Anke Hannemann

Effects of 5‐hydroxymethyl‐2‐furfural on the volume and membrane permeability of red blood cells from patients with sickle cell disease

We addressed the hypothesis that the heterocyclic aldehyde 5‐hydroxymethyl‐2‐furfural (5HMF) may act synergistically to ameliorate the complications of sickle cell disease through effects on red blood cell (RBC) membrane transport, in addition to its well‐known action of increasing the oxygen affinity of the abnormal form of haemoglobin, HbS. 5HMF was found to reduce deoxygenation‐induced dehydration of RBCs, whether in response to maintained deoxygenation or cyclical deoxygenation/re‐oxygenation. Acting at low millimolar concentrations, 5HMF reduced the activity of deoxygenation‐induced cation conductance (sometimes termed Psickle), an effect which correlated with reduction in sickling. 5HMF similarly inhibited deoxygenation‐induced activation of the Ca2+‐activated K+ channel (or Gardos channel), an effect not seen following pharmacologically mediated increases in intracellular Ca2+ via the ionophore A23187. Deoxygenation‐induced phosphatidylserine exposure, which is associated with Ca2+ entry via Psickle, was also inhibited by 5HMF. By contrast, effects of 5HMF on the K+–Cl− cotransporter (KCC) were modest, with slight inhibition following treatment with N‐ethylmaleimide (NEM) to abolish activity of its regulatory protein kinases, but stimulation in RBCs untreated with NEM. It would therefore appear that an important beneficial action of 5HMF, in addition to effects on HbS oxygen affinity, is reduction in Psickle‐mediated Ca2+ entry following RBC sickling, thereby inhibiting the deleterious sequelae of Gardos channel activation, RBC dehydration and also lipid scrambling.

A non‐electrolyte haemolysis assay for diagnosis and prognosis of sickle cell disease

Red blood cells (RBCs) from the two main genotypes of sickle cell disease (SCD) patients showed haemolysis in deoxygenated isosmotic sucrose solution, to a greater extent than those from sickle trait individuals (HbAS). RBCs from normal individuals (HbAA) did not lyse. Several treatments reduced haemolysis in RBCs from HbSS patients – cytochalasin B, aromatic aldehydes (o‐vanillin and 5‐hydroxymethylfurfural), urea, low temperature and high pH. These effects were not due to increased oxygen affinity of haemoglobin. Long‐term treatment of patients with hydroxyurea was also associated with reduced levels of haemolysis. Treatment with aromatic aldehydes and urea reduced sickling of HbSS RBCs in deoxygenated saline solutions. Further, RBCs from sickle trait individuals (HbAS) could be induced to sickle under acidic (pH 6), hypertonic (400 mosmol kg−1) conditions. When these conditions were reproduced in sucrose solutions, HbAS RBCs also showed considerable haemolysis. Results suggest that haemolysis of HbS‐containing RBCs is associated with HbS polymerisation, perhaps damaging the RBC membrane. These findings suggest that haemolysis may be used as a simple, novel technique to diagnose SCD, using the altered membrane permeability of RBCs rather than the presence of HbS per se. As extent of haemolysis varies between individuals and is reduced following hydroxyurea treatment, the method may also be useful prognostically.

The clinical significance of K-Cl cotransport activity in red cells of patients with HbSC disease

HbSC disease is the second commonest form of sickle cell disease, with poorly understood pathophysiology and few treatments. We studied the role of K-Cl cotransport activity in determining clinical and laboratory features, and investigated its potential role as a biomarker. Samples were collected from 110 patients with HbSC disease and 41 with sickle cell anemia (HbSS). K-Cl cotransport activity was measured in the oxygenated (K-Cl cotransport100) and deoxygenated (K-Cl cotransport0) states, using radioactive tracer studies. K-Cl cotransport activity was high in HbSC and decreased significantly on deoxygenation. K-Cl cotransport activity correlated significantly and positively with the formation of sickle cells. On multiple regression analysis, K-Cl cotransport increased significantly and independently with increasing reticulocyte count and age. K-Cl cotransport activity was increased in patients who attended hospital with acute pain in 2011 compared to those who did not (K-Cl cotransport100: mean 3.87 versus 3.20, P=0.009, independent samples T-test; K-Cl cotransport0: mean 0.96 versus 0.68, P=0.037). On logistic regression only K-Cl cotransport was associated with hospital attendance. Increased K-Cl cotransport activity was associated with the presence of retinopathy, but this effect was confounded by age. This study links variability in a fundamental aspect of cellular pathology with a clinical outcome, suggesting that K-Cl cotransport is central to the pathology of HbSC disease. Increased K-Cl cotransport activity is associated with increasing age, which may be of pathophysiological significance. Effective inhibition of K-Cl cotransport activity is likely to be of therapeutic benefit.

