Etiopathology of chronic tubular, glomerular and renovascular nephropathies: Clinical implications

Chronic kidney disease (CKD) comprises a group of pathologies in which the renal excretory function is chronically compromised. Most, but not all, forms of CKD are progressive and irreversible, pathological syndromes that start silently (i.e. no functional alterations are evident), continue through renal dysfunction and ends up in renal failure. At this point, kidney transplant or dialysis (renal replacement therapy, RRT) becomes necessary to prevent death derived from the inability of the kidneys to cleanse the blood and achieve hydroelectrolytic balance. Worldwide, nearly 1.5 million people need RRT, and the incidence of CKD has increased significantly over the last decades. Diabetes and hypertension are among the leading causes of end stage renal disease, although autoimmunity, renal atherosclerosis, certain infections, drugs and toxins, obstruction of the urinary tract, genetic alterations, and other insults may initiate the disease by damaging the glomerular, tubular, vascular or interstitial compartments of the kidneys. In all cases, CKD eventually compromises all these structures and gives rise to a similar phenotype regardless of etiology. This review describes with an integrative approach the pathophysiological process of tubulointerstitial, glomerular and renovascular diseases, and makes emphasis on the key cellular and molecular events involved. It further analyses the key mechanisms leading to a merging phenotype and pathophysiological scenario as etiologically distinct diseases progress. Finally clinical implications and future experimental and therapeutic perspectives are discussed

Analyse nephrotoxischer Nebenwirkungen von Calcineurininhibitoren im Rattenmodell

