Plasmin in Nephrotic Urine Activates the Epithelial Sodium Channel

Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC γ subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC.Nephrotic syndrome is characterized by proteinuria, sodium retention, and edema. Increased renal sodium reabsorption occurs in the cortical collecting duct (CCD),^1,2 where a rate-limiting step in transepithelial sodium transport is the epithelial sodium channel (ENaC), which is composed of the three homologous subunits: α, β, γ.³ENaC activity is regulated by hormones, such as aldosterone and vasopressin (AVP)^4,5; however, adrenalectomized rats and AVP-deficient Brattleboro rats are capable of developing nephrotic syndrome,^1,6 and nephrotic patients do not consistently display elevated levels of sodium-retaining hormones,^7,8 suggesting that renal sodium hyper-reabsorption is independent of systemic factors. Consistent with this, sodium retention is confined to the proteinuric kidney in the unilateral puromycin aminonucleoside (PAN) nephrotic model.^2,9,10There is evidence that proteases contribute to ENaC activation by cleaving the extracellular loops of the α- and γ-subunits.^11–13 Proteolytic activation of ENaC by extracellular proteases critically involves the cleavage of the γ subunit,^14–16 which probably leads to the release of a 43-residue inhibitory peptide from the ectodomain.¹⁷ Both cleaved and noncleaved channels are present in the plasma membrane,^18,19 allowing proteases such as channel activating protease 1 (CAP1/prostasin),²⁰ trypsin,²⁰ chymotrypsin,²¹ and neutrophil elastase²² to activate noncleaved channels from the extracellular side.^23,24 We hypothesized that the defective glomerular filtration barrier in nephrotic syndrome allows the filtration of ENaC-activating proteins into the tubular fluid, leading to stimulation of ENaC. The hypothesis was tested in the PAN nephrotic model in rats and with urine from patients with nephrotic syndrome.     

Effect of an acute medical admission unit on key quality indicators assessed by funnel plots

Objective

To determine the effect of the introduction of an acute medical admissions unit (AMAU) on key quality efficiency and outcome indicator comparisons between medical teams as assessed by funnel plots.

Methods

A retrospective analysis was performed of data relating to emergency medical patients admitted to St James'' Hospital, Dublin between 1 January 2002 and 31 December 2004, using data on discharges from hospital recorded in the hospital in‐patient enquiry system. The base year was 2002 during which patients were admitted to a variety of wards under the care of a named consultant physician. In 2003, two centrally located wards were reconfigured to function as an AMAU, and all emergency patients were admitted directly to this unit. The quality indicators examined between teams were length of stay (LOS) <30 days, LOS >30 days, and readmission rates.

Results

The impact of the AMAU reduced overall hospital LOS from 7 days in 2002 to 5 days in 2003/04 (p<0.0001). There was no change in readmission rates between teams over the 3 year period, with all teams displaying expected variability within control (95%) limits. Overall, the performance in LOS, both short term and long term, was significantly improved (p<0.0001), and was less varied between medical teams between 2002 and 2003/04.

Conclusions

Introduction of the AMAU improved performance among medical teams in LOS, both short term and long term, with no change in readmissions. Funnel plots are a powerful graphical technique for presenting quality performance indicator variation between teams over time.     

pH sensing in skin tumors: Methods to study the involvement of GPCRs,acid-sensing ion channels and transient receptor potential vanilloid channels

