The global burden of antimicrobial resistance - urinary tract infections.

Antimicrobial resistance (AMR) has emerged as a significant global healthcare problem. Antibiotic use has accelerated the physiologic process of AMR, particularly in Gram-negative pathogens. Urinary tract infections (UTIs) are predominantly of a Gram-negative nature. Uropathogens are evolutionarily highly adapted and selected strains with specific virulence factors, suggesting common mechanisms in how bacterial cells acquire virulence and AMR factors. The simultaneous increase in resistance and virulence is a complex and context-dependent phenomenon. Among known AMR mechanisms, the plenitude of different ß-lactamases is especially prominent. The risk for AMR in UTIs varies in different patient populations. A history of antibiotic consumption and the physiology of urinary flow are major factors that shape AMR prevalence. The urinary tract is in close crosstalk with the microbiome of other compartments, including the gut and genital tracts. In addition, pharmacokinetic properties and the physiochemical composition of urinary compartments can contribute to the emergence of AMR. Alternatives to antibiotic treatment and a broader approach to address bacterial infections are needed. Among the various alternatives studied, antimicrobial peptides and bacteriophage treatment appear to be highly promising approaches. We herein summarize the present knowledge of clinical and microbiological AMR in UTIs and discuss innovative approaches, namely new risk prediction tools and the use of non-antibiotic approaches to defend against uropathogenic microbes.

Free heme and hemopexin in acute kidney injury after cardiopulmonary bypass and transient renal ischemia.

Free heme is released from hemoproteins during hemolysis or ischemia reperfusion injury and can be pro-inflammatory. Most studies on nephrotoxicity of hemolysis-derived proteins focus on free hemoglobin (fHb) with heme as a prosthetic group. Measurement of heme in its free, non-protein bound, form is challenging and not commonly used in clinical routine diagnostics. In contrast to fHb, the role of free heme in acute kidney injury (AKI) after cardiopulmonary bypass (CPB) surgery is unknown. Using an apo-horseradish peroxidase-based assay, we identified free heme during CPB surgery as predictor of AKI in patients undergoing cardiac valve replacement (n = 37). Free heme levels during CPB surgery correlated with depletion of hemopexin (Hx), a heme scavenger-protein. In mice, the impact of high levels of circulating free heme on the development of AKI following transient renal ischemia and the therapeutic potential of Hx were investigated. C57BL/6 mice were subjected to bilateral renal ischemia/reperfusion injury for 15 min which did not cause AKI. However, additional administration of free heme in this model promoted overt AKI with reduced renal function, increased renal inflammation, and reduced renal perfusion on functional magnetic resonance imaging. Hx treatment attenuated AKI. Free heme administration to sham operated control mice did not cause AKI. In conclusion, free heme is a predictor of AKI in CPB surgery patients and promotes AKI in transient renal ischemia. Depletion of Hx in CPB surgery patients and attenuation of AKI by Hx in the in vivo model encourage further research on Hx therapy in patients with increased free heme levels during CPB surgery.

High RIPK3 expression is associated with a higher risk of early kidney transplant failure.

Renal ischemia-reperfusion injury (IRI) is associated with reduced allograft survival, and each additional hour of cold ischemia time increases the risk of graft failure and mortality following renal transplantation. Receptor-interacting protein kinase 3 (RIPK3) is a key effector of necroptosis, a regulated form of cell death. Here, we evaluate the first-in-human RIPK3 expression dataset following IRI in kidney transplantation. The primary analysis included 374 baseline biopsy samples obtained from renal allografts 10 minutes after onset of reperfusion. RIPK3 was primarily detected in proximal tubular cells and distal tubular cells, both of which are affected by IRI. Time-to-event analysis revealed that high RIPK3 expression is associated with a significantly higher risk of one-year transplant failure and prognostic for one-year (death-censored) transplant failure independent of donor and recipient associated risk factors in multivariable analyses. The RIPK3 score also correlated with deceased donation, cold ischemia time and the extent of tubular injury.

Kidney medullary sodium chloride concentrations induce neutrophil and monocyte extracellular DNA traps that defend against pyelonephritis in vivo.