Background: Calcineurin inhibitors (CNI) such as cyclosporine A (CsA) and tacrolimus (Tac, FK506) are commonly used as immunosuppressive drugs in patients with solid organ transplantation or autoimmune affections. With merits undisputed at short-term, their chronic use leads to nephrotoxicity in a significant proportion of renal or non-renal transplant recipients, resulting in compromised kidney function. Although attempts have been made to substitute CNIs with other immunosuppressants, the alternatives to CNIs are generally considered less effective. CsA has been considered more toxic than Tac, but the current preference for Tac over CsA needs further validation. Understanding the cellular mechanisms of their particular nephrotoxicity profiles should enable more personalized treatment protocols to minimize adverse effects. We therefore aimed to investigate whether CsA and Tac exhibit distinct renal damage patterns. Systemic and kidney parameters were studied in rats exposed to chronic CNI delivery. Methods: Wild-type male Wistar rats (group size n=6 to 12) were treated with vehicle, CsA (30 mg/kg/day) or Tac (2 mg/kg/day) via subcutaneously implanted osmotic minipumps for 28 days. Histopathology and advanced microscopy techniques such as large-scale electron microscopy and 3D-structured illumination microscopy were used tissue analyses. Standard biochemistry, RNA sequencing as well as proteomic and phosphoproteomic analyses were conducted to identify molecular changes. Results: Distinct damage patterns were observed in the CNI treatment groups compared to vehicle. Alterations were located in vasculature and nephron. Cells of the glomerular filtration barrier revealed displayed more damage by Tac than by CsA, with changes located in pore endothelium and podocytes. The VEGF/VEGFR2 pathways as well as podocyte-specific gene expression were substantially and differentially altered. Contrastingly, the renal tubule was more severely affected by CsA than by Tac with prominent lysosomal dysfunction, impaired proteostasis, oxidative stress and enhanced apoptosis chiefly observed in the early proximal nephron. Conclusion: We found that pathological changes in renal microenvironments were specific to each treatment. If our findings can be translated to the clinical context, the choice of CNI should consider individual risk factors related to renal vasculature and tubular epithelia. As a step in this direction, we present identified products and pathways from multiomics analyses with potential pathognomonic relevance.Hintergrund: Calcineurin-Inhibitoren (CNI) wie Cyclosporin A (CsA) und Tacrolimus (Tac, FK506) werden häufig als Immunsuppressiva bei Patienten mit Organtransplantation oder Autoimmunerkrankungen eingesetzt. Obwohl ihre Vorzüge kurzfristig unbestritten sind, führt ihre chronische Anwendung bei einem erheblichen Teil der Empfänger von Nieren- oder Nicht-Nierentransplantaten zu Nephrotoxizität, die zu signifikanter Beeinträchtigung der Nierenfunktion führen kann. Obwohl Versuche unternommen werden, CNI durch andere Immunsuppressiva zu ersetzen, gelten die Alternativen zu CNI im Allgemeinen als weniger wirksam. CsA gilt als toxischer als Tac, die derzeitige Bevorzugung von Tac gegenüber CsA bedarf jedoch weiterer Bestätigung. Das Verständnis der zellulären Mechanismen ihrer jeweiligen Nephrotoxizitätsprofile sollte personalisierte Behandlungsprotokolle fördern, um Nebenwirkungen zu minimieren. Unser Ziel war es daher zu untersuchen, ob CsA und Tac unterschiedliche Nierenschädigungsmuster aufweisen. Systemische und Nierenparameter wurden bei Ratten untersucht, die einer chronischen CNI-Verabreichung ausgesetzt waren. Methoden: Männliche Wildtyp-Wistaratten (Gruppengröße n=6 bis 12) wurden über subkutan implantierte osmotische Minipumpen 28 Tage lang mit Vehikel, CsA (30 mg/kg/Tag) oder Tac (2 mg/kg/Tag) behandelt. Für Gewebeanalysen wurden Histopathologie und moderne Mikroskopietechniken wie Large scale Elektronenmikroskopie und 3D-structured illumination Mikroskopie verwendet. Zur Identifizierung molekularer Veränderungen wurden grundlegende Biochemie, RNA-Sequenzierung sowie proteomische und phosphoproteomische Analysen durchgeführt. Ergebnisse: In den CNI-Behandlungsgruppen wurden im Vergleich zur Vehikelgabe deutliche Schadensmuster beobachtet. Beide Medikamente verursachten erhebliche, aber unterschiedliche Schäden im Gefäßsystem und im Nephron. Komponenten der glomerulären Filtrationsbarriere wurden durch Tac stärker beeinträchtigt als durch CsA, was zu erheblichen Beeinträchtigungen des Porenendothels und der Podozyten, vereint mit geschwächter VEGF/VEGFR2-Signalübertragung und verminderter podozytenspezifischen Genexpression, führte. Im Gegensatz dazu wurde der Nierentubulus durch CsA stärker beeinträchtigt als durch Tac, wobei ausgeprägte lysosomale Dysfunktion, beeinträchtigte Proteostase, oxidativer Stress und verstärkte Apoptose hauptsächlich im frühen proximalen Nephron beobachtet wurden. Schlussfolgerung: Wir fanden heraus, dass pathologische Veränderungen im Nierenparenchym für jede Behandlung spezifisch waren. Mit Bezug auf den klinischen Kontext sollten unsere Ergebnisse dazu verhelfen, bei der Wahl geeigneter Immunsuppressiva individuelle Risikofaktoren im Zusammenhang mit Nierengefäßen und tubulären Epithelien zu berücksichtigen. Als Schritt in diese Richtung stellen wir identifizierte Produkte und Signalwege aus den Multiomics-Analysen mit potenzieller pathognomonischer Relevanz vor

ADMA in der Pathogenese der Atherosklerose und Atherosklerose-assoziierter Krankheiten

Die Regulation der Organperfusion mittels Gefäßweitstellung und -verengung durch die L-Arginin-abhängige Stickstoffmonoxid(NO)-Bildung stellt eine der wichtigsten Funktionen unseres Gefäßsystems dar. Zu den Ursachen dieser durch NO -Mangel ausgelösten Erkrankungen gehört die Hemmung der NO-Bildung durch asymmetrisches Dimethylarginin (ADMA). In dieser Arbeit wurde die Rolle von ADMA bei der Pathogenese der Atherosklerose und Atherosklerose-assoziierter Krankheiten anhand der einschlägigen Literatur recherchiert und analysiert. Zusammenfassend konnten erhöhte ADMA-Spiegel mit dem Vorliegen von Atherosklerose, kardialen Erkrankungen, Diabetes mellitus, chronische Nierenerkrankungen, Präeklampsie, Hyperhomocysteinämie und Leberversagen in Zusammenhang gebracht werden. Neben dem antioxidativ wirkenden L-Arginin spielen reaktive Sauerstoffspezies eine zentrale Rolle im NO-/ADMA-Stoffwechsel. Eine Rolle von ADMA als Risikofaktor der Atherogenese gilt jedoch als sehr wahrscheinlich

