Identification of platelet-derived growth factor C as a mediator of both renal fibrosis and hypertension.

Platelet-derived growth factors (PDGF) have been implicated in kidney disease progression. We previously found that PDGF-C is upregulated at sites of renal fibrosis and that antagonism of PDGF-C reduces fibrosis in the unilateral ureteral obstruction model. We studied the role of PDGF-C in collagen 4A3-/- ("Alport") mice, a model of progressive renal fibrosis with greater relevance to human kidney disease. Alport mice were crossbred with PDGF-C-/- mice or administered a neutralizing PDGF-C antibody. Both PDGF-C deficiency and neutralization reduced serum creatinine and blood urea nitrogen levels and mitigated glomerular injury, renal fibrosis, and renal inflammation. Unexpectedly, systolic blood pressure was also reduced in both Alport and wild-type mice treated with a neutralizing PDGF-C antibody. Neutralization of PDGF-C reduced arterial wall thickness in the renal cortex of Alport mice. Aortic rings isolated from anti-PDGF-C-treated wildtype mice exhibited reduced tension and faster relaxation than those of untreated mice. In vitro, PDGF-C upregulated angiotensinogen in aortic tissue and in primary hepatocytes and induced nuclear factor κB (NFκB)/p65-binding to the angiotensinogen promoter in hepatocytes. Neutralization of PDGF-C suppressed transcript expression of angiotensinogen in Alport mice and angiotensin II receptor type 1 in Alport and wildtype mice. Finally, administration of neutralizing PDGF-C antibodies ameliorated angiotensin II-induced hypertension in healthy mice. Thus, in addition to its key role in mediating renal fibrosis, we identified PDGF-C as a mediator of hypertension via effects on renal vasculature and on the renin-angiotensin system. The contribution to both renal fibrosis and hypertension render PDGF-C an attractive target in progressive kidney disease.

YB-1 increases glomerular, but decreases interstitial fibrosis in CNI-induced nephropathy.

Calcineurin inhibitors (CNIs) are a cornerstone of the current treatment in solid organ transplantation and autoimmune disease. However, CNIs also bear deleterious effects as they cause glomerular and tubulointerstitial fibrosis in the kidney. We recently identified Y-box protein-1 (YB-1) as a novel downstream effector of CNI-signaling in the cytoplasm of glomerular cells. In the present study, we corroborate the pro-fibrotic role of YB-1 in glomeruli of patients under CNI-treatment. Such effects in glomeruli are significantly mitigated in CNI-treated mice with half-normal YB-1 expression (Yb1+/-). Surprisingly, in the tubulointerstitium we observe an opposite role of the CNI-YB-1 axis. Here, YB-1 is predominantly located to the nuclei and represses transcription of several extracellular matrix genes. Consistently, CNI-treatment in Yb1+/- mice markedly increases pro-fibrotic changes in the tubulointerstitium. In summary, our data provide evidence that fibrotic CNI-induced YB-1 effects in glomerular cells need to be contrasted with beneficial anti-fibrotic effects in the tubulointerstitium.

YB-1 orchestrates onset and resolution of renal inflammation via IL10 gene regulation.

The Y-box-binding protein (YB)-1 plays a non-redundant role in both systemic and local inflammatory response. We analysed YB-1-mediated expression of the immune regulatory cytokine IL-10 in both LPS and sterile inflammation induced by unilateral renal ischaemia-reperfusion (I/R) and found an important role of YB-1 not only in the onset but also in the resolution of inflammation in kidneys. Within a decisive cis-regulatory region of the IL10 gene locus, the fourth intron, we identified and characterized an operative YB-1 binding site via gel shift experiments and reporter assays in immune and different renal cells. In vivo, YB-1 phosphorylated at serine 102 localized to the fourth intron, which was paralleled by enhanced IL-10 mRNA expression in mice following LPS challenge and in I/R. Mice with half-maximal expression of YB-1 (Yb1+/- ) had diminished IL-10 expression upon LPS challenge. In I/R, Yb1+/- mice exhibited ameliorated kidney injury/inflammation in the early-phase (days 1 and 5), however showed aggravated long-term damage (day 21) with increased expression of IL-10 and other known mediators of renal injury and inflammation. In conclusion, these data support the notion that there are context-specific decisions concerning YB-1 function and that a fine-tuning of YB-1, for example, via a post-translational modification regulates its activity and/or localization that is crucial for systemic processes such as inflammation.