Solid tumors exhibit an inversed pH gradient with increased intracellular pH (pH_i) and decreased extracellular pH (pH_e). This inside-out pH gradient is generated via sodium/hydrogen antiporter 1, vacuolar-type H + ATPases, monocarboxylate transporters, (bi)carbonate (co)transporters and carboanhydrases. Our knowledge on how pH_e-signals are sensed and what the respective receptors induce inside cells is scarce. Some pH-sensitive receptors (GPR4, GPR65/TDAG8, GPR68/OGR1, GPR132/G2A, possibly GPR31 and GPR151) and ion channels (acid-sensing ion channels ASICs, transient receptor potential vanilloid receptors TRPVs) transduce signals inside cells. As little is known on the expression and function of these pH sensors, we used immunostainings to study tissue samples from common and rare skin cancers. Our current and future work is directed towards investigating the impact of all the pH-sensing receptors in different skin tumors using cell culture techniques with selective knockdown/knockout (siRNA/CRISPR-Cas9). To study cell migration and proliferation, novel impedance-based wound healing assays have been developed and are used. The field of pH sensing in tumors and wounds holds great promise for the development of pH-targeting therapies, either against pH regulators or sensors to inhibit cell proliferation and migration.     

Results of a survey of German dermatologists on the therapeutic approaches to pemphigus and bullous pemphigoid

Background: The lack of controlled trials on the treatment of pemphigus and pemphigoid diseases limits an evidence‐based therapy of these disorders.The aim of this survey was to assess the current treatment standards at German dermatological hospitals and the need for future therapeutic trials. Methods: A two‐page questionnaire was sent to 42 German academic and non‐academic dermatological hospitals and evaluated at the Department of Dermatology, University Medical Center, Freiburg. Results: The response rate was 76%. Topical clobetasol propionate treatment was regarded as first line therapy of bullous pemphigoid in 27% of the hospitals. More than 50% used systemic corticosteroids at an initial dose of ≤ 1 mg/kg prednisolone‐equivalent in pemphigus and pemphigoid. Azathioprine is used as first line adjuvant in pemphigoid and pemphigus treatment in 69% and 81% of the hospitals, respectively. Dapsone and mycophenolate mofetil represent alternative options, as well as cyclophosphamide‐dexamethasone‐pulse therapy in pemphigus. Immunosuppressive treatment of pemphigoid and pemphigus is terminated by 58% and 46% of the hospitals 1–3 months after clinical remission, respectively, but the given time points appeared to be variable. Conclusions: The high response rate to this survey demonstrates the interest in this topic. The variability of the answers regarding initial doses of corticosteroids and time points for termination of therapy indicates the need for the establishment of guidelines and for controlled therapeutic trials.     

Central hemodynamic effects of diuretic therapy in chronic heart failure

In chronic heart failure diuretic drugs improve central hemodynamic variables and cardiac pumping secondary to altered plasma and extracellular volumes; humoral markers of these changes include increased plasma renin and aldosterone levels. The latter increases are maximal over the first week but decline with chronic therapy. The plasma alpha-ANP levels show a reciprocal effect; these data are compatible with a rapid contraction of the plasma volume which is sustained during chronic therapy.The acute hemodynamic actions of diuretic agents reflect both immediate and direct vascular actions and also effects secondary to diuresis (volume redistribution). At rest substantial reductions in pulmonary wedge pressure (–29%), with a consequent fall in cardiac output (–10%), are described. Total systemic vascular resistance initially increases but reverse autoregulation over subsequent weeks returns this elevation gradually towards control values. Tolerance to these initial hemodynamic effects does not occur with maintained therapy; moreover, echocardiographic markers of contractility and exercise capacity may increase. The early venodilator effects of diuretic drugs can be attributed to prostaglandin release and the initial pressor actions to activation of the renin angiotensin system; these vascular actions may have limited relevance to long-term beneficial effects on hemodynamics. Direct pulmonary vasodilation and improved pulmonary compliance remain an interesting finding. Although most patients are both symptomatically and hemodynamically improved at rest, the actions during exercise are more varied. Some individuals with severely impaired left ventricular function show little hemodynamic improvement, whereas those with milder dysfunction usually benefit; in the main this is probably related to the latter being on a steeper cardiac function curve. The impact of diuretic therapy on the underlying disease process is unclear; however, there is little convincing evidence of remodelling or improvement in intrinsic performance (as distinct from that induced by altered loading conditions).