Brian N. Ling

Role of Janus Kinase/Signal Transducer and Activator of Transcription and Mitogen-activated Protein Kinase Cascades in Angiotensin II- and Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Proliferation

In vascular smooth muscle cells, the induction of early growth response genes involves the Janus kinase (JAK)/signal transducer and activators of transcription (STAT) and the Ras/Raf-1/mitogen-activated protein kinase cascades. In the present study, we found that electroporation of antibodies against MEK1 or ERK1 abolished vascular smooth muscle cell proliferation in response to either platelet-derived growth factor or angiotensin II. However, anti-STAT1 or -STAT3 antibody electroporation abolished proliferative responses only to angiotensin II and not to platelet-derived growth factor. AG-490, a specific inhibitor of the JAK2 tyrosine kinase, prevented proliferation of vascular smooth muscle cells, complex formation between JAK2 and Raf-1, the tyrosine phosphorylation of Raf-1, and the activation of ERK1 in response to either angiotensin II or platelet-derived growth factor. However, AG-490 had no effect on angiotensin II- or platelet-derived growth factor-induced Ras/Raf-1 complex formation. Our results indicate that: 1) STAT proteins play an essential role in angiotensin II-induced vascular smooth muscle cell proliferation, 2) JAK2 plays an essential role in the tyrosine phosphorylation of Raf-1, and 3) convergent mitogenic signaling cascades involving the cytosolic kinases JAK2, MEK1, and ERK1 mediate vascular smooth muscle cell proliferation in response to both growth factor and G protein-coupled receptors.

A Mechanism for Pentamidine-Induced Hyperkalemia: Inhibition of Distal Nephron Sodium Transport

