Framework and baseline examination of the German National Cohort (NAKO).

The German National Cohort (NAKO) is a multidisciplinary, population-based prospective cohort study that aims to investigate the causes of widespread diseases, identify risk factors and improve early detection and prevention of disease. Specifically, NAKO is designed to identify novel and better characterize established risk and protection factors for the development of cardiovascular diseases, cancer, diabetes, neurodegenerative and psychiatric diseases, musculoskeletal diseases, respiratory and infectious diseases in a random sample of the general population. Between 2014 and 2019, a total of 205,415 men and women aged 19-74 years were recruited and examined in 18 study centres in Germany. The baseline assessment included a face-to-face interview, self-administered questionnaires and a wide range of biomedical examinations. Biomaterials were collected from all participants including serum, EDTA plasma, buffy coats, RNA and erythrocytes, urine, saliva, nasal swabs and stool. In 56,971 participants, an intensified examination programme was implemented. Whole-body 3T magnetic resonance imaging was performed in 30,861 participants on dedicated scanners. NAKO collects follow-up information on incident diseases through a combination of active follow-up using self-report via written questionnaires at 2-3 year intervals and passive follow-up via record linkages. All study participants are invited for re-examinations at the study centres in 4-5 year intervals. Thereby, longitudinal information on changes in risk factor profiles and in vascular, cardiac, metabolic, neurocognitive, pulmonary and sensory function is collected. NAKO is a major resource for population-based epidemiology to identify new and tailored strategies for early detection, prediction, prevention and treatment of major diseases for the next 30 years.

Short-term monotherapy in HIV-infected patients with a virus entry inhibitor against the gp41 fusion peptide.

To infect host cells, most enveloped viruses must insert a hydrophobic fusion peptide into the host cell membrane. Thus, fusion peptides may be valuable targets for developing drugs that block virus entry. We have shown previously that a natural 20-residue fragment of α(1)-antitrypsin, designated VIRus-Inhibitory Peptide (VIRIP), that binds to the gp41 fusion peptide of HIV-1 prevents the virus from entering target cells in vitro. Here, we examine the efficacy of 10-day monotherapy with the optimized VIR-576 derivative of VIRIP in treatment-naïve, HIV-1-infected individuals with viral RNA loads of ≥10,000 copies per ml. We report that at the highest dose (5.0 grams per day), intravenous infusion of VIR-576 reduced the mean plasma viral load by 1.23 log(10) copies per ml without causing severe adverse effects. Our results are proof of concept that fusion peptide inhibitors suppress viral replication in human patients, and offer prospects for the development of a new class of drugs that prevent virus particles from anchoring to and infecting host cells.

Uroguanylin and guanylin regulate transport of mouse cortical collecting duct independent of guanylate cyclase C.

BACKGROUND: Electrolyte and water homeostasis mostly depend on differentially regulated intestinal and renal transport. Guanylin and uroguanylin were proposed as first hormones linking intestinal with renal electrolyte and water transport, which is disturbed in pathophysiology. Guanylate cyclase C is the intestinal receptor for these peptides, but in guanylate cyclase C-deficient mice renal effects are retained. Unlike for the intestine the sites of renal actions and cellular mechanisms of guanylin peptides are still unclear. METHODS: After first data on proximal tubular effects in this study their effects are examined in detail in mouse cortical collecting duct (CCD). Effects of guanylin peptides on principal cells of isolated mouse CCD were studied by slow whole-cell patch-clamp analysis, reverse transcription-polymerase chain reaction (RT-PCR), and microfluorimetric measurements of intracellular Ca2+. RESULTS: Guanylin peptides depolarized or hyperpolarized principal cells. Whereas 8-Br-cyclic guanosine monophosphate (8-Br-cGMP) hyperpolarized, 8-Br-cyclic adenosine monophosphate (8-Br-cAMP) depolarized principal cells. All effects of guanylin peptides were inhibited by Ba2+. Hyperpolarizations were blocked by clotrimazole or protein kinase G (PKG) inhibition, suggesting an involvement of basolateral Ca2+- and cGMP-dependent K+ channels. Effects remained in CCD isolated from guanylate cyclase C-deficient mice. Depolarizations were inhibited by arachidonic acid or inhibition of phospholipase A2 (PLA2), but not by protein kinase A (PKA) inhibition. Conclusion. These results suggest the existence of two signaling pathways for guanylin peptides in principal cells of mouse CCD. One pathway is cGMP- and PKG-dependent but not mediated by guanylate cyclase C, the second involves PLA2 and arachidonic acid. The first pathway most likely leads to an activation of the basolateral K+-conductance while the latter probably results in decreased activity of ROMK channels in the luminal membrane.

Acute rejection after rat renal transplantation leads to downregulation of NA+ and water channels in the collecting duct.

