[Geriatric patient with neurological symptoms and prolonged aPTT]. / Geriatrischer Patient mit neurologischer Symptomatik und verlängerter aPTT.

A 73-year-old man with dementia was referred to our clinic with hypernatremia and volume depletion. New-onset neurogenic dysphagia was likely the reason for both. The patient had chronic embolic strokes on the computed tomography (CT) images. Documentation from previous hospitalizations in different hospitals revealed a repeatedly prolonged activated partial thromboplastin time (aPTT); 5 years prior, antiphospholipid antibody syndrome had already been suspected, but the necessary workup was never completed. We diagnosed the patient with primary antiphospholipid antibody syndrome and initiated therapy with vitamin K antagonists (phenprocoumon) and aspirin.

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.

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.

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.

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.

Human CD16+ monocytes promote a pro-atherosclerotic endothelial cell phenotype via CX3CR1-CX3CL1 interaction.

AIMS: Monocytes are central for atherosclerotic vascular inflammation. The human non-classical, patrolling subtype, which expresses high levels of CD16 and fractalkine receptor CX3CR1, strongly associates with cardiovascular events. This is most marked in renal failure, a condition with excess atherosclerosis morbidity. The underlying mechanism is not understood. This study investigated how human CD16+ monocytes modulate endothelial cell function. METHODS AND RESULTS: In patients with kidney failure, CD16+ monocyte counts were elevated and dynamically decreased within a year after transplantation, chiefly due to a drop in CD14+CD16+ cells. The CX3CR1 ligand CX3CL1 was similarly elevated in the circulation of humans and mice with renal impairment. CX3CL1 up-regulation was also observed close to macrophage rich human coronary artery plaques. To investigate a mechanistic basis of this association, CD16+CX3CR1HIGH monocytes were co-incubated with primary human endothelium in vitro. Compared to classical CD14+ monocytes or transwell cocultures, CD16+ monocytes enhanced endothelial STAT1 and NF-κB p65 phosphorylation, up-regulated expression of CX3CL1 and interleukin-1ß, numerous CCL and CXCL chemokines and molecules promoting leucocyte patrolling and adhesion such as ICAM1 and VCAM1. Genes required for vasodilatation including endothelial nitric oxide synthase decreased while endothelial collagen production increased. Uraemic patients' monocytes enhanced endothelial CX3CL1 even more markedly. Their receptor CX3CR1 was required for enhanced aortic endothelial stiffness in murine atherosclerosis with renal impairment. CX3CR1 dose-dependently modulated monocyte-contact-dependent gene expression in human endothelium. CONCLUSION: By demonstrating endothelial proatherosclerotic gene regulation in direct contact with CD16+ monocytes, in part via cellular CX3CR1-CX3CL1 interaction, our data delineate a mechanism how this celltype can increase cardiovascular risk.

Notch and TLR signaling coordinate monocyte cell fate and inflammation.

Conventional Ly6Chi monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6Chi monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6Clo patrolling monocyte development from Ly6Chi monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6Clo monocyte development. At the same time, TLR7 stimulation in the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression signatures in monocytes in the blood and ectopic differentiation of Ly6Chi monocytes into macrophages and dendritic cells, which infiltrate the spleen and major blood vessels and are accompanied by aberrant systemic inflammation. Thus, Notch2 is a master regulator of Ly6Chi monocyte cell fate and inflammation in response to TLR signaling.

Effectiveness of a Fixed-Dose, Single-Pill Combination of Perindopril and Amlodipine in Patients with Hypertension: A Non-Interventional Study.