Pentamidine is an antiparasitic agent used to treat the opportunistic infection Pneumocystis carinii pneumonia [1-6]. Unfortunately, hyperkalemia is an important complication of therapy, observed in as many as 100% of patients with the acquired immunodeficiency syndrome (AIDS) receiving pentamidine for more than 6 days [1-4]. This elevation in the serum potassium level can be seen in the absence of adrenal insufficiency, hyporeninemic hypoaldosteronism, interstitial nephritis, or hyperglycemia (pentamidine-induced pancreatic islet cell dysfunction). Investigators have also reported azotemia in 25% to 95% of patients infected with the human immunodeficiency virus (HIV) who receive pentamidine [6-8]. However, hyperkalemia is usually out of proportion to the degree of coexisting renal insufficiency and is frequently associated with hyperchloremic metabolic acidosis [6, 7, 9]. These findings have led several groups to postulate that pentamidine might directly effect renal tubular K+ secretion [1, 4, 6]. Most renal K+ is excreted through secretory K+ channels located in the apical membrane of principal cells in the cortical collecting tubule [10-12]. The electrochemical driving force for distal nephron K+ secretion (that is, increased urinary lumen negativity and high intracellular K+ levels) is maintained by luminal Na+ entry through apical Na+ channels and serosal K+ uptake through the basolateral Na+/K +-adenosine triphosphatase pump [10-13]. Pentamidine is an aromatic diamidine structurally similar to potassium-sparing diuretics such as amiloride, triamterene, and trimethoprim [5, 13, 14] (Figure 1). We therefore applied transepithelial and single-channel measurement techniques to two well-established models of cortical collecting tubule ion transport (A6 amphibian cell line and primary cultured rabbit cortical collecting tubules) to investigate the effects of pentamidine on renal tubular Na+ reabsorption and, therefore, K+ secretion. Figure 1. Structures of pentamidine and potassium-sparing diuretics such as amiloride, triamterene, and trimethoprim. Methods Transepithelial Measurements on A6 Distal-Nephron Cell Line Cultures The A6 cell (American Type Culture Collection, Rockville, Maryland) subpassages 91 to 96 were grown to confluency on collagen-coated polycarbonate filters (Costar; Cambridge, Massachusetts) in the presence of 1.5 M of aldosterone. Filters were mounted in a modified Ussing chamber, and macroscopic current was measured at room temperature with a DVC-1000 voltage clamp (World Precision Instruments, Sarasota, Florida) [14-16]. We measured short-circuit current under voltage-clamp conditions and added 10 M of amiloride to the luminal bath at the end of each experiment to determine the amiloride-sensitive component of the short-circuit current. Bath solutions contained the following: 100 mM of NaCl, 4 mM of KCl, 2.5 mM of NaHCO3, 1 mM of KPo 4, 1 mM of CaCl2, 11 mM of glucose, and 10 mM of n-2-hydroxyethylpiperazine-n-2-ethanesulfonic (HEPES) (pH, 7.4). Patch Clamp Measurements on Primary Cultures of Rabbit Cortical Collecting Tubules As previously described [17], renal cortices from New Zealand white rabbits (body weight, 1 to 2 kg) were collagenase digested and subjected to Percoll density centrifugation. Rabbit cortical collecting tubule fragments were separated and grown to confluency on permeable, collagen-coated Millipore-CM filters (Millipore Corp., Bedford, Massachusetts) in the presence of 1.5 M of aldosterone. Unitary channel events were measured at 37 C with a List EPC-7 Patch Clamp (Medical Systems Corp., Greenvale, New York). Data were digitized, recorded, and analyzed as previously described [10, 14, 17]. Patch pipette and extracellular bath solutions consisted of a physiologic saline solution containing the following: 140 mM of NaCl (final NaCl concentration after titration to a pH of 7.4 with NaOH), 5 mM of KCl, 1 mM of CaCl2, 1 mM of MgCl2, and 10 mM of HEPES (pH, 7.4). Chemicals Pentamidine (1,5-bis[p-Amidinophenoxyl]-pentane bis[2-hydoxyethane-sulfonate salt]) was of the highest commercial grade available (Sigma Chemical, St. Louis, Missouri). We added appropriate solvent vehicles to control baths that by themselves did not change the Na (+) channel activity. Results The amiloride-sensitive component of the short-circuit current is a measure of the net Na+ transport across the renal epithelium [14, 16, 18]. When we applied various concentrations of pentamidine to the solution, bathing the luminal or urinary surface of A6 distal nephron cell monolayers, we observed a dose-dependent inhibition of the amiloride-sensitive, short-circuit current (five experiments) (Figure 2). This inhibition developed rapidly, and 50% blockage of the amiloride-sensitive, short-circuit current (50% inhibitory concentration) occurred with 700 M of pentamidine at a pH of 7.4. Figure 2. Effect of pentamidine on short-circuit current (Isc) in A6 distal nephron cells. In previous studies, we have