Jochen R. Hirsch

Regulation of the human organic cation transporter hOCT1

The human organic cation transporter type 1 (hOCT1) is an important transport system for small organic cations in the liver. Organic cation transporters are regulated by different signaling pathways, but the regulation of hOCT1 has not yet been studied. In this work, we have for the first time investigated the regulation of hOCT1. hOCT1 was expressed in Chinese hamster ovary cells (CHO‐hOCT1) and in human embryonic kidney cells (HEK293‐hOCT1). Its activity was monitored using microfluorimetry with the fluorescent organic cation 4‐(4‐(dimethylamino)styryl)‐N‐methylpyridinium (ASP+) as substrate. hOCT1 expressed in CHO‐cells was inhibited by protein kinase A (PKA) activation (1 µM forskolin, −58 ± 6%, n = 12), calmodulin inhibition (0.1 µM calmidazolium, −68 ± 3%, n = 6; 10 µM ophiobolin A, −48 ± 10%, n = 7), calmodulin‐dependent kinase II inhibition (1 µM KN62, −78 ± 4%, n = 12), and inhibition of p56lck tyrosine kinase (10 µM aminogenistein, −35 ± 7%, n = 12). The apparent affinities for TEA+ were lower in CHO‐hOCT1 than in HEK293‐hOCT1, while those for TPA+ and quinine were almost identical; the rank order of EC50 values (TPA+ > quinine > TEA+) was independent of the expression system. EC50 values for TEA+ in CHO‐hOCT1 or HEK293‐hOCT1 were increased under calmidazolium incubation (6.3 and 1.4 mM, respectively). hOCT1 was inhibited by PKA and endogenously activated by calmodulin, calmodulin‐dependent kinase II, and p56lck tyrosine kinase. Regulation pathways were the same in the two expression systems. Since apparent substrate affinities depend on activity of regulatory pathways, the expression system plays a role in determining the substrate affinities.

Short-Term Monotherapy in HIV-Infected Patients with a Virus Entry Inhibitor Against the gp41 Fusion Peptide

An optimized derivative of a natural HIV-1 entry inhibitor targeting the gp41 fusion peptide shows antiviral potency and minimal side effects in a Phase I/II clinical trial. Anchors Away: Blocking HIV Entry Combination antiretroviral therapy has been very successful for treating infection with the human immunodeficiency virus (HIV-1), which causes AIDS. However, drug resistance is emerging and there is a need to develop new antiretroviral drugs that work earlier in the virus life cycle, for example, by preventing HIV-1 from entering host cells. Two such virus entry inhibitors, maraviroc and T-20, are in clinical use, but both have drawbacks. Forssmann, Kirchhoff and their colleagues have now developed a new virus entry inhibitor called VIRIP (VIRus-Inhibitory Peptide), a 20-peptide fragment of α1-antitrypsin, an abundant circulating serine protease inhibitor. VIRIP and its optimized derivative VIR-576 are so-called anchoring inhibitors because they prevent the gp41 fusion peptide of HIV-1 from inserting itself into the host cell membrane. This then blocks the next step in the virus life cycle, which is fusion of the virion envelope with the host cell membrane. Forssmann and co-workers now report on a Phase I/II clinical trial in which 18 HIV-1–infected patients who were not on any other antiretroviral therapy were treated for 10 days with three different doses of VIR-576 (0.5, 1.5, 5.0 g/day). They show that VIR-576 reduced the viral load in the plasma of patients on the highest dose by an order of magnitude and that the drug was well tolerated. Previous studies have shown that the gp41 fusion peptide is essential for HIV-1 entry into host cells, and suggest that it may be difficult for HIV-1 to develop resistance to VIR-576 because the fusion peptide is highly conserved and hardly tolerates changes without loss of function. This anchoring inhibitor, unlike other HIV entry inhibitors, is also active against many different HIV strains and has a different target (the gp41 fusion peptide). Thus, VIR-576 represents a potential new class of HIV entry inhibitor. However, VIR-576 does have some drawbacks too. Because VIR-576 is a peptide, it will be costly and time-consuming to produce and it must be administered intravenously. This has prompted Kirchhoff and colleagues to start searching for a small molecule that would block the gp41 fusion peptide in the same way as VIR-576 but would have the advantage that it could be made cheaper and given orally. 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 α1-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 log10 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.

pH dependence of K+ conductances of rat cortical collecting duct principal cells.