Macrophage Migration Inhibitory Factor Mediates Proliferative GN via CD74.

Pathologic proliferation of mesangial and parietal epithelial cells (PECs) is a hallmark of various glomerulonephritides. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that mediates inflammation by engagement of a receptor complex involving the components CD74, CD44, CXCR2, and CXCR4. The proliferative effects of MIF may involve CD74 together with the coreceptor and PEC activation marker CD44. Herein, we analyzed the effects of local glomerular MIF/CD74/CD44 signaling in proliferative glomerulonephritides. MIF, CD74, and CD44 were upregulated in the glomeruli of patients and mice with proliferative glomerulonephritides. During disease, CD74 and CD44 were expressed de novo in PECs and colocalized in both PECs and mesangial cells. Stress stimuli induced MIF secretion from glomerular cells in vitro and in vivo, in particular from podocytes, and MIF stimulation induced proliferation of PECs and mesangial cells via CD74. In murine crescentic GN, Mif-deficient mice were almost completely protected from glomerular injury, the development of cellular crescents, and the activation and proliferation of PECs and mesangial cells, whereas wild-type mice were not. Bone marrow reconstitution studies showed that deficiency of both nonmyeloid and bone marrow-derived Mif reduced glomerular cell proliferation and injury. In contrast to wild-type mice, Cd74-deficient mice also were protected from glomerular injury and ensuing activation and proliferation of PECs and mesangial cells. Our data suggest a novel molecular mechanism and glomerular cell crosstalk by which local upregulation of MIF and its receptor complex CD74/CD44 mediate glomerular injury and pathologic proliferation in GN.

IL-6 Trans-Signaling Drives Murine Crescentic GN.

The role of IL-6 signaling in renal diseases remains controversial, with data describing both anti-inflammatory and proinflammatory effects. IL-6 can act via classic signaling, engaging its two membrane receptors gp130 and IL-6 receptor (IL-6R). Alternatively, IL-6 trans-signaling requires soluble IL-6R (sIL-6R) to act on IL-6R-negative cells that express gp130. Here, we characterize the role of both pathways in crescentic nephritis. Patients with crescentic nephritis had significantly elevated levels of IL-6 in both serum and urine. Similarly, nephrotoxic serum-induced nephritis (NTN) in BALB/c mice was associated with elevated serum IL-6 levels. Levels of serum sIL-6R and renal downstream signals of IL-6 (phosphorylated signal transducer and activator of transcription 3, suppressor of cytokine signaling 3) increased over time in this model. Simultaneous inhibition of both IL-6 signaling pathways using anti-IL-6 antibody did not have a significant impact on NTN severity. In contrast, specific inhibition of trans-signaling using recombinant sgp130Fc resulted in milder disease. Vice versa, specific activation of trans-signaling using a recombinant IL-6-sIL-6R fusion molecule (Hyper-IL-6) significantly aggravated NTN and led to increased systolic BP in NTN mice. This correlated with increased renal mRNA synthesis of the Th17 cell cytokine IL-17A and decreased synthesis of resistin-like alpha (RELMalpha)-encoding mRNA, a surrogate marker of lesion-mitigating M2 macrophage subtypes. Collectively, our data suggest a central role for IL-6 trans-signaling in crescentic nephritis and offer options for more effective and specific therapeutic interventions in the IL-6 system.

Therapeutic nuclear shuttling of YB-1 reduces renal damage and fibrosis.