characterized the amiloride-sensitive, 4-picosiemen Na+ channel that is responsible for physiologic, mineralocorticoid-dependent Na+ reabsorption in the mammalian distal nephron [17]. We therefore examined the effect of pentamidine on 4-picosiemen Na+ channel activity in apical, cell-attached patches on principal cells of primary cultured rabbit cortical collecting tubules. The results, summarized in Table 1, show that addition of 1.0 M of pentamidine to the serosal bath or luminal bath (outside the cell-attached pipette) did not significantly affect Na+ channel activity within the unexposed patch membrane (no pentamidine was added to the pipette solution). In contrast, when pentamidine was placed in direct contact with the luminal surface of the patch membrane (1.0 M of pentamidine in the pipette solution), Na+ channel activity (measured as the number of channels times the open probability) decreased to 40% of control values. Table 1. Effects of Pentamidine on Principal-Cell Na+ Channels The pentamidine-induced inhibition of Na+ channel activity appeared to be primarily caused by a decrease in open probability (percentage of time an individual channel is open) rather than the number of channels per patch (membrane channel density) (Table 1). We confirmed this impression in four cell-attached patches that contained only one Na+ channel and therefore permitted us to directly calculate open probability (Figure 3). In these latter experiments, we could also show reversibility of the inhibitory effects of luminal pentamidine on Na+ channel kinetics. Figure 3. Effect of intrapipette pentamidine on Na+ channel activity in rabbit cortical collecting tubule primary cultures. Top. Bottom. Discussion The electrochemical driving force for K+ secretion in the cortical collecting tubule is maintained by luminal Na+ entry through apical Na+ channels and by serosal K+ uptake through the basolateral Na+/K+-adenosine triphosphatase pump [10-12]. We have previously shown that the mechanism of action for potassium-sparing diuretics, such as amiloride, trimethoprim, and triamterene, is direct blockade of these apical Na+ channels [13, 14, 17, 18]. Therefore, unlike most diuretics, which promote both natriuresis and kaliuresis, these latter drugs actually inhibit kaliuresis [13, 16]. In this study, we showed that luminal exposure to pentamidine inhibits Na+ reabsorption in both mammalian and amphibian distal nephron cells. Luminal pentamidine inhibited both amiloride-sensitive, macroscopic short-circuit currents and individual Na+ channels at concentrations greater than 50 M and 1.0 M, respectively. Differences in the apparent inhibitory constant of pentamidine for the Na+ channel in rabbit principal cells and in amphibian A6 cells may reflect either subtle variabilities within the structure of Na+ channels expressed in mammalian and amphibian renal cells or the different techniques used to examine Na+ transport [14, 19]. Little information is available on the pharmacokinetics of pentamidine in humans [5]. Pentamidine has a serum half-life of 5 to 6 hours after one parenteral dose, but the half-life increases to a mean of 52 89 hours after multiple doses [20]. Only 15% to 20% of a single intramuscular dose is excreted daily in the urine, yielding urinary concentrations of 20 to 25 g/mL (1 g/mL M) [5]. In patients with impaired renal function, parenteral pentamidine is excreted in the urine at a rate of 1.85 to 7.13 mg/d [20]. Urinary concentrations after daily administration of aerosolized pentamidine range from 1.3 to 778 ng/mg of creatinine per milliliter [21]. Therefore, most of the administered pentamidine becomes protein- and tissue-bound, with an estimated volume of distribution of 3 L/kg body weight and the greatest accumulation occurring in the kidney [5]. Urinary levels are detectable for months after therapy is discontinued. In patients with AIDS, autopsy studies have found detectable levels of pentamidine in renal tissue for as long as 1 year after the last dose [1]. Tissue depot release probably accounts for the wide range of reported urinary concentrations and the observation that pentamidine-induced hyperkalemia can persist for days after the drug is withdrawn [2]. In conclusion, pentamidine therapy for treating HIV-infected patients with P. carinii pneumonia can be associated with life-threatening hyperkalemia. We have shown that pentamidine, at concentrations found clinically in the urine, directly and reversibly blocks apical Na+ channels in a manner similar to potassium-sparing diuretics. The result is a decrease in the electrochemical driving force for both K+ and H+ secretion in the cortical collecting tubule. It is therefore not surprising that hyperkalemia and hyperchloremic metabolic acidosis are observed in patients treated with pentamidine, amiloride, triamterene, or trimethoprim [6, 7, 9, 14, 22]. These renal tubular effects provide a mechanism for pentamidine-induced hyperkalemia in patients without sever