INTRODUCTION: We conducted a prospective, non-interventional, multicenter study to examine the effect of a fixed-dose combination of perindopril/amlodipine in patients with arterial hypertension. METHODS: Patients who were previously untreated or required a change in medication were treated with a fixed combination of perindopril/amlodipine (3.5/2.5 or 7.0/5.0 mg) for 12 weeks. Changes in office, home and ambulatory blood pressure (BP) were recorded. Adherence was assessed by the Hill-Bone medication adherence scale. RESULTS: Overall, 1814 patients (mean age 60.0 ± 13.4 years) were included in 614 German practices, and data of 1770 patients were analyzed. At study entry, 97.7% of patients received perindopril/amlodipine at a daily dose of 3.5 mg/2.5 mg, and 47.9% of patients remained on this dose during the study period. Treatment with perindopril/amlodipine decreased mean office BP from 163.7/95.4 to 133.6/80.3 mmHg (p < 0.0001), resulting in a hypertension control rate of 69.1%. Blood pressure control was comparable in previously untreated and treated patients (70.3 vs. 68.1%), and in younger and older patients (70.6 < 65 vs. 66.3% ≥ 65 years). Ambulatory BP measurements were available in a subgroup of patients (n = 167), and mean 24 h ambulatory BP decreased from 150.6 ± 12.6/88.9 ± 8.8 to 132.4 ± 11.9/79.4 ± 8.5 mmHg (p < 0.0001). Furthermore, the proportion of patients with severe hypertension European Society of Hypertension/European Society of Cardiology (ESH/ESC) grade II or III decreased from 64.4 to 3.9%, and patients with pre-existing isolated systolic hypertension (n = 284) converted to normal BP in 67.6% of cases. Nearly half of the patients (47.2%) were perfectly adherent during the study. In previously treated patients, the percentage of patients with perfect adherence increased from 20.6% prior to study to 43.5% at final visit (p < 0.0001). Adverse drug reactions were documented for 4.9% of patients. CONCLUSION: A fixed-dose combination of perindopril/amlodipine shows significant blood pressure reduction and improvement in medication adherence in a primary care setting. TRIAL REGISTRATION: ISRCTN26323538. FUNDING: Servier Deutschland GmbH.

Mixed leukocyte cell-derived chemotaxin 2 and amyloid A renal amyloidosis in a Kazakh-German patient.

Leukocyte cell-derived chemotaxin 2 (LECT2)-related amyloidosis (ALECT2) constitutes a subtype of systemic amyloidosis affecting the kidney. This is the first case describing mixed ALECT2 and Amyloid A renal amyloidosis in a Kazakh-German patient. Genetic analysis shows a polymorphism in the LECT2 gene and a homozygous mutation in the SAA1 gene. Notably, our patient has a body mass index of 61 kg/m2 and a pathological glucose tolerance test. ALECT2 was found in certain ethnic groups with a high incidence of diabetes. In our case, morbid obesity may have played a significant role in clinical manifestation of ALECT2 amyloidosis.

Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis.

Chronic kidney disease is characterized by interstitial fibrosis and proliferation of scar-secreting myofibroblasts, ultimately leading to end-stage renal disease. The hedgehog (Hh) pathway transcriptional effectors GLI1 and GLI2 are expressed in myofibroblast progenitors; however, the role of these effectors during fibrogenesis is poorly understood. Here, we demonstrated that GLI2, but not GLI1, drives myofibroblast cell-cycle progression in cultured mesenchymal stem cell-like progenitors. In animals exposed to unilateral ureteral obstruction, Hh pathway suppression by expression of the GLI3 repressor in GLI1+ myofibroblast progenitors limited kidney fibrosis. Myofibroblast-specific deletion of Gli2, but not Gli1, also limited kidney fibrosis, and induction of myofibroblast-specific cell-cycle arrest mediated this inhibition. Pharmacologic targeting of this pathway with darinaparsin, an arsenical in clinical trials, reduced fibrosis through reduction of GLI2 protein levels and subsequent cell-cycle arrest in myofibroblasts. GLI2 overexpression rescued the cell-cycle effect of darinaparsin in vitro. While darinaparsin ameliorated fibrosis in WT and Gli1-KO mice, it was not effective in conditional Gli2-KO mice, supporting GLI2 as a direct darinaparsin target. The GLI inhibitor GANT61 also reduced fibrosis in mice. Finally, GLI1 and GLI2 were upregulated in the kidneys of patients with high-grade fibrosis. Together, these data indicate that GLI inhibition has potential as a therapeutic strategy to limit myofibroblast proliferation in kidney fibrosis.

Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis.