Renal transplantation is associated with alterations of tubular functions and of the renin-angiotensin-aldosterone system. The underlying cellular and molecular mechanisms are unclear. We used an allogeneic rat renal transplantation model of acute rejection with and without immunosuppression by cyclosporine A (CsA) and a syngeneic model as control. Uninephrectomized Lewis or Lewis-Brown-Norway (LBN) rats received a kidney from LBN-rats. Renal transporters and receptors were analyzed by immunohistochemistry, semiquantitative RT-PCR and Western-blot analysis. Intracellular Na(+) was analyzed microfluorimetrically in isolated cortical collecting ducts. mRNA expression and function of the epithelial Na(+)-channel (ENaC) and mRNA and protein expression of the water-channel AQP2 were downregulated in transplanted kidneys undergoing rejection. Expression of the serum- and glucocorticoid-kinase (Sgk1) was decreased and that of the ubiquitin-protein ligase Nedd4-2 was increased. These changes were absent under CsA-therapy and in syngeneic model. Expression and function of the Na(+)-K(+)-ATPase, expression of the secretory K(+)-channel and of the mineralocorticoid receptor remained unchanged. Reduced ENaC function is likely due to decreased Sgk1- and increased Nedd4-2 mRNA expression leading to reduced ENaC expression in the membrane. These acute downregulations of ENaC and AQP2 may be triggered to reduce energy consumption in the distal nephron to protect the kidney immediately after transplantation.

Guanylin and uroguanylin regulate electrolyte transport in isolated human cortical collecting ducts.

BACKGROUND: Guanylin and uroguanylin link intestinal and renal electrolyte and water transport. Their function in intestine is well studied, but renal actions are less understood. Uroguanylin concentrations are increased in patients with chronic renal failure, nephrotic syndrome, or those on dialysis. Guanylate cyclase C (GC-C) is the receptor first described for these peptides. In guanylate cyclase C-deficient mice guanylin- and uroguanylin-induced renal natriuresis, kaliuresis, and diuresis are retained. METHODS: Effects of guanylin and uroguanylin on principal cells of human cortical collecting ducts (CCD) isolated from kidneys after tumor nephrectomy were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), slow whole-cell patch-clamp, and microfluorimetric analysis of intracellular Ca(2+) were used. Here we present first functional measurements of isolated human CCD. RESULTS: Principal cells of CCD were identified by the amiloride-induced hyperpolarization of principal cells (-3.8 +/- 0.3 mV) (N= 52). Cells depolarized upon guanylin or uroguanylin (each 10 nmol/L) by 3.3 +/- 0.8 mV (N= 12) and 3.4 +/- 0.5 mV (N= 18), respectively, but were hyperpolarized by 8Br-cyclic guanosine monophosphate (cGMP) (100 micromol/L) (-3.0 +/- 0.2 mV) (N= 4). mRNA for GC-C was not detected in CCD. Effects of both peptides were inhibited by Ba(2+) (1 mmol/L) or phospholipase A(2) (PLA(2)) inhibition (AACOCF(3)) (5 micromol/L). CONCLUSION: These findings suggest a new cGMP- and GC-C-independent but PLA(2)-dependent signaling pathway for these peptides in the kidney. Most likely guanylin and uroguanylin inhibit luminal K(+) channels of principal cells of human CCD via this pathway. This depolarization of principal cells consequently reduces the driving force of Na(+) and water reabsorption, explaining natriuresis and diuresis caused by these peptides.

Up-regulation of early growth response gene-1 via the CXCR3 receptor induces reactive oxygen species and inhibits Na+/K+-ATPase activity in an immortalized human proximal tubule cell line.

The CXCR3 chemokine receptor, a member of the CXCR family, has been linked to a pathological role in autoimmune disease, inflammatory disease, allograft rejection, and ischemia. In the kidney, expression of the CXCR3 receptor and its ligands is up-regulated in states of glomerulonephritis and in allograft rejection, but little is known about the expression and functional role the CXCR3 receptor might play. Here, we study the function of the CXCR3 chemokine receptor in an immortalized human proximal tubular cell line (IHKE-1). Stimulation of the CXCR3 receptor by its selective agonist monokine induced by IFN-gamma leads via a Ca(2+)-dependent mechanism to an up-regulation of early growth response gene (EGR)-1. Overexpression of EGR-1 induces down-regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase and stimulates the generation of reactive oxygen species (ROS) via the NADH/NADPH-oxidase system. EGR-1 overexpression or treatment with monokine induced by IFN-gamma resulted in a ROS-dependent inhibition of basolateral Na(+)/K(+)-ATPase activity, compromising sodium transport in these cells. Thus, activation of the CXCR3 receptor in proximal tubular cells might disturb natriuresis during inflammatory and ischemic kidney disease via EGR-1-mediated imbalance of ROS.

Regulation of human organic cation transporter hOCT2 by PKA, PI3K, and calmodulin-dependent kinases.