Die Bedeutung des Asymmetrischen Dimethylarginins für die Pathogenese der Endorganschäden bei Angiotensin II-induzierter Hypertonie der Maus

2.1 Background and aim of the study Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of NO-synthase. Elevated ADMA levels are predictive of cardiovascular risk and are associated with cardiovascular disease. The aim of the present study was to elucidate a possible link between ADMA and angiotensin II (Ang II) in the pathogenesis of hypertension and resulting target organ damage. 2.2 Methods Transgenic (TG) mice overexpressing the human isoform 1 of the ADMA degrading enzyme DDAH as well as wildtype (WT) littermates were treated with Ang II (1.0µg/kg/min or 3.0µg/kg/min) or phosphate buffered saline solution (PBS) via subcutaneous infusion using osmotic minipumps over a period of four weeks. 2.3 Results Infusion of Ang II caused hypertension and target organ damage in all mice. Except for renal interstitial fibrosis and vascular oxidative stress that were less pronounced in TG mice no differences between the two genotypes were observed. As expected, TG mice had markedly lower ADMA levels. Interestingly, infusion of Ang II had no effect on plasma ADMA levels. RT-PCR revealed an increased renal expression of ADMA degrading (DDAH2) and ADMA generating (PRMT1) enzymes following Ang II infusion. 2.4 Conclusions The data presented herein do not support a clear link between ADMA and Ang II in the pathogenesis of hypertension or hypertensive target organ damage. Interestingly, TG mice were resistant to Ang II induced renal interstitial fibrosis and vascular oxidative stress. This observation needs further exploration in future studies to investigate the pathophysiological relevance of ADMA.1.1 Hintergrund und Ziele Asymmetrisches Dimethylarginin (ADMA) ist ein endogen gebildeter, kompetitiver Hemmstoff der NO-Synthase. Erhöhte ADMA-Spiegel gelten als kardiovaskulärer Risikofaktor und sind mit Herz-Kreislauferkrankungen assoziiert. Ziel der vorliegenden Arbeit war es, im Tierversuch einen möglichen Zusammenhang von ADMA und Angiotensin II (Ang II) bei der Pathogenese der Hypertonie und ihrer Endorganschäden zu untersuchen. 1.2 Methoden Die Versuche wurden an transgenen (TG) Mäusen durchgeführt, die die humane Form des ADMA abbauenden Enzyms DDAH1 überexprimieren. Die TG Mäuse sowie Wildtyp (WT) Geschwistertiere erhielten Ang II (1.0µg/kg/min oder 3.0µg/kg/min) bzw. gepufferte Kochsalzlösung (PBS) als subkutane Infusion über eine Dauer von vier Wochen. 1.3 Ergebnisse und Beobachtungen Die Infusion von Ang II erzeugte bei allen Mäusen eine Hypertonie sowie hypertoniebedingte Endorganschäden. Allerdings bestanden keine signifikanten Unterschiede zwischen den Genotypen, lediglich das Ausmaß der renalen interstitiellen Fibrose und des oxidativen Stress in der Aorta war bei DDAH1 transgenen Mäusen vermindert. Die transgenen Mäuse wiesen insgesamt deutlich niedrigere ADMA-Spiegel auf. Die Infusion von Ang II hatte keinerlei Einfluss auf die ADMA-Spiegel. In der RT-PCR zeigte sich eine gesteigerte Expression der Enzyme DDAH2 und PRMT1 in der Niere nach Ang II Gabe. 1.4 Praktische Schlussfolgerungen Die in dieser Arbeit erhobenen Daten sprechen insgesamt gegen einen engen Zusammenhang zwischen ADMA und Ang II bei der Genese hypertensiver Organschäden. Interessant scheint der Einfluss von ADMA auf die renale interstitielle Fibrose sowie den vaskulären oxidativen Stress. Hierin liegen Ansatzpunkte für weiterführende Experimente zur Aufklärung der pathophysiologischen Relevanz von ADMA

