regulatory T cells and enhanced expression of anaphylatoxin receptors. Our investigations do not corroborate a role for C3aR/C5aR1 axis in Ang II-induced hypertension hence challenging the concept of anaphylatoxin receptor targeting in the treatment of hypertensive disease.

An integrated organoid omics map extends modeling potential of kidney disease.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

Cold Shock Domain Protein DbpA Orchestrates Tubular Cell Damage and Interstitial Fibrosis in Inflammatory Kidney Disease.

DNA-binding protein A (DbpA) belongs to the Y-box family of cold shock domain proteins that exert transcriptional and translational activities in the cell via their ability to bind and regulate mRNA. To investigate the role of DbpA in kidney disease, we utilized the murine unilateral ureter obstruction (UUO) model, which recapitulates many features of obstructive nephropathy seen in humans. We observed that DbpA protein expression is induced within the renal interstitium following disease induction. Compared with wild-type animals, obstructed kidneys from Ybx3-deficient mice are protected from tissue injury, with a significant reduction in the number of infiltrating immune cells as well as in extracellular matrix deposition. RNAseq data from UUO kidneys show that Ybx3 is expressed by activated fibroblasts, which reside within the renal interstitium. Our data support a role for DbpA in orchestrating renal fibrosis and suggest that strategies targeting DbpA may be a therapeutic option to slow disease progression.

Complement component C3 as a new target to lower albuminuria in hypertensive kidney disease.

BACKGROUND AND PURPOSE: Complement activation may drive hypertension through its effects on immunity and tissue integrity. EXPERIMENTAL APPROACH: We examined expression of C3, the central protein of the complement cascade, in hypertension. KEY RESULTS: Increased C3 expression was found in kidney biopsies and micro-dissected glomeruli of patients with hypertensive nephropathy. Renal single cell RNA sequence data from normotensive and hypertensive patients confirmed expression of C3 in different cellular compartments of the kidney. In angiotensin II (Ang II) induced hypertension renal C3 expression was up-regulated. C3-/- mice revealed a significant lower albuminuria in the early phase of hypertension. However, no difference was found for blood pressure, renal injury (histology, glomerular filtration rate, inflammation) and cardiac injury (fibrosis, weight, gene expression) between C3-/- and wildtype mice after Ang II infusion. Also, in deoxycorticosterone acetate (DOCA) salt hypertension, a significantly lower albuminuria was found in the first weeks of hypertension in C3 deficient mice but no significant difference in renal and cardiac injury. Down-regulation of C3 by C3 targeting GalNAc (n-acetylgalactosamine) small interfering RNA (siRNA) conjugate decreased C3 in the liver by 96% and lowered albuminuria in the early phase but showed no effect on blood pressure and end-organ damage. Inhibition of complement C5 by siRNA showed no effect on albuminuria. CONCLUSION AND IMPLICATIONS: Increased C3 expression is found in the kidneys of hypertensive mice and men. Genetic and therapeutic knockdown of C3 improved albuminuria in the early phase of hypertension but did not ameliorate arterial blood pressure nor renal and cardiac injury.

Down-regulation of human long non-coding RNA LINC01187 is associated with nephropathies.

Chronic kidney diseases affect a substantial percentage of the adult population worldwide. This observation emphasizes the need for novel insights into the molecular mechanisms that control the onset and progression of renal diseases. Recent advances in genomics have uncovered a previously unanticipated link between the non-coding genome and human kidney diseases. Here we screened and analysed long non-coding RNAs (lncRNAs) previously identified in mouse kidneys by genome-wide transcriptomic analysis, for conservation in humans and differential expression in renal tissue from healthy and diseased individuals. Our data suggest that LINC01187 is strongly down-regulated in human kidney tissues of patients with diabetic nephropathy and rapidly progressive glomerulonephritis, as well as in murine models of kidney diseases, including unilateral ureteral obstruction, nephrotoxic serum-induced glomerulonephritis and ischemia/reperfusion. Interestingly, LINC01187 overexpression in human kidney cells in vitro inhibits cell death indicating an anti-apoptotic function. Collectively, these data suggest a negative association of LINC01187 expression with renal diseases implying a potential protective role.

CD4+ T cells produce GM-CSF and drive immune-mediated glomerular disease by licensing monocyte-derived cells to produce MMP12.