Milk Fat Globule‐Epidermal Growth Factor 8 (MFG‐E8) is a Novel Anti‐inflammatory Factor in Rheumatoid Arthritis in Mice and Humans

Milk fat globule-epidermal growth factor 8 (MFG-E8) is an anti-inflammatory glycoprotein that mediates the clearance of apoptotic cells and is implicated in the pathogenesis of autoimmune and inflammatory diseases. Because MFG-E8 also controls bone metabolism, we investigated its role in rheumatoid arthritis (RA), focusing on inflammation and joint destruction. The regulation of MFG-E8 by inflammation was assessed in vitro using osteoblasts, in arthritic mice and in patients with RA. K/BxN serum transfer arthritis (STA) was applied to MFG-E8 knock-out mice to assess its role in the pathogenesis of arthritis. Stimulation of osteoblasts with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α downregulated the expression of MFG-E8 by 30% to 35%. MFG-E8-deficient osteoblasts responded to LPS with a stronger production of pro-inflammatory cytokines. In vivo, MFG-E8 mRNA levels were 52% lower in the paws of collagen-induced arthritic (CIA) mice and 24% to 42% lower in the serum of arthritic mice using two different arthritis models (CIA and STA). Similarly, patients with RA (n = 93) had lower serum concentrations of MFG-E8 (-17%) compared with healthy controls (n = 140). In a subgroup of patients who had a moderate to high disease activity (n = 21), serum concentrations of MFG-E8 rose after complete or partial remission had been achieved (+67%). Finally, MFG-E8-deficient mice subjected to STA exhibited a stronger disease burden, an increased number of neutrophils in the joints, and a more extensive local and systemic bone loss. This was accompanied by an increased activation of osteoclasts and a suppression of osteoblast function in MFG-E8-deficient mice. Thus, MFG-E8 is a protective factor in the pathogenesis of RA and subsequent bone loss. Whether MFG-E8 qualifies as a novel biomarker or therapeutic target for the treatment of RA is worth addressing in further studies.

The Properties of Red Blood Cells from Patients Heterozygous for HbS and HbC (HbSC Genotype)

Sickle cell disease (SCD) is one of the commonest severe inherited disorders, but specific treatments are lacking and the pathophysiology remains unclear. Affected individuals account for well over 250,000 births yearly, mostly in the Tropics, the USA, and the Caribbean, also in Northern Europe as well. Incidence in the UK amounts to around 12–15,000 individuals and is increasing, with approximately 300 SCD babies born each year as well as with arrival of new immigrants. About two thirds of SCD patients are homozygous HbSS individuals. Patients heterozygous for HbS and HbC (HbSC) constitute about a third of SCD cases, making this the second most common form of SCD, with approximately 80,000 births per year worldwide. Disease in these patients shows differences from that in homozygous HbSS individuals. Their red blood cells (RBCs), containing approximately equal amounts of HbS and HbC, are also likely to show differences in properties which may contribute to disease outcome. Nevertheless, little is known about the behaviour of RBCs from HbSC heterozygotes. This paper reviews what is known about SCD in HbSC individuals and will compare the properties of their RBCs with those from homozygous HbSS patients. Important areas of similarity and potential differences will be emphasised.

Functional expression of the Na-K-2Cl cotransporter NKCC2 in mammalian cells fails to confirm the dominant negative effect of the AF splice variant

The renal bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) is the major salt transport pathway in the apical membrane of the mammalian thick ascending limb. It is differentially spliced and the three major variants (A, B, and F) differ in their localization and transport characteristics. Most knowledge about its regulation comes from experiments in Xenopus oocytes as NKCC2 proved difficult to functionally express in a mammalian system. Here we report the cloning and functional expression of untagged and unmodified versions of the major splice variants from ferret kidney (fNKCC2A, -B, and -F) in human embryonic kidney (HEK) 293 cells. Many NKCC2 antibodies used in this study detected high molecular weight forms of the transfected proteins, probably NKCC2 dimers, but not the monomers. Interestingly, monomers were strongly detected by phosphospecific antibodies directed against phosphopeptides in the regulatory N terminus. Bumetanide-sensitive 86Rb uptake was significantly higher in transfected HEK-293 cells and could be stimulated by incubating cells in a medium containing a low chloride concentration prior the uptake measurements. fNKCC2 was less sensitive to the reduction in chloride concentration than NKCC1. Using HEK-293 cells stably expressing fNKCC2A we also show that co-expression of variant NKCC2AF does not have the dominant-negative effect on NKCC2A activity that was seen in Xenopus oocytes, nor is it trafficked to the cell surface. In addition, fNKCC2AF is neither complex glycosylated nor phosphorylated in its N terminus regulatory region like other variants.

Cation Homeostasis in Red Cells From Patients With Sickle Cell Disease Heterologous for HbS and HbC (HbSC Genotype)

Sickle cell disease (SCD) in patients of HbSC genotype is considered similar, albeit milder, to that in homozygous HbSS individuals — but with little justification. In SCD, elevated red cell cation permeability is critical as increased solute loss causes dehydration and encourages sickling. Recently, we showed that the KCl cotransporter (KCC) activity in red cells from HbSC patients correlated significantly with disease severity, but that in HbSS patients did not. Two transporters involved in red cell dehydration, the conductive channels Psickle and the Gardos channel, behaved similarly in red cells from the two genotypes, but were significantly less active in HbSC patients. By contrast, KCC activity was quantitatively greater in HbSC red cells. Results suggest that KCC is likely to have greater involvement in red cell dehydration in HbSC patients, which could explain its association with disease severity in this genotype. This work supports the hypothesis that SCD in HbSC patients is a distinct disease entity to that in HbSS patients. Results suggest the possibility of designing specific treatments of particular benefit to HbSC patients and a rationale for the development of prognostic markers, to inform early treatment of children likely to develop more severe complications of the disease.