Virtually all chronic kidney diseases progress towards tubulointerstitial fibrosis. In vitro, Y-box protein-1 (YB-1) acts as a central regulator of gene transcription and translation of several fibrosis-related genes. However, it remains to be determined whether its pro- or antifibrotic propensities prevail in disease. Therefore, we investigated the outcome of mice with half-maximal YB-1 expression in a model of renal fibrosis induced by unilateral ureteral obstruction. Yb1+/- animals displayed markedly reduced tubular injury, immune cell infiltration and renal fibrosis following ureteral obstruction. The increase in renal YB-1 was limited to a YB-1 variant nonphosphorylated at serine 102 but phosphorylated at tyrosine 99. During ureteral obstruction, YB-1 localized to the cytoplasm, directly stabilizing Col1a1 mRNA, thus promoting fibrosis. Conversely, the therapeutic forced nuclear compartmentalization of phosphorylated YB-1 by the small molecule HSc025 mediated repression of the Col1a1 promoter and attenuated fibrosis following ureteral obstruction. Blunting of these effects in Yb1+/- mice confirmed involvement of YB-1. HSc025 even reduced tubulointerstitial damage when applied at later time points during maximum renal damage. Thus, phosphorylation and subcellular localization of YB-1 determines its effect on renal fibrosis in vivo. Hence, induced nuclear YB-1 shuttling may be a novel antifibrotic treatment strategy in renal diseases with the potential of damage reversal.

Interaction of atypical cadherin Fat1 with SoHo adaptor proteins CAP/ponsin and ArgBP2.

Mammalian Fat1 is a giant atypical cadherin/tumor suppressor involved in the regulation of cellular orientation, migration, and growth. Fat1 is implicated in the development of the brain, eye, and kidney. Altered expression or mutations of FAT1 are also associated with cancer and facioscapulohumeral muscular dystrophy (FSHD). Yet, the mechanistic functions of this pathway remain incompletely understood. Here, we report the identification of Sorbin-homology (SoHo) proteins as novel interaction partners of Fat1 by virtue of a yeast-two-hybrid screen. SoHo proteins play diverse roles as adaptor proteins in cell signaling, cell adhesion and sarcomere architecture, including altered expression in cancer and FSHD. Specifically, we found SoHo proteins CAP/ponsin-1 and -2 (Sorbs1) and ArgBP2 (Sorbs2) to interact with the cytoplasmic domain of Fat1. We mapped the interaction to a prolin-rich classic type II PXXP motif within Fat1 and to the three Src-homology (SH3) domains within SoHo proteins using mutant expression in yeast, pulldown assays, and cell culture. Functionally, endogenous ponsin-2 expression of NRK-52E cells at cellular leading edges was lost upon knockdown of Fat1. In summary, our data point to an interaction of Fat1 with SoHo proteins that is able to recruit SoHo proteins to sites of Fat1 expression.

The atypical cadherin Dachsous1 localizes to the base of the ciliary apparatus in airway epithelia.

Mucociliary clearance requires the distinct orientation and coordinated movement of airway cilia, which is established through planar cell polarity signaling (PCP). The atypical cadherin Dachsous1 (Dchs1) is a transmembrane protein that regulates PCP in D. melanogaster. However, little is known about Dchs1 expression and its potential role in PCP in mammalian adult tissues. Here, we show that Dchs1 is ubiquitously expressed in mouse embryos, but exhibits a highly restricted expression to lung tissues in the adult stage. Strikingly, human Dchs1 localized exclusively to the base of the ciliary apparatus in cultured human respiratory epithelial cells with differentiated motile 9 + 2 cilia. This localization could be functionally important as we observed aberrant DCHS1 mRNA expression in human non-small cell lung cancer tissue. In sum, we establish Dchs1 as a component of the membrane domain surrounding the ciliary base. This suggests a specific role of Dchs1 in PCP-dependent organization of ciliary function and a possible role in lung disease.

Gp130-dependent signaling in the podocyte.

Renal inflammation, in particular glomerular, is often characterized by increased IL-6 levels. The in vivo relevance of IL-6 signaling in glomerular podocytes, which play central roles in most glomerular diseases, is unknown. Here, we show that in normal mice, podocytes express gp130, the common signal-transducing receptor subunit of the IL-6 family of cytokines. Following systemic IL-6 or LPS injection in mice, podocyte IL-6 signaling was evidenced by downstream STAT3 phosphorylation. Next, we generated mice deficient for gp130 in podocytes. Expectedly, these mice exhibited abrogated IL-6 downstream signaling in podocytes. At the age of 40 wk, they did not show spontaneous renal pathology or abnormal renal function. The mice were then challenged using two LPS injury models as well as nephrotoxic serum to induce crescentic nephritis. Under all conditions, circulating IL-6 levels increased markedly and the mice developed the pathological hallmarks of the corresponding injury models such as proteinuria and development of glomerular crescents, respectively. However, despite the capacity of normal podocytes to transduce IL-6 family signals downstream, there were no significant differences between mice bearing the podocyte-specific gp130 deletion and their control littermates in any of these models. In conclusion, under the different conditions tested, gp130 signaling was not a critical component of the (patho-)biology of the podocyte in vivo.