GM-CSF in glomerulonephritisDespite glomerulonephritis being an immune-mediated disease, the contributions of individual immune cell types are not clear. To address this gap in knowledge, Paust et al. characterized pathological immune cells in samples from patients with glomerulonephritis and in samples from mice with the disease. The authors found that CD4+ T cells producing granulocyte-macrophage colony-stimulating factor (GM-CSF) licensed monocytes to promote disease by producing matrix metalloproteinase 12 and disrupting the glomerular basement membrane. Targeting GM-CSF to inhibit this axis reduced disease severity in mice, implicating this cytokine as a potential therapeutic target for patients with glomerulonephritis. -CM.

Single-cell transcriptomics reveals a mechanosensitive injury signaling pathway in early diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of end-stage renal disease, and histopathologic glomerular lesions are among the earliest structural alterations of DN. However, the signaling pathways that initiate these glomerular alterations are incompletely understood. METHODS: To delineate the cellular and molecular basis for DN initiation, we performed single-cell and bulk RNA sequencing of renal cells from type 2 diabetes mice (BTBR ob/ob) at the early stage of DN. RESULTS: Analysis of differentially expressed genes revealed glucose-independent responses in glomerular cell types. The gene regulatory network upstream of glomerular cell programs suggested the activation of mechanosensitive transcriptional pathway MRTF-SRF predominantly taking place in mesangial cells. Importantly, activation of MRTF-SRF transcriptional pathway was also identified in DN glomeruli in independent patient cohort datasets. Furthermore, ex vivo kidney perfusion suggested that the regulation of MRTF-SRF is a common mechanism in response to glomerular hyperfiltration. CONCLUSIONS: Overall, our study presents a comprehensive single-cell transcriptomic landscape of early DN, highlighting mechanosensitive signaling pathways as novel targets of diabetic glomerulopathy.

Molecular consequences of SARS-CoV-2 liver tropism.

Extrapulmonary manifestations of COVID-19 have gained attention due to their links to clinical outcomes and their potential long-term sequelae1. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) displays tropism towards several organs, including the heart and kidney. Whether it also directly affects the liver has been debated2,3. Here we provide clinical, histopathological, molecular and bioinformatic evidence for the hepatic tropism of SARS-CoV-2. We find that liver injury, indicated by a high frequency of abnormal liver function tests, is a common clinical feature of COVID-19 in two independent cohorts of patients with COVID-19 requiring hospitalization. Using autopsy samples obtained from a third patient cohort, we provide multiple levels of evidence for SARS-CoV-2 liver tropism, including viral RNA detection in 69% of autopsy liver specimens, and successful isolation of infectious SARS-CoV-2 from liver tissue postmortem. Furthermore, we identify transcription-, proteomic- and transcription factor-based activity profiles in hepatic autopsy samples, revealing similarities to the signatures associated with multiple other viral infections of the human liver. Together, we provide a comprehensive multimodal analysis of SARS-CoV-2 liver tropism, which increases our understanding of the molecular consequences of severe COVID-19 and could be useful for the identification of organ-specific pharmacological targets.

Studies on the Role of the Transcription Factor Tcf21 in the Transdifferentiation of Parietal Epithelial Cells into Podocyte-Like Cells.

BACKGROUND/AIMS: Podocyte differentiation is essential for proper blood filtration in the kidney. It is well known that transcription factors play an essential role to maintain the differentiation of podocytes. The present study is focused on the basic helix-loop-helix (bHLH) transcription factor Tcf21 (Pod1) which is essential for the development of podocytes in vivo. Since parietal epithelial cells (PECs) are still under debate to be progenitor cells which can differentiate into podocytes, we wanted to find out whether the expression of Tcf21 induces a transition of PECs into podocytes. METHODS: We transfected PECs with Tcf21-GFP and analyzed the expression of PEC- and podocyte-specific markers. Furthermore, we performed ChIP-Seq analysis to identify new putative interaction partners and target genes of Tcf21. RESULTS: By gene arrays analysis, we found that podocytes express high levels of Tcf21 in vivo in contrast to cultured podocytes and parietal epithelial cells (PECs) in vitro. After the expression of Tcf21 in PECs, we observed a downregulation of specific PEC markers like caveolin­1, ß-catenin and Pax2. Additionally, we found that the upregulation of Tcf21 induced multi-lobulation of cell nuclei, budding and a formation of micronuclei (MBM). Furthermore, a high number of PECs showed a tetraploid set of chromosomes. By qRT-PCR and Western blot analysis, we revealed that the transcription factor YY1 is downregulated by Tcf21. Interestingly, co-expression of YY1 and Tcf21 rescues MBM and reduced tetraploidy. By ChIP-Seq analysis, we identified a genome-wide Tcf21-binding site (CAGCTG), which matched the CANNTG sequence, a common E-box binding motif used by bHLH transcription factors. Using this technique, we identified additional Tcf21 targets genes that are involved in the regulation of the cell cycle (e.g. Mdm2, Cdc45, Cyclin D1, Cyclin D2), on the stability of microtubules (e.g. Mapt) as well as chromosome segregation. CONCLUSION: Taken together, we demonstrate that Tcf21 inhibits the expression of PEC-specific markers and of the transcription factor YY1, induces MBM as well as regulates the cell cycle suggesting that Tcf21 might be important for PEC differentiation into podocyte-like cells.

