Enhanced pulmonary expression of the TrkB neurotrophin receptor in hypoxic rats is associated with increased acetylcholine-induced airway contractility

AIM We have recently reported that hypoxia stimulates transcription of the TrkB neurotrophin receptor in cultured cells via stabilization of hypoxia-inducible factor-1alpha. Here we investigated whether the expression of TrkB and other neurotrophin receptors is oxygen-sensitive also in vivo, and explored the functional consequences of an oxygen-regulated TrkB expression. METHODS Rats were exposed either to 21% O(2) or 8% O(2) for 6 h and TrkB was analysed by reverse transcription real-time PCR, in situ mRNA hybridization, and immunological techniques. The importance of the brain-derived neurotrophic factor (BDNF)-TrkB pathway in the control of mechanical airway function was assessed on isolated tracheal segments from normoxic and hypoxic rats. RESULTS TrkB transcripts were increased approx. 15-fold in the lungs of hypoxic rats, and the respiratory epithelium was identified as the site of enhanced TrkB expression in hypoxia. The TrkB ligand, BDNF, significantly increased the contractile response to acetylcholine (ACh) of isolated tracheal segments from hypoxic but not from normoxic rats. This effect of BDNF was prevented by pre-incubation of the tissue specimens with the tyrosine kinase inhibitor K252a and by mechanical removal of the TrkB containing airway epithelium. Likewise, the nitric oxide (NO) synthase inhibitor l-NAME abrogated the influence of BDNF on ACh-induced contractions of isolated tracheal segments from hypoxic rats. CONCLUSION These results demonstrate that systemic hypoxia stimulates expression of the TrkB neurotrophin receptor in the airway epithelium. Furthermore, activation of TrkB signalling by BDNF in hypoxia enhances mechanical airway contractility to ACh through a mechanism that requires NO

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

Impact of primary kidney disease on the effects of empagliflozin in patients with chronic kidney disease: secondary analyses of the EMPA-KIDNEY trial

Background: The EMPA KIDNEY trial showed that empagliflozin reduced the risk of the primary composite outcome of kidney disease progression or cardiovascular death in patients with chronic kidney disease mainly through slowing progression. We aimed to assess how effects of empagliflozin might differ by primary kidney disease across its broad population. Methods: EMPA-KIDNEY, a randomised, controlled, phase 3 trial, was conducted at 241 centres in eight countries (Canada, China, Germany, Italy, Japan, Malaysia, the UK, and the USA). Patients were eligible if their estimated glomerular filtration rate (eGFR) was 20 to less than 45 mL/min per 1·73 m2, or 45 to less than 90 mL/min per 1·73 m2 with a urinary albumin-to-creatinine ratio (uACR) of 200 mg/g or higher at screening. They were randomly assigned (1:1) to 10 mg oral empagliflozin once daily or matching placebo. Effects on kidney disease progression (defined as a sustained ≥40% eGFR decline from randomisation, end-stage kidney disease, a sustained eGFR below 10 mL/min per 1·73 m2, or death from kidney failure) were assessed using prespecified Cox models, and eGFR slope analyses used shared parameter models. Subgroup comparisons were performed by including relevant interaction terms in models. EMPA-KIDNEY is registered with ClinicalTrials.gov, NCT03594110. Findings: Between May 15, 2019, and April 16, 2021, 6609 participants were randomly assigned and followed up for a median of 2·0 years (IQR 1·5–2·4). Prespecified subgroupings by primary kidney disease included 2057 (31·1%) participants with diabetic kidney disease, 1669 (25·3%) with glomerular disease, 1445 (21·9%) with hypertensive or renovascular disease, and 1438 (21·8%) with other or unknown causes. Kidney disease progression occurred in 384 (11·6%) of 3304 patients in the empagliflozin group and 504 (15·2%) of 3305 patients in the placebo group (hazard ratio 0·71 [95% CI 0·62–0·81]), with no evidence that the relative effect size varied significantly by primary kidney disease (pheterogeneity=0·62). The between-group difference in chronic eGFR slopes (ie, from 2 months to final follow-up) was 1·37 mL/min per 1·73 m2 per year (95% CI 1·16–1·59), representing a 50% (42–58) reduction in the rate of chronic eGFR decline. This relative effect of empagliflozin on chronic eGFR slope was similar in analyses by different primary kidney diseases, including in explorations by type of glomerular disease and diabetes (p values for heterogeneity all >0·1). Interpretation: In a broad range of patients with chronic kidney disease at risk of progression, including a wide range of non-diabetic causes of chronic kidney disease, empagliflozin reduced risk of kidney disease progression. Relative effect sizes were broadly similar irrespective of the cause of primary kidney disease, suggesting that SGLT2 inhibitors should be part of a standard of care to minimise risk of kidney failure in chronic kidney disease. Funding: Boehringer Ingelheim, Eli Lilly, and UK Medical Research Council