Properties and regulation of the human organic cation (OC) transporter type 2 (hOCT2) expressed in HEK-293 cells were extensively characterized using the fluorescent OC 4-[4-(dimethylamino)styryl]-N-methylpyridinium (ASP(+)). ASP(+) uptake was electrogenic and inhibited by TPA(+) (EC(50) = 2.7 microM), tetraethylammonium (EC(50) = 35 microM), cimetidine (EC(50) = 36 microM), or quinine (EC(50) = 6.7 microM). Stimulation with carbachol or ATP decreased initial uptake by 44 +/- 3 (n = 14) and 34 +/- 4% (n = 21), respectively, independently of PKC but dependent on phosphatidylinositol 3-kinase (PI3K). PKA stimulation decreased uptake by 18 +/- 4% (n = 40). Inhibition of calmodulin (CaM), Ca(2+)/CaM-dependent kinase II, or myosin light chain kinase decreased uptake by 63 +/- 2 (n = 15), 40 +/- 4 (n = 30), and 31 +/- 4% (n = 16), respectively. Inhibition of CaM resulted in a significant change in the EC(50) value for the inhibition of ASP(+) uptake by tetraethylammonium. In conclusion, we demonstrate that the hOCT2 is inhibited by PI3K and PKA and activated by a CaM-dependent signaling pathway, probably via a change in substrate affinity.

Inhibition of Na(+)-dependent transporters in cystine-loaded human renal cells: electrophysiological studies on the Fanconi syndrome of cystinosis.

Cystinosis is the most common cause of the renal Fanconi syndrome in children, leading to severe electrolyte disturbances and growth failure. A defective lysosomal transporter, cystinosin, results in intralysosomal accumulation of cystine. Loading cells with cystine dimethyl ester (CDME) is the only available model for this disease. This model was used to present electrophysiologic studies on immortalized human kidney epithelial (IHKE-1) cells that had been derived from the proximal tubule with the slow whole-cell patch clamp technique. Basal membrane voltages (V(m)) of IHKE-1 cells were -30.7 +/- 0.4 mV (n = 151). CDME concentration-dependently altered V(m) with an initial depolarization (2.7 +/- 0.2 mV;n = 76; 1 mM CDME) followed by a more pronounced hyperpolarization (-9.9 +/- 1.0 mV;n = 49). Three Na(+)-dependent transporters were examined. Alanine (1 mM) depolarized IHKE-1 cells by 17.6 +/- 0.7 mV (n = 59), and phosphate (1.8 mM) depolarized by 9.7 +/- 1.1 mV (n = 18). Acidification of IHKE-1 cells with propionate (20 mM) resulted in a depolarization of V(m) by 7.1 +/- 0.3 mV (n = 21) followed by a repolarization by 2.9 +/- 0.3 mV/min (n = 17), reflecting Na(+)/H(+)-exchanger activity. Acute addition of 1 mM CDME did not alter the alanine- and propionate-induced changes in V(m), but it reduced the phosphate-induced depolarization by 37 +/- 9% (n = 10). Incubation with 1 mM CDME reduced the activity of all three transporters. Depolarizations by alanine and phosphate and the repolarization after propionate were inhibited by 57 +/- 4% (n =30), 45 +/- 9% (n = 9), and 78 +/- 15% (n = 8), respectively. In conclusion, this study demonstrates that CDME acutely alters V(m) of IHKE-1 cells and that at least three Na(+)-dependent transporters are inhibited, the Na(+)-phosphate cotransporter most sensitively. This might suggest that phosphate depletion and dissipation of the Na(+)-gradient are involved in the development of the Fanconi syndrome of cystinosis.

Guanylin, uroguanylin, and heat-stable euterotoxin activate guanylate cyclase C and/or a pertussis toxin-sensitive G protein in human proximal tubule cells.

Membrane guanylate cyclase C (GC-C) is the receptor for guanylin, uroguanylin, and heat-stable enterotoxin (STa) in the intestine. GC-C-deficient mice show resistance to STa in intestine but saluretic and diuretic effects of uroguanylin and STa are not disturbed. Here we describe the cellular effects of these peptides using immortalized human kidney epithelial (IHKE-1) cells with properties of the proximal tubule, analyzed with the slow-whole-cell patch clamp technique. Uroguanylin (10 or 100 nm) either hyperpolarized or depolarized membrane voltages (V(m)). Guanylin and STa (both 10 or 100 nm), as well as 8-Br-cGMP (100 microm), depolarized V(m). All peptide effects were absent in the presence of 1 mm Ba(2+). Uroguanylin and guanylin changed V(m) pH dependently. Pertussis toxin (1 microg/ml, 24 h) inhibited hyperpolarizations caused by uroguanylin. Depolarizations caused by guanylin and uroguanylin were blocked by the tyrosine kinase inhibitor, genistein (10 microm). All three peptides increased cellular cGMP. mRNA for GC-C was detected in IHKE-1 cells and in isolated human proximal tubules. In IHKE-1 cells GC-C was also detected by immunostaining. These findings suggest that GC-C is probably the receptor for guanylin and STa. For uroguanylin two distinct signaling pathways exist in IHKE-1 cells, one involves GC-C and cGMP as second messenger, the other is cGMP-independent and connected to a pertussis toxin-sensitive G protein.