A simple clinical score to reduce unnecessary testing for Puumala hantavirus.

BACKGROUND: Puumala hantavirus (PUUV) causes nephropathia epidemica (NE), an endemic form of transient acute renal injury (AKI). Serological testing is the mainstay of diagnosis. It was the aim of the present study to assist decision-making for serological testing by constructing a simple tool that predicts the likelihood of PUUV positivity. METHODS: We conducted a comparative cohort study of all PUUV-tested cases at Aachen University tertiary care center in Germany between mid-2013 and mid-2021. N = 293 qualified for inclusion; N = 30 had a positive test result and clinical NE; N = 263 were negative. Two predictive point scores, the Aachen PUUV Score (APS) 1 and 2, respectively, were derived with the aid of logistic regression and receiver operating characteristic (ROC) analysis by determining the presence of four admission parameters. For internal validation, the internal Monte Carlo method was applied. In addition, partial external validation was performed using an independent historic cohort of N = 41 positive cases of NE. RESULTS: APS1 is recommended for clinical use as it estimated the probability of PUUV positivity in the entire medical population tested. With a range from 0 to 6 points, it yielded an area under the curve of 0.94 by allotting 2 points each for fever or headache and 1 point each for AKI or LDH>300 U/L. A point sum of 0-2 safely predicted negativity for PUUV, as was confirmed in the NE validation cohort. CONCLUSION: Here, we present a novel, easy-to-use tool to guide the diagnostic management of suspected PUUV infection/NE and to safely avoid unnecessary serological testing, as indicated by point sum class 0-2. Since 67% of the cohort fell into this stratum, half of the testing should be avoidable in the future.

Electrical forces determine glomerular permeability.

There is ongoing controversy about the mechanisms that determine the characteristics of the glomerular filter. Here, we tested whether flow across the glomerular filter generates extracellular electrical potential differences, which could be an important determinant of glomerular filtration. In micropuncture experiments in Necturus maculosus, we measured a potential difference across the glomerular filtration barrier that was proportional to filtration pressure (-0.045 mV/10 cm H2O). The filtration-dependent potential was generated without temporal delay and was negative within Bowman's space. Perfusion with the cationic polymer protamine abolished the potential difference. We propose a mathematical model that considers the relative contributions of diffusion, convection, and electrophoretic effects on the total flux of albumin across the filter. According to this model, potential differences of -0.02 to -0.05 mV can induce electrophoretic effects that significantly influence the glomerular sieving coefficient of albumin. This model of glomerular filtration has the potential to provide a mechanistic theory, based on experimental data, about the filtration characteristics of the glomerular filtration barrier. It provides a unique approach to the microanatomy of the glomerulus, renal autoregulation, and the pathogenesis of proteinuria.

Time on previous renal replacement therapy is associated with worse outcomes of COVID-19 in a regional cohort of kidney transplant and dialysis patients.

ABSTRACT: Chronic renal replacement therapy by either a kidney transplant (KTX) or hemodialysis (HD) predisposes patients to an increased risk for adverse outcomes of COVID-19. However, details on this interaction remain incomplete. To provide further characterization, we undertook a retrospective observational cohort analysis of the majority of the hemodialysis and renal transplant population affected by the first regional outbreak of severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) in Germany. In a region of 250,000 inhabitants we identified a total of 21 cases with SARS-CoV-2 among 100 KTX and 260 HD patients, that is, 7 KTX with COVID-19, 14 HD with COVID-19, and 3 HD with asymptomatic carrier status. As a first observation, KTX recipients exhibited trends for a higher mortality (43 vs 18%) and a higher proportion of acute respiratory distress syndrome (ARDS) (57 vs 27%) when compared to their HD counterparts. As a novel finding, development of ARDS was significantly associated with the time spent on previous renal replacement therapy (RRT), defined as the composite of dialysis time and time on the transplant (non-ARDS 4.3 vs ARDS 10.6 years, P = .016). Multivariate logistic regression analysis showed an OR of 1.7 per year of RRT. The association remained robust when analysis was confined to KTX patients (5.1 vs 13.2 years, P = .002) or when correlating the time spent on a renal transplant alone (P = .038). Similarly, longer RRT correlated with death vs survival (P = .0002). In conclusion our data suggest renal replacement vintage as a novel risk factor for COVID-19-associated ARDS and death. The findings should be validated by larger cohorts.