Urinary tract infections are common. Here, we delineate a role of extracellular DNA trap (ET) formation in kidney antibacterial defense and determine mechanisms of their formation in the hyperosmotic environment of the kidney medulla. ET of granulocytic and monocytic origin were present in the kidneys of patients with pyelonephritis along with systemically elevated citrullinated histone levels. Inhibition of the transcription coregulatory, peptidylarginine deaminase 4 (PAD4), required for ET formation, prevented kidney ET formation and promoted pyelonephritis in mice. ETs predominantly accumulated in the kidney medulla. The role of medullary sodium chloride and urea concentrations in ET formation was then investigated. Medullary-range sodium chloride, but not urea, dose-, time- and PAD4-dependently induced ET formation even in the absence of other stimuli. Moderately elevated sodium chloride promoted myeloid cell apoptosis. Sodium gluconate also promoted cell death, proposing a role for sodium ions in this process. Sodium chloride induced myeloid cell calcium influx. Calcium ion-free media or -chelation reduced sodium chloride-induced apoptosis and ET formation while bacterial lipopolysaccharide amplified it. Autologous serum improved bacterial killing in the presence of sodium chloride-induced ET. Depletion of the kidney sodium chloride gradient by loop diuretic therapy diminished kidney medullary ET formation and increased pyelonephritis severity. Thus, our data demonstrate that ETs may protect the kidney against ascending uropathogenic E. coli and delineate kidney medullary range sodium chloride concentrations as novel inducers of programmed myeloid cell death.

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.

NT-proBNP as predictor of major cardiac events after renal transplantation in patients with preserved left ventricular ejection fraction.

BACKGROUND: For the improvement of outcome after renal transplantation it is important to predict future risk of major adverse cardiac events as well as all-cause mortality. We aimed to determine the relationship of pre-transplant NT-proBNP with major adverse cardiac events and all-cause mortality after transplant in patients on the waiting-list with preserved left ventricular ejection fraction. PATIENTS AND METHODS: We included 176 patients with end-stage renal disease and preserved left ventricular ejection fraction who received a kidney transplant. MACE was defined as myocardial infarction (ST-segment elevation [STEMI] or non-ST-segment elevation [NSTEMI]), stroke or transient ischemic attack), coronary artery disease requiring intervention or bypass or death from cardiovascular causes. RESULTS: MACE occurred in 28/176 patients. Patients with NT-proBNP levels above 4350 pg/ml had 1- and 5-year survival rates of 90.67% and 68.20%, whereas patients with NT-proBNP levels below 4350 pg/ml had 1- and 5-year survival rates of 100% and 90.48% (p < 0.01). 1- and 5-year MACE-free survival rates were calculated as 78.82% and 74.68% for patients with NT-proBNP > 4350 pg/ml and 93.33% and 91.21% for patients with NT-proBNP < 4350 pg/ml (p < 0.01). CONCLUSIONS: Pre-transplant NT-proBNP might identify renal transplant candidates at risk for MACE after transplant.

Pre-transplant serum Beta Trace Protein indicates risk for post-transplant major cardiac adverse events.

BACKGROUND: Beta Trace Protein (BTP) is a biomarker for residual kidney function which has been linked to cardiovascular and all-cause mortality in haemodialysis patients. Following renal transplantation, recipients remain at increased risk for cardiovascular events compared with the general population. We aimed to determine the relationship of pre-transplant BTP to major adverse cardiac events (MACE) in patients following kidney transplantation. METHODS: We included 384 patients with end-stage renal disease who received a kidney transplant. MACE was defined as myocardial infarction (ST-segment elevation or non-ST-segment elevation, stroke or transient ischemic attack), coronary artery disease requiring intervention or bypass or death for cardiovascular reason. The association between pre-transplant serum BTP concentration and post-transplant MACE was evaluated by Kaplan-Meier and Cox regression analyses. RESULTS: Post-transplant MACE occurred in 70/384 patients. Pre-transplant BTP was significantly higher in patients with post-transplant MACE (14.36 ± 5.73 mg/l vs. 11.26 ± 5.11 mg/l; p < .01). Next to smoking (HR 1.81), age > 56.38 years (HR 1.97) and pre-existing coronary heart disease (HR 8.23), BTP above the cut off value of 12.7 mg/l was confirmed as independent risk factor for MACE (HR 2.02, all p < .05). MACE-free survival inversely correlated with pre-transplant BTP levels. CONCLUSIONS: Pre-transplant serum BTP concentration may identify renal transplant recipients with higher risk of post-transplant MACE.