Angiotensin II signalling events mediated by tyrosine phosphorylation

Angiotensin II is a potent vasoconstrictor that is important in the control of systemic blood pressure. All the hemodynamic effects of angiotensin II result from the AT1 receptor which has the structural features of a seven transmembrane receptor. Both in cultured rat aortic smooth muscle cells and rat glomerular mesangial cells, angiotensin II stimulates the rapid tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1). Tyrosine kinase inhibitors that block this phosphorylation also block the angiotensin II-mediated production of 1,4,5 inositol trisphosphate (1,4,5-IP3) and the intracellular release of Ca2+. The cellular tyrosine kinase c-src appears to play a critical role in the angiotensin II-stimulated tyrosine phosphorylation of PLC-gamma 1 and the generation of 1,4,5-IP3. We have also found that angiotensin II stimulates the tyrosine phosphorylation and activation of the JAK family of intracellular kinases. This in turn activates the STAT family of transcription factors. Angiotensin II, working through the AT1 receptor, uses tyrosine phosphorylation as a mechanism to convey signals from the cell surface to the cell nucleus.

Comparison of Fine-Needle Aspiration Biopsy, Doppler Ultrasound, and Radionuclide Scintigraphy in the Diagnosis of Acute Allograft Dysfunction in Renal Transplant Recipients: Sensitivity, Specificity, and Cost Analysis

150 episodes of allograft dysfunction in 128 renal transplant recipients, 77 due to acute rejection, 32 secondary to acute-on-chronic rejection, 33 due to either prerenal factors, acute tubular necrosis, or ciclosporin A nephrotoxicity, and 8 secondary to multiple causes, were evaluated by fine-needle aspiration biopsy (FNAB), Doppler ultrasound (DUS), and radionuclide scintigraphy (RS), each performed within a 24-hour period and prior to any specific therapeutic intervention. Tests were interpreted by appropriate specialists in a large transplant center without access to clinical information. The final diagnosis was based primarily upon response to therapeutic maneuvers with histological (core biopsy) confirmation in 123 episodes. RS was the most sensitive (70%) test for the diagnosis of acute rejection during the early posttransplant period, exceeding both FNAB (52%) and DUS (43%). The predictive accuracy of either FNAB, DUS, RS, or core biopsy in the detection of a steroid-responsive component to acute rejection when superimposed upon chronic rejection was low at approximately 50%. When the underlying cause of renal dysfunction was either prerenal, acute tubular necrosis, or ciclosporin A nephrotoxicity, FNAB, DUS, and RS each gave an erroneous diagnosis of acute rejection in about 50% of the episodes. Cost analysis revealed that core biopsy was the most expensive test, but only 9% more than RS, with FNAB the least costly. In conclusion, the lack of ideal sensitivity and specificity combined with the expense of present-day FNAB, DUS, RS, and core biopsy in the diagnosis of a therapeutically reversible component to acute-on-chronic rejection and of FNAB, DUS, and RS in the diagnosis of acute rejection during the early posttransplant period should prompt research into ways to improve their diagnostic yield or alternate modalities.

Regulation of the amiloride-blockable sodium channel from epithelial tissue.

The first step in net active transepithelial transport of sodium in tight epithelia is mediated by the amiloride-blockable sodium channel in the apical membrane. This sodium channel is the primary site for discretionary control of total body sodium and, therefore, investigating its regulatory mechanisms is important to our understanding of the physiology of fluid and electrolyte balance. Because essentially all of the regulatory sites on the channel are on the intracellular surface, patch clamp methods have proven extremely useful in the electrophysiological characterization of the sodium channel by isolating it from other channel proteins in the epithelial membrane and by allowing access to the intracellular surface of the protein. We have examined three different regulatory mechanisms. (1) Inhibition of channel activity by activation of protein kinase C; (2) activation of the channel by agents which activate G-proteins; and (3) modulation of channel kinetics and channel number by mineralocorticoids. Activation of protein kinase C by phorbol esters or synthetic diacylglycerols reduces the open probability of sodium channels. Protein kinase C can be activated in a physiological context by enhancing apical sodium entry. Actions which reduce sodium entry (low luminal sodium concentrations or the apical application of amiloride) increase channel open probability. The link between sodium entry and activation of protein kinase C appears to be mediated by intracellular calcium activity linked to sodium via a sodium/calcium exchange system. Thus, the intracellular sodium concentration is coupled to sodium entry in a negative feedback loop which promotes constant total entry of sodium. Activation of G-proteins by pertussis toxin greatly increases the open probability of sodium channels. Since channels can also be activated by pertussis toxin or GTP gamma S in excised patches, the G-protein appears to be closely linked in the apical membrane to the sodium channel protein itself. The mechanism for activation of this apical G-protein, when most hormonal and transmitter receptors are physically located on the basolateral membrane, is unclear. Mineralocorticoids such as aldosterone have at least two distinct effects. First, as expected, increasing levels of aldosterone increase the density of functional channels detectable in the apical membrane. Second, contrary to expectations, application of aldosterone increases the open probability of sodium channels. Thus aldosterone promotes the functional appearance of new sodium channels and promotes increased sodium entry through both new and pre-existant channels.