The K+ channels of the principal cells of rat cortical collecting duct (CCD) are pH sensitive in excised membranes. K+ secretion is decreased with increased H+ secretion during acidosis. We examined whether the pH sensitivity of these K+ channels is present also in the intact cell and thus could explain the coupling between K+ and H+ secretion. Membrane voltages (Vm), whole-cell conductances (gc), and single-channel currents of K+ channels were recorded from freshly isolated CCD cells or isolated CCD segments with the patch-clamp method. Intracellular pH (pHi) was measured using the pH-sensitive fluorescent dye 2′-7′-bis(carboxyethyl)-5-6-carboxyfluorescein (BCECF). Acetate (20 mmol/l) had no effect on Vm, gc, or the activity of the K+ channels in these cells. Acetate, however, acidified pHi slightly by 0.17±0.04 pH units (n=19). Vm depolarized by 12±3 mV (n=26) and by 23±2 mV (n=66) and gc decreased by 26±5% (n=13) and by 55±5% (n=12) with 3–5 or 8–10% CO2, respectively. The same CO2 concentrations decreased pHi by 0.49±0.07 (n=15) and 0.73±0.11 pH units (n=12), respectively. Open probability (Po) of all four K+ channels in the intact rat CCD cells was reversibly inhibited by 8–10% CO2. pHi increased with the addition of 20 mmol/l NH4+/NH3 by a maximum of 0.64±0.08 pH units (n=33) and acidified transiently by 0.37±0.05 pH units (n=33) upon NH4+/NH3 removal. In the presence of NH4+/NH3Vm depolarized by 16±2 mV (n=66) and gc decreased by 26±7% (n=16). The activity of all four K+ channels was also strongly inhibited in the presence of NH4+/NH3. The effect of NH4+/NH3 on Vm and gc was markedly increased when the pH of the NH4+/NH3-containing solution was set to 8.5 or 9.2. From these data we conclude that cellular acidification in rat CCD principal cells down-regulates K+ conductances, thus reduces K+ secretion by direct inhibition of K+ channel activity. This pH dependence is present in all four K+ channels of the rat CCD. The inhibition of K+ channels by NH4+/NH3 is independent of changes in pHi and rather involves an effect of NH3.

The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP.

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 ± 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP + . ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 ± 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.

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.

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.

Renal Transplantation Modulates Expression and Function of Receptors and Transporters of Rat Proximal Tubules

Kidney transplantation often leads to disturbances of solute and volume maintenance in humans. To investigate underlying mechanisms, expression and function of renal transporters and receptors of the proximal tubule (PT) were analyzed in an acute rejection model of rat kidney transplantation. Semiquantitative RT-PCR and Western blot, histology, immunohistochemistry, and microfluorometry were performed on whole kidneys and isolated PT. With acute rejection, Na+/H+-exchanger type-3 (NHE-3) was markedly downregulated. Na+-HCO(3)(-)-cotransporter (NBC-1) and Na+-glucose transporter type-2 (SGLT2) were upregulated after transplantation. Expressions of Na+/H+-exchanger type-1 (NHE-1), Na+/K+-ATPase (NKA), angiotensin II (AngII) receptor (AT-1), or natriuretic peptide receptor (GC-A) were unaltered. Microfluorometric analyses of intracellular pH, Na+, and Ca2+ demonstrated a decrease in NHE-3 function and AngII-mediated stimulation of NHE-3. AngII-mediated inhibition of NHE-1 and function of all other transporters tested remained unaltered. Function of AT-1 and GC-A were unaffected. Reduced expression of NHE-3 was also confirmed by semiquantitative immunohistochemistry. These findings suggest that expression and function of transmembrane proteins involved in Na+-transport after transplantation and rejection is specifically modulated. The local renin-angiotensin-system is apparently not altered. Downregulation of NHE-3 may be a protective mechanism occurring in the graft.

Cellular localization, membrane distribution, and possible function of guanylyl cyclases A and 1 in collecting ducts of rat

BACKGROUND Natriuretic peptides regulate Na+ and H(2)O transport in the cortical collecting duct (CCD). We have shown that natriuretic peptides have no effect on ion conductances or water transport of principal cells (PC) even though a cGMP-regulated K+ channel is located in the basolateral membrane of these cells. METHODS RT-PCR was used to screen for different guanylyl cyclases (GC) in CCD and to look for the expression of GC-1 and GC-A mRNA in CCD of male and female Wistar and Sprague-Dawley rats. Polyclonal antibodies were raised against the detected GC. BCECF was used to investigate the effects of ANP on intracellular pH in intercalated cells (IC). RESULTS GC-A and GC-1 were detected. GC-A was immunolocalized in the luminal membrane of IC while GC-1 was mainly found in the luminal membrane of PC. GC-1 is expressed in Sprague-Dawley and Wistar rats except for male Sprague-Dawley rats, while GC-A is expressed in all strains. ANP (160 nM, n=11), urodilatin (140 nM, n=6), which had no effect in PC, significantly decreased pH(i) by 0.02+/-0.01 and 0.03 +/- 0.01 Units in IC, respectively. ANP as well as urodilatin and 8-Br-cGMP decreased the pH(i) recovery after acidification by 30 +/- 6% (n=12), 37 +/- 7% (n=8), and 19 +/- 3% (n=8), respectively. CONCLUSION GC-A is located in the luminal membrane of IC of rat CCD and ANP acts through this receptor when regulating pH(i) via an inhibition of the Na+/H+-exchanger. PC do not possess GC-A. GC-1 seems to be the only GC in these cells of most rat strains tested and therefore, it could be responsible for the regulation of K+ channels in the basolateral membrane via cGMP-dependent protein kinase.