Validation of a Prospective Urinalysis-Based Prediction Model for ICU Resources and Outcome of COVID-19 Disease: A Multicenter Cohort Study.

In COVID-19, guidelines recommend a urinalysis on hospital admission as SARS-CoV-2 renal tropism, post-mortem, was associated with disease severity and mortality. Following the hypothesis from our pilot study, we now validate an algorithm harnessing urinalysis to predict the outcome and the need for ICU resources on admission to hospital. Patients were screened for urinalysis, serum albumin (SA) and antithrombin III activity (AT-III) obtained prospectively on admission. The risk for an unfavorable course was categorized as (1) "low", (2) "intermediate" or (3) "high", depending on (1) normal urinalysis, (2) abnormal urinalysis with SA ≥ 2 g/dL and AT-III ≥ 70%, or (3) abnormal urinalysis with SA or AT-III abnormality. Time to ICU admission or death served as the primary endpoint. Among 223 screened patients, 145 were eligible for enrollment, 43 falling into the low, 84 intermediate, and 18 into high-risk categories. An abnormal urinalysis significantly elevated the risk for ICU admission or death (63.7% vs. 27.9%; HR 2.6; 95%-CI 1.4 to 4.9; p = 0.0020) and was 100% in the high-risk group. Having an abnormal urinalysis was associated with mortality, a need for mechanical ventilation, extra-corporeal membrane oxygenation or renal replacement therapy. In conclusion, our data confirm that COVID-19-associated urine abnormalities on admission predict disease aggravation and the need for ICU (ClinicalTrials.gov number NCT04347824).

Occurrence of HSV-1-induced pneumonitis in patients under standard immunosuppressive therapy for rheumatic, vasculitic, and connective tissue disease.

BACKGROUND: Herpes simplex virus type-1 (HSV-1) has been described to cause respiratory tract infections in critically ill patients or in individuals that are immunocompromised. It is a continuing matter of debate under which circumstances HSV-1 is a relevant pathogen for pneumonitis. While its role during critical illness has been investigated by prospective interventional studies, comparatively little systematic data is available on the role of HSV-1 for pneumonitis in outpatients with autoimmune disease under a maintenance regimen of immunosuppression. METHODS: We retrospectively reviewed the charts of approximately 1400 patients with rheumatoid arthritis, vasculitis, and systemic lupus erythematosus (SLE) that were followed at the outpatient clinic of a German University hospital during the years 2000-2007. Episodes of admission to a ward resulting in the diagnosis of pneumonia/pneumonitis were identified, and the type of pneumonia and clinical features retrospectively studied. RESULTS: 63 patients with rheumatoid arthritis, vasculitis, or SLE were admitted to a ward and diagnosed to have pneumonia/pneumonitis. Using bronchoscopy a total of 6 cases of pulmonary infection associated with HSV-1 in the lower respiratory tract were identified. Among those, 2 cases suggested a causative role of HSV-1 as the sole agent causing pneumonitis that proved clinically responsive to antiviral treatment. In the remaining 4 cases HSV-1 appeared as a bystander of bacterial infection. Maintenance therapy with leflunomide, which inhibits HSV-1 assembly in vitro, was associated with a milder course of pneumonitis in one patient. Detection of HSV-1 was associated with stronger immunosuppressive regimens and vasculitic disease. CONCLUSION: The present study analyzed the frequency and hallmarks of cases of HSV-1 associated pneumonitis that occurred in a comparatively large cohort of patients with rheumatologic autoimmune diseases. In an area of controversy, this study provides further evidence that HSV-1 causes isolated pneumonitis in the immunocompromised. The study may provide an estimate on the frequency of relevant HSV-1 infection and bacterial agents in outpatients with autoimmune disease.