Pro-cachectic factors link experimental and human chronic kidney disease to skeletal muscle wasting programs.

Skeletal muscle wasting is commonly associated with chronic kidney disease (CKD), resulting in increased morbidity and mortality. However, the link between kidney and muscle function remains poorly understood. Here, we took a complementary interorgan approach to investigate skeletal muscle wasting in CKD. We identified increased production and elevated blood levels of soluble pro-cachectic factors, including activin A, directly linking experimental and human CKD to skeletal muscle wasting programs. Single-cell sequencing data identified the expression of activin A in specific kidney cell populations of fibroblasts and cells of the juxtaglomerular apparatus. We propose that persistent and increased kidney production of pro-cachectic factors, combined with a lack of kidney clearance, facilitates a vicious kidney/muscle signaling cycle, leading to exacerbated blood accumulation and, thereby, skeletal muscle wasting. Systemic pharmacological blockade of activin A using soluble activin receptor type IIB ligand trap as well as muscle-specific adeno-associated virus-mediated downregulation of its receptor ACVR2A/B prevented muscle wasting in different mouse models of experimental CKD, suggesting that activin A is a key factor in CKD-induced cachexia. In summary, we uncovered a crosstalk between kidney and muscle and propose modulation of activin signaling as a potential therapeutic strategy for skeletal muscle wasting in CKD.

Perspectives in systems nephrology.

Chronic kidney diseases (CKD) are a major health problem affecting approximately 10% of the world's population and posing increasing challenges to the healthcare system. While CKD encompasses a broad spectrum of pathological processes and diverse etiologies, the classification of kidney disease is currently based on clinical findings or histopathological categorizations. This descriptive classification is agnostic towards the underlying disease mechanisms and has limited progress towards the ability to predict disease prognosis and treatment responses. To gain better insight into the complex and heterogeneous disease pathophysiology of CKD, a systems biology approach can be transformative. Rather than examining one factor or pathway at a time, as in the reductionist approach, with this strategy a broad spectrum of information is integrated, including comprehensive multi-omics data, clinical phenotypic information, and clinicopathological parameters. In recent years, rapid advances in mathematical, statistical, computational, and artificial intelligence methods enable the mapping of diverse big data sets. This holistic approach aims to identify the molecular basis of CKD subtypes as well as individual determinants of disease manifestation in a given patient. The emerging mechanism-based patient stratification and disease classification will lead to improved prognostic and predictive diagnostics and the discovery of novel molecular disease-specific therapies.

Effects of Environmental Conditions on Nephron Number: Modeling Maternal Disease and Epigenetic Regulation in Renal Development.

A growing body of evidence suggests that low nephron numbers at birth can increase the risk of chronic kidney disease or hypertension later in life. Environmental stressors, such as maternal malnutrition, medication and smoking, can influence renal size at birth. Using metanephric organ cultures to model single-variable environmental conditions, models of maternal disease were evaluated for patterns of developmental impairment. While hyperthermia had limited effects on renal development, fetal iron deficiency was associated with severe impairment of renal growth and nephrogenesis with an all-proximal phenotype. Culturing kidney explants under high glucose conditions led to cellular and transcriptomic changes resembling human diabetic nephropathy. Short-term high glucose culture conditions were sufficient for long-term alterations in DNA methylation-associated epigenetic memory. Finally, the role of epigenetic modifiers in renal development was tested using a small compound library. Among the selected epigenetic inhibitors, various compounds elicited an effect on renal growth, such as HDAC (entinostat, TH39), histone demethylase (deferasirox, deferoxamine) and histone methyltransferase (cyproheptadine) inhibitors. Thus, metanephric organ cultures provide a valuable system for studying metabolic conditions and a tool for screening for epigenetic modifiers in renal development.

The role of filamins in mechanically stressed podocytes.