The measurement, biological variation and response to acute inflammation of asymmetric dimethylarginine (ADMA)

Introduction and methods Asymmetric dimethylarginine (ADMA) is a potent endogenous competitive inhibitor of nitric oxide synthases, which has attracted considerable attention as a marker and mediator of atherosclerotic disease and as a potential mediator of multiple organ failure in critical illness due to endothelial dysfunction. However, data regarding basic aspects of its biology such as biological variation and its response to acute inflammation are lacking. Moreover, significant methodological variability has been a barrier to collating the burgeoning data available. Therefore, this thesis describes the development and validation of a reliable assay for measurement of ADMA and related compounds in plasma, urine and other biological fluids based on isocratic reverse phase high performance liquid chromatography (HPLC). This method was used to determine the biological variation of ADMA in human plasma, and its response to acute inflammation using a model of elective knee arthroplasty. Further HPLC methods for measurement of dimethylamine (DMA), the main metabolite of ADMA, and nitrate were developed and used to determine excretion of these compounds in acute inflammation to complement the observed changes in plasma ADMA concentration. Results Complete chromatographic separation of arginine, homoarginine, monomethyl-arginine, ADMA and its structural isomer SDMA was achieved, permitting their accurate quantification using a novel, non-endogenous, internal standard. The intra-individual biological variation of ADMA was found to be low at 7.4%, imposing a tight imprecision goal for analytical methods. Plasma ADMA concentration decreases rapidly during the acute inflammatory response, with a median decrease of around 30%, and a significant change already evident as little as 12 hours following the onset of inflammation. No similar change was seen in the concentration of the closely related compound SDMA. No significant increase in the urine excretion of DMA was noted during the early phase of the response, with a significant increase seen 5 days following the insult by which point the plasma ADMA concentration had returned to baseline levels. A small, but significant, decrease in nitrate excretion during the inflammatory response was seen, mirroring the observed changes in plasma ADMA. Conclusion The low biological variation of ADMA suggests physiological regulation. The rapid and significant decrease in plasma concentration during inflammation does not appear due to increased catabolism, but rather is more likely to represent increased cellular partitioning. This may be associated with an impairment in NOS activity. It is unclear whether this is of pathological significance, or represents a physiological response to regulate NO production in inflammation. Further study is warranted in relevant models, particularly with attention to intracellular concentrations

Die Bedeutung des Asymmetrischen Dimethylarginins zur Prädiktion der Gesamtmortalität nach erfolgreicher kardiopulmonaler Reanimation

von Conrad Friedrich Gen

Glyoxalase 1 inducer therapy for the treatment of experimental diabetic kidney disease