Interactions in the juxtaglomerular apparatus of the kidney

Titelblatt und Inhaltsverzeichnis Einleitung Herleitung der Aufgabenstellung Material und Methoden Ergebnisse Diskussion Zusammenfassung LiteraturverzeichnisEinleitung: Im juxtaglomerulären Apparat der Niere (JGA) werden die neuronale Isoform der Stickstoffmonoxid-Synthase (NOS1) und die induzierbare Isoform der Cyclooxygenase (COX-2) exprimiert. Beide Enzyme weisen Ähnlichkeiten im Expressionsmuster auf und sind an der Langzeit-Regulation der Nierenfunktion und an der Steuerung der Aktivität des Renin-Angiotensin-Aldosteronsystems (RAAS) beteiligt. Versuche zur Aufklärung der Mechanismen einer eventuellen Interaktion erbrachten jedoch widersprüchliche Resultate. Gegenstand der vorliegenden Arbeit war die Charakterisierung der JGA-Enzymexpression in NOS1 defizienten (NOS1-/-) bzw. COX-2 defizienten (COX-2-/-) Mäusen. Ein weiterer Teil der Studie diente der Untersuchung der Auswirkungen einer Behandlung mit dem NADPH-Oxidase-Inhibitor Apocynin auf Blutdruck, oxidativen Stress und die Expression der JGA-Enzyme in spontan hypertensiven Ratten (SHR) und Wistar- Kyoto-Kontrolltieren (WKY). Resultate: Der Vergleich zwischen Kontrolltieren und NOS1-/- Mäusen zeigte keine Unterschiede der basalen JGA-assoziierten COX-2 Immunreaktivität (IR). Auch die basale Renin IR war nicht unterschiedlich. Im Gegensatz dazu waren die basale Renin mRNA und Plasma- Reninaktivität (PRA) bei NOS1-/- Mäusen auf 64% bzw. 53% verringert. Eine Diät mit niedrigem Salzgehalt führte bei Kontrolltieren und NOS1-/- Mäusen gleichermassen zu einer Steigerung, eine Diät mit hohem Salzgehalt hingegen bei beiden Gruppen zu einer Verminderung der COX-2 IR. Der Vergleich zwischen Kontrollmäusen und COX-2-/- Mäusen zeigte in erster Linie erhebliche renale Strukturveränderungen. In Übereinstimmung mit publizierten Daten waren die Nieren der COX-2-/- Mäuse kleiner und beinhalteten weniger Glomeruli als die der Kontrolltiere. Besonders in der subkapsulären Region des renalen Kortex waren die Glomeruli unreif bzw. hypoplastisch und enthielten weniger Zellen. Im Gegensatz dazu waren die Glomeruli der tieferen Regionen deutlich hypertrophiert. Die Quantifizierung von NOS1 IR und NADPH-Diaphoraseaktivität zeigte im Vergleich zu den Kontrolltieren signifikant gesteigerte Mengen in den normal entwickelten Glomeruli (1,6- bzw. 1,2-fache Steigerung subkapsulär, und 2,7 bzw. 3,4-fache Steigerung juxtamedullär). Die Gesamtanzahl der Renin- positiven JGA war auf 62% vermindert. Ältere COX-2-/- Mäuse zeigten eine signifikante Zunahme glomerulosklerotischer Läsionen in den hypertrophierten Glomeruli. Der Vergleich von SHR und WKY zeigte erhöhte Blutdruckwerte, eine auf das 2,2-Fache gesteigerte Ausscheidung von 8-Isoprostan F2 im Urin (IP), eine auf das 1,5-Fache vermehrte NOS1 IR, sowie eine auf 35% bzw. 51% verringerte COX-2 bzw. Renin IR bei den hypertensiven Tieren. Renin mRNA Expression und PRA waren auf 43% bzw. auf 7% reduziert. Die Gabe von Apocynin führte bei den SHR zu einer Reduktion der IP-Ausscheidung und NOS1 IR auf 52% bzw. 69%. COX-2 IR, Renin mRNA Expression und PRA blieben dabei unverändert. Die Renin IR war auf 62% vermindert. Schlussfolgerungen: Die vorliegende Arbeit liefert Aufschlüsse über das Zusammenwirken von NOS1- und COX-2-Produkten im JGA. Physiologische und pathologische Zusammenhänge der Funktion des JGA werden hierdurch klarer. Im einzelnen konnte gezeigt werden, dass die Regulation der Expression von COX-2 im JGA der Maus unabhängig von der Wirkung von NOS1 erfolgt. Hingegen wirken COX-2 Produkte inhibitorisch auf die Synthese von NOS1 hin. Bei Abwesenheit von COX-2 hat NOS1 offenbar fördernde Wirkung auf glomerulosklerotische Veränderungen. Eine gesteigerte NOS1 Expression bei experimenteller Hypertonie hängt von örtlicher