Annexin A1 exerts renoprotective effects in experimental crescentic glomerulonephritis.

Non-resolving inflammation plays a critical role during the transition from renal injury towards end-stage renal disease. The glucocorticoid-inducible protein annexin A1 has been shown to function as key regulator in the resolution phase of inflammation, but its role in immune-mediated crescentic glomerulonephritis has not been studied so far. Methods: Acute crescentic glomerulonephritis was induced in annexin A1-deficient and wildtype mice using a sheep serum against rat glomerular basement membrane constituents. Animals were sacrificed at d5 and d10 after nephritis induction. Renal leukocyte abundance was studied by immunofluorescence and flow cytometry. Alterations in gene expression were determined by RNA-Seq and gene ontology analysis. Renal levels of eicosanoids and related lipid products were measured using lipid mass spectrometry. Results: Histological analysis revealed an increased number of sclerotic glomeruli and aggravated tubulointerstitial damage in the kidneys of annexin A1-deficient mice compared to the wildtype controls. Flow cytometry analysis confirmed an increased number of CD45+ leukocytes and neutrophil granulocytes in the absence of annexin A1. Lipid mass spectrometry showed elevated levels of prostaglandins PGE2 and PGD2 and reduced levels of antiinflammatory epoxydocosapentaenoic acid regioisomers. RNA-Seq with subsequent gene ontology analysis revealed induction of gene products related to leukocyte activation and chemotaxis as well as regulation of cytokine production and secretion. Conclusion: Intrinsic annexin A1 reduces proinflammatory signals and infiltration of neutrophil granulocytes and thereby protects the kidney during crescentic glomerulonephritis. The annexin A1 signaling cascade may therefore provide novel targets for the treatment of inflammatory kidney disease.

Generation of an alpaca serum that induces immune-mediated crescentic glomerulonephritis in mice.

Crescentic glomerulonephritis (cGN) is the most aggressive form of glomerulonephritis in humans. A widely studied mouse model is induced by sheep or rabbit antisera raised against murine renal cortical antigens. We here, report that Alpaca readily produce ample amounts of antisera that induces pathology in mice, resembling human disease regarding crescent formation, proteinuria, infiltrating immune cells and a significant Th1, but not Th17 immune response. Alpaca antiserum did not cause end-stage kidney failure, neither in a passive nor in an accelerated experimental setting, which may be advantageous for long term studies of crescentic glomerulonephritis.

Myeloid CCR2 Promotes Atherosclerosis after AKI.

BACKGROUND: The risk of cardiovascular events rises after AKI. Leukocytes promote atherosclerotic plaque growth and instability. We established a model of enhanced remote atherosclerosis after renal ischemia-reperfusion (IR) injury and investigated the underlying inflammatory mechanisms. METHODS: Atherosclerotic lesions and inflammation were investigated in native and bone marrow-transplanted LDL receptor-deficient (LDLr-/- ) mice after unilateral renal IR injury using histology, flow cytometry, and gene expression analysis. RESULTS: Aortic root atherosclerotic lesions were significantly larger after renal IR injury than in controls. A gene expression screen revealed enrichment for chemokines and their cognate receptors in aortas of IR-injured mice in early atherosclerosis, and of T cell-associated genes in advanced disease. Confocal microscopy revealed increased aortic macrophage proximity to T cells. Differential aortic inflammatory gene regulation in IR-injured mice largely paralleled the pattern in the injured kidney. Single-cell analysis identified renal cell types that produced soluble mediators upregulated in the atherosclerotic aorta. The analysis revealed a marked early increase in Ccl2, which CCR2+ myeloid cells mainly expressed. CCR2 mediated myeloid cell homing to the post-ischemic kidney in a cell-individual manner. Reconstitution with Ccr2-/- bone marrow dampened renal post-ischemic inflammation, reduced aortic Ccl2 and inflammatory macrophage marker CD11c, and abrogated excess aortic atherosclerotic plaque formation after renal IR. CONCLUSIONS: Our data introduce an experimental model of remote proatherogenic effects of renal IR and delineate myeloid CCR2 signaling as a mechanistic requirement. Monocytes should be considered as mobile mediators when addressing systemic vascular sequelae of kidney injury.