Mesenchymal stem cells (MSCs) reside in the perivascular niche of many organs, including kidney, lung, liver, and heart, although their roles in these tissues are poorly understood. Here, we demonstrate that Gli1 marks perivascular MSC-like cells that substantially contribute to organ fibrosis. In vitro, Gli1(+) cells express typical MSC markers, exhibit trilineage differentiation capacity, and possess colony-forming activity, despite constituting a small fraction of the platelet-derived growth factor-ß (PDGFRß)(+) cell population. Genetic lineage tracing analysis demonstrates that tissue-resident, but not circulating, Gli1(+) cells proliferate after kidney, lung, liver, or heart injury to generate myofibroblasts. Genetic ablation of these cells substantially ameliorates kidney and heart fibrosis and preserves ejection fraction in a model of induced heart failure. These findings implicate perivascular Gli1(+) MSC-like cells as a major cellular origin of organ fibrosis and demonstrate that these cells may be a relevant therapeutic target to prevent solid organ dysfunction after injury.

Rationale of mesenchymal stem cell therapy in kidney injury.

Hundreds of clinical trials are currently investigating the potential for mesenchymal stem cells (MSC) to treat human disease, including kidney disease. There is tremendous excitement over the therapeutic potential of this form of stem cell therapy and an improving understanding of how MSC act. This review will summarize our current knowledge concerning the mechanisms by which MSC accelerate kidney repair after acute injury. It will also survey the current MSC clinical trial landscape in nephrology. Finally, future challenges to a widespread application of MSC therapies for patients with kidney injury will be outlined.

Translational profiles of medullary myofibroblasts during kidney fibrosis.

Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)-tagged L10a ribosomal subunit protein under control of the collagen1α1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1α1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies.

"Zebrafishing" for novel genes relevant to the glomerular filtration barrier.

Data for genes relevant to glomerular filtration barrier function or proteinuria is continually increasing in an era of microarrays, genome-wide association studies, and quantitative trait locus analysis. Researchers are limited by published literature searches to select the most relevant genes to investigate. High-throughput cell cultures and other in vitro systems ultimately need to demonstrate proof in an in vivo model. Generating mammalian models for the genes of interest is costly and time intensive, and yields only a small number of test subjects. These models also have many pitfalls such as possible embryonic mortality and failure to generate phenotypes or generate nonkidney specific phenotypes. Here we describe an in vivo zebrafish model as a simple vertebrate screening system to identify genes relevant to glomerular filtration barrier function. Using our technology, we are able to screen entirely novel genes in 4-6 weeks in hundreds of live test subjects at a fraction of the cost of a mammalian model. Our system produces consistent and reliable evidence for gene relevance in glomerular kidney disease; the results then provide merit for further analysis in mammalian models.

Best supportive care and therapeutic plasma exchange with or without eculizumab in Shiga-toxin-producing E. coli O104:H4 induced haemolytic-uraemic syndrome: an analysis of the German STEC-HUS registry.

BACKGROUND: May 22nd marks the beginning of a Shiga-toxin-producing Escherichia coli (STEC) O104:H4 outbreak in Northern Germany. By its end on 27 July, it had claimed 53 deaths among 2987 STEC and 855 confirmed haemolytic-uraemic syndrome (HUS) cases. METHODS: To describe short-term effectiveness of best supportive care (BSC), therapeutic plasma exchange (TPE) and TPE with eculizumab (TPE-Ecu) in 631 patients with suspected HUS treated in 84 hospitals in Germany, Sweden and the Netherlands using the web-based registry of the DGfN (online since 27 May). RESULTS: Of 631 entries, 491 fulfilled the definition of HUS (median age 46 years; 71% females). The median (inter-quartile range) hospital stay was 22 (14-31) days. Two hundred and eighty-one (57%) patients underwent dialysis and 114 (23%) mechanical ventilation. Fifty-seven patients received BSC, 241 TPE and 193 TPE-Ecu. Treatment strategy was dependent on disease severity (laboratory signs of haemolysis, thrombocytopenia, peak creatinine level, need for dialysis, neurological symptoms, frequency of seizures) which was lower in BSC than in TPE and TPE-Ecu patients. At study endpoint (hospital discharge or death), the median creatinine was lower in BSC [1.1 mg/dL (0.9-1.3)] than in TPE [1.2 mg/dL (1.0-1.5), P < 0.05] and TPE-Ecu [1.4 mg/dL (1.0-2.2), P < 0.001], while need for dialysis was not different between BSC (0.0%, n = 0), TPE (3.7%; n = 9) and TPE-Ecu (4.7%, n = 9). Seizures were absent in BSC and rare in TPE (0.4%; n = 1) and TPE-Ecu (2.6%; n = 5) patients. Total hospital mortality in HUS patients was 4.1% (n = 20) and did not differ significantly between the TPE and TPE-Ecu groups. CONCLUSIONS: Despite frequent renal impairment, advanced neurological disorders and severe respiratory failure, short-term outcome was better than expected when compared with previous reports. Within the limitations of a retrospective registry analysis, our data do not support the notion of a short-term benefit of Ecu in comparison to TPE alone in the treatment of STEC-HUS. A randomized trial comparing BSC, TPE and Ecu seems to be prudent and necessary prior to establishing new treatment guidelines for STEC-HUS.