Sauerstoffradikalbildung ab. Wird ein Antioxidans verabreicht, normalisiert sich dieser Parameter. Allerdings wird der Gedanke, dass reaktive Sauerstoffspezies Ursache für juxtaglomerulär veränderte COX-2- und Reninwerte sowie für vom JGA ausgehende Blutdruckverschiebungen sind, durch die Befunde nicht gerechtfertigt.Study outline: The juxtaglomerular apparatus (JGA) of the kidney constitutively expresses the cyclooxygenase-2 (COX-2) and the neuronal isoform of nitric oxide synthase (NOS1). These enzymes act to regulate renal function and renin biosynthesis. Aim of this study was to corroborate the hypothesis that interactive aspects between NOS1 and COX-2 play a role in JGA function. To do so the expression of these enzymes was determined in NOS1 deficient (NOS1-/-) and COX-2 deficient (COX-2-/-) mice. I have further investigated whether reactive oxygen species (ROS) may modify the expression of NOS1 and COX-2 in the JGA, and whether such an effect is relevant to juxtaglomerular signalling. To this end, the NADPH-oxidase inhibitor, apocynin, was systemically applied in spontaneously hypertensive rats (SHR) and Wistar Kyoto control rats (WKY). Effects of the treatment on oxidative stress markers, blood pressure, and on the expression of JGA enzymes were studied. Study protocol and results: Under baseline condition, NOS1-/- mice revealed an attenuation of renal renin mRNA expression to 64% and plasma activity of the enzyme (PRA) to 53%, whereas COX-2 and renin protein were unchanged compared to control littermates. Dietary sodium depletion increased COX-2 protein levels to similar extent in both strains, whereas sodium loading had opposite effects. In agreement with earlier data, COX-2-/- mice showed an abnormal renal morphology. The overall kidney size and number of glomeruli was reduced as compared to wildtype littermates. Glomeruli in the subcapsular portion of the renal cortex of COX-2-/- were smaller and displayed an immature phenotype. Juxtamedullary glomeruli were intact, but hypertrophied and showed sclerosis with advanced age. JGA-NOS1 expression and activity were increased 1.6- and 1.2-fold in the subcapsular and 2.7- and 3.4-fold in the juxtamedullary layer of the renal cortex. Renin biosynthesis was diminished (62%) as compared to controls. To study the role of ROS, young SHR and WKY received the specific NADPH oxidase inhibitor, apocynin, during 3 weeks. Strains were tested for renal functional and JGA histochemical parameters, plasma renin activity (PRA), and 24h urinary isoprostane excretion (IP) as a measure of ROS activity. IP in untreated SHR was 2.2-fold higher and NOS1 immunoreactiviy (IR) of JGA 1.5-fold higher, than in WKY. COX-2 IR was reduced to 35%, renin IR to 51%, and PRA to 7% of WKY levels. Apocynin treatment reduced IP in SHR to 52%, NOS1 IR to 69%, and renin IR to 62% of untreated SHR, whereas renin mRNA, COX-2 IR, GFR, PRA. and systolic blood pressure remained unchanged. WKY generally revealed no changes under apocynin treatment. Conclusions: These studies have permitted to gain insight into the interactions between NOS1 and COX-2 and may thus contribute to a better understanding of JGA function. Expression of COX-2 in the JGA of the mouse kidney does not depend on the activity of NOS1, whereas COX-2-derived metabolites have an inhibitory effect on the synthesis of NOS1. In agreement with this, COX-2-/- deficient mice reveal an increased juxtaglomerular expression of NOS1 in association with glomerular pathology, suggesting a causative role of NO in local hyperperfusion. Increased levels of NOS1 in the JGA are related with local ROS production in hypertension. Antioxidant-induced normalization of NOS1 indicates a role for juxtaglomerular NADPH oxidase expression but does not support a systemic impact for local ROS in raising blood pressure

