32 research outputs found
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 Ă„hnlichkeiten 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 widersprĂĽchliche 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
fĂĽhrte 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 AufschlĂĽsse ĂĽber 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 fĂĽr 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
Calcineurin homologous protein 1 interferes with calcineurin-dependent regulation of the renal Na-K-2Cl-cotransporter
Collagen-binding proteins in age-dependent changes in renal collagen turnover: microarray analysis of mRNA expression
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