Sonic hedgehog is an autocrine viability factor for myofibroblastic hepatic stellate cells.

BACKGROUND/AIMS: Factors released during liver injury, such as platelet derived growth factor-BB (PDGF), promote accumulation of myofibroblastic hepatic stellate cells (MFB) that drive the pathogenesis of cirrhosis. The hedgehog (Hh) pathway regulates remodeling of other injured tissues. This study evaluates the hypothesis that autocrine production of Sonic hedgehog (Shh) promotes MFB growth. METHODS: Primary rat hepatic stellate cells (HSC) were treated without or with PDGF, a pharmacologic inhibitor of PDGF-regulated kinases, adenovirus expressing activated or dominant negative AKT, or Hh signaling inhibitors. Shh production, expression of Hh inhibitors and target genes, and HSC growth were assessed. RESULTS: HSC expressed Shh, Hh pathway components, and the Hh inhibitor, Hip. During culture Hip expression fell, Shh production increased, and Hh target gene expression was induced. Neutralizing Shh antibodies promoted apoptosis. Adding PDGF increased Shh expression and MFB growth. Both processes followed activation of AKT and were abrogated by AKT inhibitors. Adenoviral delivery of activated AKT up-regulated Shh expression, demonstrating a direct role for AKT in regulating Shh expression. Shh-neutralizing antibodies and other Hh pathway inhibitors blocked the mitogenic effects of PDGF. CONCLUSIONS: These results identify Shh as an autocrine growth factor for MFB and suggest a role for Hh signaling in the pathogenesis of cirrhosis.

Hepatic accumulation of Hedgehog-reactive progenitors increases with severity of fatty liver damage in mice.

Progenitors regenerate fatty livers but the mechanisms involved are uncertain. The Hedgehog pathway regulates mesendodermal progenitors and modulates mesenchymal-epithelial interactions during tissue remodeling. To determine if Hedgehog signaling increases in liver progenitors during fatty liver injury, we compared expression of Hedgehog ligands and target genes across a spectrum of injury. Leptin-deficient ob/ob mice with fatty livers and their healthy lean littermates were studied before and after exposure to the hepatotoxin, ethionine. At baseline, ob/ob mice had greater liver damage than controls. Ethionine induced liver injury in both ob/ob and lean mice, with greater injury occurring in ob/ob mice. After ethionine, the ob/ob mice developed liver atrophy and fibrosis. Liver injury increased hepatic accumulation of progenitors, including ductular cells that produced and responded to Hedgehog ligands. A dose-response relationship was demonstrated between liver injury and expansion of Hedgehog-responsive progenitors. In severely damaged, atrophic livers, nuclei in mature-appearing hepatocytes accumulated the Hedgehog-regulated mesenchymal transcription factor, Gli2 and lost expression of the liver epithelial transcription factor, hepatocyte nuclear factor 6 (HNF-6). Hepatic levels of collagen mRNA and pericellular collagen fibrils increased concomitantly. Hence, fatty liver injury increases Hedgehog activity in liver progenitors, and this might promote epithelial-mesenchymal transitions that result in liver fibrosis.