Regulation of renal epithelial sodium channels

The high selectivity, low conductance, amiloride-blockable, sodium channel of the mammalian distal nephron (i.e. cortical collecting tubule) is the site of discretionary regulation which allows maintainance of total body sodium balance. In order to understand the physiological events that participate in this regulation, we have used the patch-clamp technique which allows us to measure individual Na+ channel currents and permits access to the cytosolic side of the channel-protein as well as its associated regulatory components. Most of our experiments have utilized the A6 amphibian renal cell line, which when grown on permeable supports is an excellent model for the mammalian distal nephron. Different mechanisms have been examined: (1) regulation by hormonal factors such as Anti-Diuretic Hormone (ADH) and aldosterone, (2) regulation by G-proteins, (3) modulation by protein kinase C (PK-C), and (4) modulation by products of arachidonic acid metabolism. Consistent with noise analysis of tight epithelial tissues, ADH treatment increased the number of active channels in apical membrane patches of A6 cells, without any apparent change in the open probability (Po) of the individual channels. Agents that increased intracellular cAMP mimicked the effects of ADH. In contrast, aldosterone was found to act through a dramatic increase in Po rather than through changes in channel density. Inhibition of methylation by deazaadenosine antagonizes the stimulatory effect of aldosterone. In excised inside-out patches GTPγS inhibits channel activity, whereas GDPβS or pertussis toxin stimulates activity suggesting regulatory control by G-proteins. PK-C has been shown to contribute to ‘feed-back inhibition’ of apical Na+ conductance in tight epithelia. Raising luminal bath sodium and therefore intracellular Na+ inhibits sodium channel activity, an effect that is prevented by PK-C inhibitors and mimicked by PK-C agonists. Cyclooxygenase metabolites of arachidonic acid have an inhibitory effect on channel activity. Finally, a possible role for tyrosine kinase as well as membrane cytoskeleton in the regulation of sodium channel function is also suggested.

SYMPOSIUM: Experimental Biology 1995 Role of Mesangial Cell Ion Transport in Glomerular Physiology and Disease: ANGIOTENSIN II-INDUCED TYROSINE PHOSPHORYLATION IN MESANGIAL AND VASCULAR SMOOTH MUSCLE CELLS