Induction of ferroptosis selectively eliminates senescent tubular cells.

The accumulation of senescent cells is an important contributor to kidney aging, chronic renal disease, and poor outcome after kidney transplantation. Approaches to eliminate senescent cells with senolytic compounds have been proposed as novel strategies to improve marginal organs. While most existing senolytics induce senescent cell clearance by apoptosis, we observed that ferroptosis, an iron-catalyzed subtype of regulated necrosis, might serve as an alternative way to ablate senescent cells. We found that murine kidney tubular epithelial cells became sensitized to ferroptosis when turning senescent. This was linked to increased expression of pro-ferroptotic lipoxygenase-5 and reduced expression of anti-ferroptotic glutathione peroxidase 4 (GPX4). In tissue slice cultures from aged kidneys low dose application of the ferroptosis-inducer RSL3 selectively eliminated senescent cells while leaving healthy tubular cells unaffected. Similar results were seen in a transplantation model, in which RSL3 reduced the senescent cell burden of aged donor kidneys and caused a reduction of damage and inflammatory cell infiltration during the early post-transplantation period. In summary, these data reveal an increased susceptibility of senescent tubular cells to ferroptosis with the potential to be exploited for selective reduction of renal senescence in aged kidney transplants.

Single cell versus single nucleus: transcriptome differences in the murine kidney after ischemia-reperfusion injury.

The kidney is a complex organ, which consists of multiple components with highly diverse cell types. A detailed understanding of these cell types in health and disease is crucial for the future development of preventive and curative treatment strategies. In recent years, single-cell RNA sequencing (scRNAseq) and single-nucleus RNA sequencing (snRNAseq) technology has opened up completely new possibilities in investigating the variety of renal cell populations in physiological and pathological states. Here, we systematically assessed differences between scRNAseq and snRNAseq approaches in transcriptome analysis of murine kidneys after ischemia-reperfusion injury. We included tissues from control kidneys and from kidneys harvested 1 wk after mild (17-min clamping time) and severe (27-min clamping time) transient unilateral ischemia. Our findings revealed important methodological differences in the discovery of inflammatory cells, tubular cells, and other specialized cell types. Although the scRNAseq approach was advantageous for investigating immune cells, the snRNAseq approach allowed superior insights into healthy and damaged tubular cells. Apart from differences in the quantitative discovery rate, we found important qualitative discrepancies in the captured transcriptomes with crucial consequences for the interpretation of cell states and molecular functions. Together, we provide an overview of method-dependent differences between scRNAseq and snRNAseq results from identical postischemic kidney tissues. Our results highlight the importance of choosing the right approach for specific research questions.NEW & NOTEWORTHY Single-cell and single-nucleus RNA sequencing technologies provide powerful new tools to examine complex tissues such as the kidney. This research reference paper provides practical information on the differences between the two technologies when examining murine kidneys after ischemia-reperfusion injury. The results will serve those who are debating which protocols to use in their given study.

Renal medullary osmolytes NaCl and urea differentially modulate human tubular cell cytokine expression and monocyte recruitment.

Renal immune cells serve as sentinels against ascending bacteria but also promote detrimental inflammation. The kidney medulla is characterized by extreme electrolyte concentrations. We here address how its main osmolytes, NaCl and urea, regulate tubular cell cytokine expression and monocyte chemotaxis. In the healthy human kidney, more monocytes were detected in medulla than cortex. The monocyte gradient was attenuated in patients with medullary NaCl depletion by loop diuretic therapy and in the nephrotic syndrome. Renal tubular epithelial cell gene expression responded similarly to NaCl and tonicity control mannitol, but not urea. NaCl significantly upregulated chemotactic cytokines, most markedly CCL26, CCL2, and CSF1. This induction was inhibited by the ROS scavenger n-acetylcysteine. In contrast, urea, the main medullary osmolyte in catabolism, dampened tubular epithelial CCL26 and CSF1 expression. Renal medullary chemokine and monocyte marker expression decreased in catabolic mice. NaCl-, but not urea-stimulated tubular epithelium or CCL2 and CCL26, promoted human classical monocyte migration. CCL26 improved bactericidal function. In the human kidney medulla, monocyte densities correlated with tubular CCL26 protein abundance. In summary, medullary-range NaCl, but not urea, promotes tubular cytokine expression and monocyte recruitment. This may contribute to the pyelonephritis vulnerability in catabolism but can possibly be harnessed against pathologic inflammation.

Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures.

Tertiary lymphoid structures (TLS) are lymph node-like immune cell clusters that emerge during chronic inflammation in non-lymphoid organs like the kidney, but their origin remains not well understood. Here we show, using conditional deletion strategies of the canonical Notch signaling mediator Rbpj, that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona fide TLS in the kidney, liver and lung, based on molecular, cellular and structural criteria. These TLS form in a stereotypical manner around parenchymal arteries, while secondary lymphoid structures remained largely unchanged. This effect is mediated by endothelium of blood vessels, but not lymphatics, since a lymphatic endothelial-specific targeting strategy did not result in TLS formation, and involves loss of arterial specification and concomitant acquisition of a high endothelial cell phenotype, as shown by transcriptional analysis of kidney endothelial cells. This indicates a so far unrecognized role for vascular endothelial cells and Notch signaling in TLS initiation.

Renal Denervation Exacerbates LPS- and Antibody-induced Acute Kidney Injury, but Protects from Pyelonephritis in Mice.

BACKGROUND: Renal denervation (RDN) is an invasive intervention to treat drug-resistant arterial hypertension. Its therapeutic value is contentious. Here we examined the effects of RDN on inflammatory and infectious kidney disease models in mice. METHODS: Mice were unilaterally or bilaterally denervated, or sham operated, then three disease models were induced: nephrotoxic nephritis (NTN, a model for crescentic GN), pyelonephritis, and acute endotoxemic kidney injury (as a model for septic kidney injury). Analytical methods included measurement of renal glomerular filtration, proteinuria, flow cytometry of renal immune cells, immunofluorescence microscopy, and three-dimensional imaging of optically cleared kidney tissue by light-sheet fluorescence microscopy followed by algorithmic analysis. RESULTS: Unilateral RDN increased glomerular filtration in denervated kidneys, but decreased it in the contralateral kidneys. In the NTN model, more nephritogenic antibodies were deposited in glomeruli of denervated kidneys, resulting in stronger inflammation and injury in denervated compared with contralateral nondenervated kidneys. Also, intravenously injected LPS increased neutrophil influx and inflammation in the denervated kidneys, both after unilateral and bilateral RDN. When we induced pyelonephritis in bilaterally denervated mice, both kidneys contained less bacteria and neutrophils. In unilaterally denervated mice, pyelonephritis was attenuated and intrarenal neutrophil numbers were lower in the denervated kidneys. The nondenervated contralateral kidneys harbored more bacteria, even compared with sham-operated mice, and showed the strongest influx of neutrophils. CONCLUSIONS: Our data suggest that the increased perfusion and filtration in denervated kidneys can profoundly influence concomitant inflammatory diseases. Renal deposition of circulating nephritic material is higher, and hence antibody- and endotoxin-induced kidney injury was aggravated in mice. Pyelonephritis was attenuated in denervated murine kidneys, because the higher glomerular filtration facilitated better flushing of bacteria with the urine, at the expense of contralateral, nondenervated kidneys after unilateral denervation.

Long-term B cell depletion associates with regeneration of kidney function.