Glomerular hypertension induces mechanical load to podocytes, often resulting in podocyte detachment and the development of glomerulosclerosis. Although it is well known that podocytes are mechanosensitive, the mechanosensors and mechanotransducers are still unknown. Since filamin A, an actin-binding protein, is already described to be a mechanosensor and mechanotransducer, we hypothesized that filamins could be important for the outside-in signaling as well as the actin cytoskeleton of podocytes under mechanical stress. In this study, we demonstrate that filamin A is the main isoform of the filamin family that is expressed in cultured podocytes. Together with filamin B, filamin A was significantly up-regulated during mechanical stretch (3 days, 0.5 Hz, and 5% extension). To study the role of filamin A in cultured podocytes under mechanical stress, filamin A was knocked down (Flna KD) by specific siRNA. Additionally, we established a filamin A knockout podocyte cell line (Flna KO) by CRISPR/Cas9. Knockdown and knockout of filamin A influenced the expression of synaptopodin, a podocyte-specific protein, focal adhesions as well as the morphology of the actin cytoskeleton. Moreover, the cell motility of Flna KO podocytes was significantly increased. Since the knockout of filamin A has had no effect on cell adhesion of podocytes during mechanical stress, we simultaneously knocked down the expression of filamin A and B. Thereby, we observed a significant loss of podocytes during mechanical stress indicating a compensatory mechanism. Analyzing hypertensive mice kidneys as well as biopsies of patients suffering from diabetic nephropathy, we found an up-regulation of filamin A in podocytes in contrast to the control. In summary, filamin A and B mediate matrix-actin cytoskeleton interactions which are essential for the adaptation of cultured podocyte to mechanical stress.

Endothelial-Specific Deletion of CD146 Protects Against Experimental Glomerulonephritis in Mice.

[Figure: see text].

TrkC Is Essential for Nephron Function and Trans-Activates Igf1R Signaling.

BACKGROUND: Injury to kidney podocytes often results in chronic glomerular disease and consecutive nephron malfunction. For most glomerular diseases, targeted therapies are lacking. Thus, it is important to identify novel signaling pathways contributing to glomerular disease. Neurotrophic tyrosine kinase receptor 3 (TrkC) is expressed in podocytes and the protein transmits signals to the podocyte actin cytoskeleton. METHODS: Nephron-specific TrkC knockout (TrkC-KO) and nephron-specific TrkC-overexpressing (TrkC-OE) mice were generated to dissect the role of TrkC in nephron development and maintenance. RESULTS: Both TrkC-KO and TrkC-OE mice exhibited enlarged glomeruli, mesangial proliferation, basement membrane thickening, albuminuria, podocyte loss, and aspects of FSGS during aging. Igf1 receptor (Igf1R)-associated gene expression was dysregulated in TrkC-KO mouse glomeruli. Phosphoproteins associated with insulin, erb-b2 receptor tyrosine kinase (Erbb), and Toll-like receptor signaling were enriched in lysates of podocytes treated with the TrkC ligand neurotrophin-3 (Nt-3). Activation of TrkC by Nt-3 resulted in phosphorylation of the Igf1R on activating tyrosine residues in podocytes. Igf1R phosphorylation was increased in TrkC-OE mouse kidneys while it was decreased in TrkC-KO kidneys. Furthermore, TrkC expression was elevated in glomerular tissue of patients with diabetic kidney disease compared with control glomerular tissue. CONCLUSIONS: Our results show that TrkC is essential for maintaining glomerular integrity. Furthermore, TrkC modulates Igf-related signaling in podocytes.

Pathogen-induced tissue-resident memory TH17 (TRM17) cells amplify autoimmune kidney disease.

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (TRM) cells persist in peripheral organs and provide immune protection against reinfection. However, whether TRM cells participate in responses unrelated to the primary infection, such as autoimmune inflammation, is unknown. By using high-dimensional single-cell analysis, we identified CD4+ TRM cells with a TH17 signature (termed TRM17 cells) in kidneys of patients with ANCA-associated glomerulonephritis. Experimental models demonstrated that renal TRM17 cells were induced by pathogens infecting the kidney, such as Staphylococcus aureus, Candida albicans, and uropathogenic Escherichia coli, and persisted after the clearance of infections. Upon induction of experimental glomerulonephritis, these kidney TRM17 cells rapidly responded to local proinflammatory cytokines by producing IL-17A and thereby exacerbate renal pathology. Thus, our data show that pathogen-induced TRM17 cells have a previously unrecognized function in aggravating autoimmune disease.