Effects of diadenosine polyphosphates, ATP and angiotensin II on membrane voltage and membrane conductances of rat mesangial cells.

Diadenosine polyphosphates have been shown to influence renal perfusion pressure. As mesangial cells may contribute to these effects we investigated the effects of diadenosine triphosphate (Ap3A), diadenosine tetraphosphate (Ap4A), diadenosine pentaphosphate (Ap5A) and diadenosine hexaphosphate (Ap6A) on membrane voltage (Vm) and membrane conductance (gm) in mesangial cells (MC) of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats in primary and long-term culture. We applied the patch-clamp technique in the fast-whole-cell configuration to measure Vm and gm. To compare the effects of diadenosine polyphosphates with hitherto known agonists we also tested adenosine 5′-triphosphate (ATP) and angiotensin II (Ang II). As there was no significant difference in the Vm values in MC of WKY (−42±1 mV, n=70) and SHR rats (−45±2 mV, n=99) as well as in the agonist-induced changes of Vm, all data were pooled. The Vm of all the cells was −44±1 mV (n=169) and gm was 15.9±1.8 nS (n=141). Ion-exchange experiments showed the presence of a K+ and a non-selective cation conductance in resting MC whereas a Cl− conductance or a Na+selective conductance could not be observed. Ap3A, Ap4A, Ap5A, AP6A and ATP each at a concentration of 5 μmol/l, led to a significant depolarization of Vm by 5±2 mV (n=14), 7±1 mV (n=25), 3±1 mV (n=23), 2±1 mV (n=16), and 14±2 mV (n=23), respectively. For Ap4A, the most potent diadenosine polyphosphate, we determined the half-maximally effective concentration (EC50) as 6 μmol/l (n=5–25), for ATP as 2 μmol/l (n=9–37), and for Ang II as 8 nmol/l (n=6–18). Ap4A 100 μmol/l increased gm significantly by 55±20% (n=16), 100 μmol/l ATP by 135±60% (n=18). The diadenosine polyphosphates examined were able to depolarize Vm (Ang II >ATP> Ap4A>Ap3A>Ap5A>Ap6A) by activation of a Cl− conductance and a non-selective cation conductance, as do ATP or Ang II.

Genistein Potentiates the ANP Effect on a K+-Conductance in HEK-293 Cells

HEK-293 cells are known to reflect many features of the late distal tubule. Furthermore, they have the ability to release urodilatin, the structural analog to ANP. RT-PCR was performed to test for the expression of natriuretic peptide receptors. While the mRNA for the human ANP receptor (NPR-A, GC-A) could be amplified, the CNP-specific receptor NPR-B (GC-B) and the receptor specific for guanylins, GC-C, could not be detected. In patch clamp experiments the effects of ANP (10 nM) on membrane voltage (Vm) were monitored and HEK-293 cells depolarized by 2.3 ± 0.5 mV (n=14). In the presence of the EGF receptor blocker genistein (10 µM) the effect of ANP was increased by 65% to 3.9 ± 0.8 mV (n=14). After removal of genistein the ANP-mediated depolarization further increased by 147% to 5.7 ± 1.0 mV (n=14). ANP given repetitively without genistein had no increasing depolarizing effect in HEK-293 cells with time. The ANP effect could be fully blocked by 1 mM Ba2+ and by 1 µM of the specific PKG inhibitor KT5823 indicating that ANP inhibits a K+-conductance via a cGMP-dependent protein kinase. Genistein itself hyperpolarized the membrane voltage of HEK-293 cells by –3.9 ± 0.6 mV (n=11) and this effect could also be fully blocked by Ba2+ (-0.3 ± 0.1 mV, n=5), indicating that genistein activates a K+-conductance which contributes significantly to the membrane potential of HEK-293 cells.