Effects of o-vanillin on K+ transport of red blood cells from patients with sickle cell disease

Aromatic aldehydes like o-vanillin were designed to reduce the complications of sickle cell disease (SCD) by interaction with HbS, to reduce polymerisation and RBC sickling. Present results show that o-vanillin also directly affects RBC membrane permeability. Both the K+–Cl− cotransporter (KCC) and the Ca2 +-activated K+ channel (or Gardos channel) were inhibited with IC50 of about 0.3 and 1 mM, respectively, with activities almost completely abolished by 5 mM. Similar effects were observed in RBCs treated with the thiol reacting reagent N-ethylmaleimide or with the Ca2 + ionophore A23187, to circumvent any action via HbS polymerisation. The deoxygenation-induced cation conductance (sometimes termed Psickle) was partially inhibited, whilst deoxygenation-induced exposure of phosphatidylserine was completely abrogated. Na+/K+ pump activity was also reduced. Notwithstanding, o-vanillin stimulated K+ efflux through an unidentified pathway and resulted in reduction in cell volume (as measured by wet weight − dry weight). These actions are relevant to understanding how aromatic aldehydes may affect RBC membrane permeability per se as well as HbS polymerisation and thereby inform design of compounds most efficacious in ameliorating the complications of SCD.

The Use of Radioisotopes to Characterise the Abnormal Permeability of Red Blood Cells from Sickle Cell Patients

1.1 Membrane transport and radioisotopes In order to function, cells of necessity must transport a variety of substances across their plasma membranes. Aside from simple non-polar entities, most of these will require the presence of special transport proteins. Radioisotopes, used as tracers for these substrates, have provided an invaluable tool for understanding the mechanism and regulation of such transport pathways. In this chapter, we will cover some theoretical and practical issues concerning the measurement of transport with radioactive tracers. As an illustration, we will focus on membrane transport in red blood cells, a tissue much used in the study of membrane permeability. In particular, we will look at the abnormal cation permeability of red blood cells from sickle cell patients to show how radioactive tracer methodologies can be used to investigate the pathophysiology of membrane permeability. Chemical analysis of transport is feasible, but can often be tedious and slow. That by radioactive tracers has the advantage of being relatively immediate whilst retaining comparative simplicity. The first artificial radioisotopes were produced by Curie and Juliot in 1934 when they synthesised phosphorus-30 by exposure of aluminium-27 to Ǐ particles. Over the next 50 years or so, numerous different radioisotopes became available and were widely used to follow transport across biological membranes. Seminal examples include the use of 24Na+ to investigate active Na+ transport across the giant axon of cuttlefish in the mid 1950s (Hodgkin & Keynes, 1955a, 1955b; as elegantly retold by Boyd, 2011, following Keynes’ death last year). The perceptive analysis of these studies underpinned the hypothesis and identification of the Na+/K+ pump by Skou, Post, Jolly and colleagues (Skou, 1957; Post & Jolly, 1957; Glynn, 2002; Skou, 2003), whilst in the late 1960s onwards Glynn and colleagues also used 24Na+ and 42K+ to study the pump (Garrahan & Glynn, 1967) and especially the existence of occluded ions (Glynn et al., 1984; Glynn, 2002). With more recent methodogical advances and the more restrictive practices surrounding use of radioactive compounds, other non-radioactive methods (such as ion-sensitive

Serum protease activity in chronic kidney disease patients: The GANI_MED renal cohort

Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the “Greifswald Approach to Individualized Medicine” (GANI_MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR < 15 mL/min/1.73 m2 or <45 mL/min/1.73 m2, respectively. Results with a false discovery rate below 0.05 were deemed statistically significant. Among the 10 proteases investigated, only the activities of ACE2 and DPP4 were correlated with eGFR. Patients with lowest eGFR exhibited highest DPP4 and ACE2 activities. DPP4 and PEP were correlated with age, but all other serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly, resulting in increased morbidity and mortality. Understanding of molecular mechanisms linking both diseases is limited, available fragmentary data point to a role of the renin–angiotensin system (RAS) and, in particular, Ras-related peptidases. • Here, a comprehensive analysis of serum peptidase activities in patients with different stages of chronic kidney disease (CKD) is presented, with special emphasis given to RAS peptidases • The serum activities of the peptidases angiotensin I-converting enzyme 2 and dipeptidyl peptidase 4 were identified as closely associated with kidney function, specifically with the estimated glomerular filtration rate. The findings are discussed in the context of available data suggesting protective roles for both enzymes in reno-cardiac diseases. • The data add to our understanding of pathomechanisms underlying development and progression of CKD and indicate that both enzymes might represent potential pharmacological targets for the preservation of renal function.