BACKGROUND: Chronic kidney disease (CKD) is a common condition that increases mortality and the risk of cardiovascular and other morbidities regardless of underlying renal condition. Chronic inflammation promotes renal fibrosis. Recently, renal B cell infiltrates were described in chronic kidney disease of various etiologies beyond autoimmunity. METHODS: We here investigated B cells and indicators of tertiary lymphoid structure formation in human renal biopsies. Renal function was studied during long-term B cell depletion in human patients with membranous nephropathy and with CKD of unknown origin. RESULTS: Cytokine profiles of tertiary lymphoid structure formation were detected in human renal interstitium in a range of kidney diseases. Complex B cell structures consistent with tertiary lymphoid organ formation were evident in human membranous nephropathy. Here, B cell density did not significantly associate with proteinuria severity, but with worse excretory renal function. Proteinuria responses mostly occurred within the first 6 months of B cell depletion. In contrast, recovery of excretory kidney function was observed only after 18 months of continuous therapy, consistent with a structural process. Renal tertiary lymphatic structures were also detected in the absence of autoimmune kidney disease. To start to address whether B cell depletion may affect CKD in a broader population, we assessed kidney function in neurologic patients with CKD of unknown origin. In this cohort, eGFR significantly increased within 24 months of B cell depletion. CONCLUSION: Long-term B cell depletion associated with significant improvement of excretory kidney function in human CKD. Kinetics and mechanisms of renal B cell aggregation should be investigated further to stratify the impact of B cells and their aggregates as therapeutic targets.

A flow cytometry approach reveals heterogeneity in conventional subsets of murine renal mononuclear phagocytes.

Mononuclear phagocytes (MNPs) participate in inflammation and repair after kidney injury, reflecting their complex nature. Dissection into refined functional subunits has been challenging and would benefit understanding of renal pathologies. Flow cytometric approaches are limited to classifications of either different MNP subsets or functional state. We sought to combine these two dimensions in one protocol that considers functional heterogeneity in each MNP subset. We identified five distinct renal MNP subsets based on a previously described strategy. In vitro polarization of bone marrow-derived macrophages (BMDM) into M1- and M2-like cells suggested functional distinction of CD86 + MHCII + CD206- and CD206 + cells. Combination of both distinction methods identified CD86 + MHCII + CD206- and CD206 + cells in all five MNP subsets, revealing their heterologous nature. Our approach revealed that MNP composition and their functional segmentation varied between different mouse models of kidney injury and, moreover, was dynamically regulated in a time-dependent manner. CD206 + cells from three analyzed MNP subsets had a higher ex vivo phagocytic capacity than CD86 + MHCII + CD206- counterparts, indicating functional uniqueness of each subset. In conclusion, our novel flow cytometric approach refines insights into renal MNP heterogeneity and therefore could benefit mechanistic understanding of renal pathology.

Regulation and function of CX3CR1 and its ligand CX3CL1 in kidney disease.

Attraction, retention, and differentiation of leukocytes to and within the kidney are governed by chemokines. The chemokine CX3CL1 (fractalkine) and its receptor CX3CR1 are exemplary in this regard as they are highly expressed and further upregulated in a range of kidney diseases. CX3CL1 is chiefly produced by renal endothelium and tubular epithelium, where it promotes leukocyte attraction. Recent data suggest that in addition to established soluble mediators, cellular interactions may enhance CX3CL1 expression. The receptor CX3CR1 is essential in myeloid phagocyte homing to the kidney at homeostasis, after acute cell depletion and in inflammation. CX3CR1 and its ligand are highly regulated in human kidney diseases such as IgA nephritis, systemic lupus erythematosus, and inflammatory conditions such as transplant rejection. A mechanistic role of CX3CR1 has been established in experimental models of nephrotoxic nephritis and renal candidiasis. It is debated in fibrosis. Recent publications demonstrate a role for CX3CR1+ myeloid cells in radio-contrast-agent and sepsis-induced kidney damage. Systemically, circulating CX3CR1+ monocytes reversibly increase in individuals with renal impairment and correlate with their cardiovascular risk. In this review, we discuss role and regulatory mechanisms of the CX3CL1-CX3CR1 axis in both localized and systemic effects of renal inflammation.