Cryoglobulinaemic vasculitis: classification and clinical and therapeutic aspects.

Cryoglobulinaemia may cause cutaneous vasculitis and glomerulonephritis, potentially leading to end stage renal failure. An important proportion of cryoglobulinaemias are secondary to hepatitis C virus infection. Emerging antiviral treatment options offer a chance for causal therapy of these cases of cryoglobulinaemia. This review summarises the classification and clinical and therapeutic aspects of cryoglobulinaemic vasculitis and glomerulonephritis.

Mycoplasma pneumoniae: usual suspect and unsecured diagnosis in the acute setting.

Mycoplasma pneumoniae is one of the most common known bacterial pathogens of the respiratory tract, especially in patients between 5 and 30 years of age. It may be encountered at a relatively high rate in the non-life-threatened fraction of Emergency Department (ED) patients presenting with upper respiratory symptoms or cough. Yet its hallmarks are very non-specific, including a great variety of presentations from mild pharyngitis to potentially life-threatening complications such as the Stevens-Johnson Syndrome. Here, we describe a typical case of pneumonia due to Mycoplasma pneumoniae in a young adult with mild pharyngitis as the leading symptom. Disease presentation, complications, diagnostic means, therapeutic options, and suspicious clinical settings are discussed to provide a review on the clinical aspects of the disease that are important in the ED setting.

Anti-interleukin-6 therapy through application of a monogenic protein inhibitor via gene delivery.

Anti-cytokine therapies have substantially improved the treatment of inflammatory and autoimmune diseases. Cytokine-targeting drugs are usually biologics such as antibodies or other engineered proteins. Production of biologics, however, is complex and intricate and therefore expensive which might limit therapeutic application. To overcome this limitation we developed a strategy that involves the design of an optimized, monogenic cytokine inhibitor and the protein producing capacity of the host. Here, we engineered and characterized a receptor fusion protein, mIL-6-RFP-Fc, for the inhibition of interleukin-6 (IL-6), a well-established target in anti-cytokine therapy. Upon application in mice mIL-6-RFP-Fc inhibited IL-6-induced activation of the transcription factor STAT3 and ERK1/2 kinases in liver and kidney. mIL-6-RFP-Fc is encoded by a single gene and therefore most relevant for gene transfer approaches. Gene transfer through hydrodynamic plasmid delivery in mice resulted in hepatic production and secretion of mIL-6-RFP-Fc into the blood in considerable amounts, blocked hepatic acute phase protein synthesis and improved kidney function in an ischemia and reperfusion injury model. Our study establishes receptor fusion proteins as promising agents in anti-cytokine therapies through gene therapeutic approaches for future targeted and cost-effective treatments. The strategy described here is applicable for many cytokines involved in inflammatory and other diseases.

Development of renal function.

The mammalian metanephric kidney develops following a general principle of organogenesis of epithelial organs, i.e., along the tree-like structure of an arborizing ductal system (the ureteric bud and cortical collecting duct). In parallel, the proximal portions of the uriniferous tubule develop by mesenchymal-to-epithelial transition of the neighbouring mesenchyme. On one hand, vectorial transport systems in nephrogenesis should be functional at the onset of glomerular filtration in any of the newly formed nephron generations to prevent loss of salt, water and metabolites. On the other hand, developing nephron epithelia must serve the needs of organ-formation such as cell proliferation and fluid-secretion for morphogenic purposes. This review intends to summarize current data and concepts on the development of renal epithelial functions with an emphasis on ion channels. Current model systems are introduced, such as ureteric bud cell monolayer culture, in vitro nephron culture, HEK293 cell culture, and the dissection of tubular cells for direct analysis. The current data on the developmental expression and functions of ENaC Na(+) channels, the CFTR, ClC-2 Cl(ndash;) channels, L-type Ca(2+) channels, P2 purinoceptors, and the Kir6.1/SUR2, ROMK (Kir1.1), and Kv K(+) channels are presented.