Diabetic kidney disease (DKD), one of the most common diabetic complications, is identified as a progressive kidney disease in patients with diabetes resulting from angiopathy of the capillaries in renal glomeruli. DKD affects nearly 40% of patients with diabetes and is characterised by increased albuminuria and decreased glomerular filtration rate (GFR). Hyperglycaemia and inflammation enhance DKD through increased glycation formation. Current treatments address only ca. 15% of the risk of DKD development. Development of DKD and other microvascular complications of diabetes is linked to glycemic control. In hyperglycaemia, dysregulation of glucose metabolism drives the activation of multiple metabolic pathways potentially damaging to the kidney, for example mitochondrial dysfunction with NADPH oxidase-driven increased formation of reactive oxygen species, the polyol pathway, the hexosamine pathway, and increased formation advanced glycation endproducts (AGEs). Driving the latter process leads to increased formation and decreased metabolism of the reactive dicarbonyl metabolite, methylglyoxal (MG), leading to increased concentrations of MG – an abnormal metabolic state called dicarbonyl stress. MG is the precursor of the major quantitative and damaging AGE in physiological systems, arginine residue derived hydroimidazolone, MG-H1. Most MG, ca. 99%, formed in the body is metabolised by glyoxalase 1 (Glo1) of the glyoxalase system. Glo1 activity decreased in the kidney in diabetes. The host research team have recently developed a new strategy for treatment of DKD: induction of Glo1 expression by small molecule activators of transcription factor Nrf2; Nrf2 binds a functional antioxidant response element in the GLO1 gene and increase expression. The optimised Glo1 inducer is a combination of trans-resveratrol and hesperetin (tRSV-HESP) this coformulation increases Glo1 expression and might drive improvements in microvascular complications of diabetes linked to hyperglycemia. The aim of this project is to evaluate the effect of Glo1 inducer treatment on human renal cell dysfunction in high glucose concentration primary cultures in vitro. Thesis work set out to characterise the glyoxalase system in primary human renal cells (Tubular epithelial cells and Mesangial cells) in models of hyperglycaemia in vitro and investigate the effects of the Glo1 inducer treatment, tRSV-HESP, on human renal cell function and protein expression in high glucose cultures in vitro. Data indicated that Glo1 inducer treatment had a significant impact on MG metabolism, as determined by measuring D-lactate flux, in both primary proximal tubular epithelial cells and primary mesangial cells, with evidence of treatment-induced increases in Glo1 levels shown via Western blot. Also, data showed significant, quantitative changes in proteomic 15 profiles between treatment groups in mesangial cells, but much less so in proximal tubular epithelial cells. Where detected, tRSV-HESP affected expression of key functional clusters involved in energy metabolism. Paradoxically, changes in Glo1 levels did not reach statistical significance between experimental groups. A striking discovery was that the impact of tRSV-HESP on proteomic profile for both sets of cells was greatly diminished by the presence of high glucose (25 mM) in the cultures. Taken together, these findings suggest that tRSV-HESP has potential to modulate therapeutically the Glo1 system and reduce dicarbonyl stress, but that any potential pharmacological effects would require greater glucose control prior to starting treatment

The role of iron in oxidative stress accelerated endothelial dysfunction in chronic kidney disease

Chronic kidney disease (CKD) is growing global public health problem affecting 1 in 10 adults in developed countries and recognised as an important risk factor for cardiovascular disease (CVD) development. CVD is the main cause of death among CKD patients. Endothelial injury and dysfunction are critical steps in atherosclerosis, a major CVD. Oxidative stress (increased level of reactive oxygen species, ROS) has been associated with CVD development. Intravenous (IV) iron preparations are widely used in the management of CKD mediated anaemia, and have been associated with increased oxidative stress and cellular dysfunction. This study examined the effect of pharmacologically-relevant concentrations of IV Venofer (iron sucrose) or IV Ferinject (Ferric carboxymaltose, FCM) on primary human umbilical vein endothelial cell (HUVEC) activation/damage and on intracellular ROS generation as well as studying the potential mechanisms responsible. Data from TUNEL assay and Annexin V-FITC/PI staining showed that, IV FCM had no effect, but IV iron sucrose increased HUVEC apoptosis at 24hr. IV iron sucrose inhibited cell proliferation and reduced cell viability. Both compounds induced EC activation through sustained activation of p38 MAPK and up-regulation of ICAM-1 and VCAM-1. Additionally, the compounds induced significant increase in total ROS and superoxide anion production, which was attenuated by the anti-oxidant N-acetylcysteine (NAC). P38 MAPK showed up-regulation of pro-apoptotic protein Bax and down-regulation of antiapoptotic Bcl-2 protein in HUVEC treated with IV iron sucrose and p38 inhibition reversed these effects. In summary, these results suggest that IV iron sucrose causes more severe EC injury than IV FCM. However, both IV iron preparations induced intracellular ROS and superoxide anion generation in HUVEC leading to EC activation/dysfunction, providing a potential explanation for vascular damage in CKD patients

Smoking and Second Hand Smoking in Adolescents with Chronic Kidney Disease: A Report from the Chronic Kidney Disease in Children (CKiD) Cohort Study

The goal of this study was to determine the prevalence of smoking and second hand smoking [SHS] in adolescents with CKD and their relationship to baseline parameters at enrollment in the CKiD, observational cohort study of 600 children (aged 1-16 yrs) with Schwartz estimated GFR of 30-90 ml/min/1.73m2. 239 adolescents had self-report survey data on smoking and SHS exposure: 21 [9%] subjects had “ever” smoked a cigarette. Among them, 4 were current and 17 were former smokers. Hypertension was more prevalent in those that had “ever” smoked a cigarette (42%) compared to non-smokers (9%),