1 Angiotensin II (AngII)‐induced, activation of phospholipase C (PLC) and Ca2+‐dependent Cl− channels is an important signal transduction pathway for the regulation of vascular smooth muscle cell (VSMC) and glomerular mesangial cell contraction and growth. While AT receptors are traditionally thought to be G‐protein coupled to the β isoform of PLC, recent evidence suggests that in some tissues AT receptors may also activate the PLC‐γ isoform via tyrosine phosphorylation. 2 By western analysis, we identified PLC‐γ1 in the above cell types. We found that within 3 min of exposure to 10−7 mol/L AngII, tyrosine phosphorylation of PLC‐γ1 was observed; however, peak response (> 3‐fold increase) occurred within 0.5 min. In addition, pre‐incubation of these cells with the tyrosine kinase inhibitor genistein blocked the tyrosine phosphorylation of PLC‐γ1 by AngII. In contrast, preincubation with the tyrosine phosphatase inhibitor sodium vanadate increased the levels of tyrosine phosphorylation of PLC‐γ1. Similar results were found when intracellular levels of 1,4,5‐IP3 were measured after AngII exposure. 3 By using patch clamp techniques on cultured rat mesangial cells exposed to AngII, we found that the release of 1,4,5‐IP3‐sensitive intracellular Ca2+ stores stimulated low conductance Cl− channels. Preincubation with genistein, abolished the usual 10‐fold increase in Cl− channel activity observed with AngII. 4 Therefore, we conclude that in VSMC and glomerular mesangial cells (i) AngII transiently stimulates PLC activity via tyrosine phosphorylation of the γ1 isoenzyme, (ii) tyrosine phosphorylation of PLC‐γ1 and production of 1,4,5‐IP3 in response to AngII is dramatically inhibited by tyrosine kinase inhibition and stimulated by tyrosine phosphatase inhibition, (iii) activation of Ca2+‐dependent Cl− channels by AngII‐induced release of 1,4,5‐IP3‐dependent intracellular Ca2+ stores is also abolished by tyrosine kinase inhibition. In summary, this AngII‐induced signal transduction cascade provides a possible mechanism for both the contractile and growth‐stimulating effects of AngII on VSMC and glomerular mesangial cells.

Naproxen-Induced Nephropathy in Systemic Lupus erythematosus

A 34-year-old female with an 8-month history of systemic lupus erythematosus and intermittent naproxen use presented with acute oliguric renal failure, hypoalbuminemia, 4+ proteinuria, and an active urinary sediment. The clinical picture suggested a rapidly progressive lupus glomerulonephritis. Renal biopsy, however, demonstrated chronic, active interstitial nephritis without evidence of immune deposits by immunofluorescence or electron microscopy. Nonsclerotic glomeruli revealed diffuse foot process fusion without cellular proliferation. These findings were consistent with nonsteroidal anti-inflammatory drug induced nephropathy. Discontinuation of naproxen and institution of corticosteroid therapy was followed by improvement in renal function and remission of nephrotic syndrome. This case represents the first report of nonsteroidal antiinflammatory drug nephropathy associated with systemic lupus erythematosus.

SYMPOSIUM: Experimental Biology 1995 Role of Mesangial Cell Ion Transport in Glomerular Physiology and Disease: REGULATION OF MESANGIAL CHLORIDE CHANNELS BY INSULIN AND GLUCOSE: ROLE IN DIABETIC NEPHROPATHY

1 In response to vasoactive peptides (e.g. angiotensin II (AngII), vasopressin, endothelin‐1, platelet‐activating factor), glomerular mesangial cell contraction is mediated through activation of a Ca2+‐dependent Cl− conductance that, in turn, promotes membrane depolarization and voltage‐activated Ca2+ entry. 2 Using patch clamp technology, our laboratory was the first to characterize a candidate Ca2+‐dependent, 4pS Cl− channel that is stimulated by vasoactive peptides in cultured rat mesangial cells. In the absence of extracellular insulin, the activation of Cl− channels by AngII is abolished. We find that Cl− channel sensitivity to intracellular Ca2+ and the membrane density of AngII receptors is also dependent on the presence of insulin. 3 Our studies also show that high extracellular glucose interferes with mesangial cell IP3 generation and Cl− channel stimulation. Importantly, we find that the insulin‐dependency of Cl− channels occurs within the range of plasma insulin concentrations observed in normal, obese, hypertensive and diabetic humans (i.e. 1–100μU/mL). Similarly, normal regulation of Cl− channel activity is also modulated by glucose concentrations commonly observed in the plasma of diabetic humans (5–30 mmol/L). 4 There is substantial evidence, both in diabetic humans and animal models, that the provision of insulin and improved glycaemic control corrects or prevents glomerular hyperfiltration. The requirement for normal insulin and glucose levels, for the proper regulation of the 4pS Cl− channel, provides a mechanism for impaired Ca2+ uptake and contraction observed in glomerular mesangial cells in association with insulin deficiency and hyperglocaemia.