Kidney transplant monitoring by urinary flow cytometry: Biomarker combination of T cells, renal tubular epithelial cells, and podocalyxin-positive cells detects rejection

Creatinine and proteinuria are used to monitor kidney transplant patients. However, renal biopsies are needed to diagnose renal graft rejection. Here, we assessed whether the quantification of different urinary cells would allow non-invasive detection of rejection. Urinary cell numbers of CD4+ and CD8+ T cells, monocytes/macrophages, tubular epithelial cells (TEC), and podocalyxin(PDX)-positive cells were determined using flow cytometry and were compared to biopsy results. Urine samples of 63 renal transplant patients were analyzed. Patients with transplant rejection had higher amounts of urinary T cells than controls; however, patients who showed worsening graft function without rejection had similar numbers of T cells. T cells correlated with histological findings (interstitial inflammation p = 0.0005, r = 0.70; tubulitis p = 0.006, r = 0.58). Combining the amount of urinary T cells and TEC, or T cells and PDX+ cells, yielded a significant segregation of patients with rejection from patients without rejection (all p < 0.01, area under the curve 0.89–0.91). Urinary cell populations analyzed by flow cytometry have the potential to introduce new monitoring methods for kidney transplant patients. The combination of urinary T cells, TEC, and PDX-positive cells may allow non-invasive detection of transplant rejection

The pathological features of regulated necrosis

Necrosis of a cell is defined by the loss of its plasma membrane integrity. Morphologically, necrosis occurs in several forms such as coagulative necrosis, colliquative necrosis, caseating necrosis, fibrinoid necrosis, and others. Biochemically, necrosis was demonstrated to represent a number of genetically determined signalling pathways. These include (i) kinase-mediated necroptosis, which depends on receptor interacting protein kinase 3 (RIPK3)-mediated phosphorylation of the pseudokinase mixed lineage kinase domain like (MLKL); (ii) gasdermin-mediated necrosis downstream of inflammasomes, also referred to as pyroptosis; and (iii) an iron-catalysed mechanism of highly specific lipid peroxidation named ferroptosis. Given the molecular understanding of the nature of these pathways, specific antibodies may allow direct detection of regulated necrosis and correlation with morphological features. Necroptosis can be specifically detected by immunohistochemistry and immunofluorescence employing antibodies to phosphorylated MLKL. Likewise, it is possible to generate cleavage-specific antibodies against epitopes in gasdermin protein family members. In ferroptosis, however, specific detection requires quantification of oxidative lipids by mass spectrometry (oxylipidomics). Together with classical cell death markers, such as TUNEL staining and detection of cleaved caspase-3 in apoptotic cells, the extension of the arsenal of necrosis markers will allow pathological detection of specific molecular pathways rather than isolated morphological descriptions. These novel pieces of information will be extraordinarily helpful for clinicians as inhibitors of necroptosis (necrostatins), ferroptosis (ferrostatins), and inflammasomes have emerged in clinical trials. Anatomical pathologists should embrace these novel ancillary tests and the concepts behind them and test their impact on diagnostic precision, prognostication, and the prediction of response to the upcoming anti-necrotic therapies. Copyright (c) 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd