James B Wade

University of Maryland, Baltimore, Baltimore, Maryland, United States

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Publications (72)384.19 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H+-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.
    The Journal of clinical investigation 04/2015; 125(5). DOI:10.1172/JCI78558 · 13.77 Impact Factor
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    ABSTRACT: The NaCl cotransporter (NCC) of the renal distal convoluted tubule is stimulated by low K+ diet by an unknown mechanism. Since recent work has shown that the STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) can function to stimulate NCC by phosphorylation of specific N-terminal sites, we investigated if the NCC response to low K+ diet is mediated by SPAK. Using phospho-specific antibodies in Western blot and immunolocalization studies of wild-type (WT) and SPAK knock-out (SPAK-/-) mice fed a low K+ or control diet for 4 days, we found that low K+ diet strongly increased total NCC expression and phosphorylation of NCC. This was associated with an increase in total SPAK expression in cortical homogenates and an increase in phosphorylation of SPAK at the S383 activation site. The increased pNCC in response to low K+ diet was blunted but not completely inhibited in SPAK-/- mice. These findings reveal that SPAK is an important mediator of the increased NCC activation by phosphorylation that occurs in the DCT in response to a low K+ diet, but other low potassium-activated kinases are likely to be involved. Copyright © 2014, American Journal of Physiology - Renal Physiology.
    American journal of physiology. Renal physiology 01/2015; 308(8):ajprenal.00388.2014. DOI:10.1152/ajprenal.00388.2014 · 3.30 Impact Factor
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    ABSTRACT: The renal outer medullary potassium channel (ROMK, KCNJ1) mediates potassium recycling and facilitates sodium reabsorption through the Na(+)/K(+)/2Cl(-) cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Human genetic studies indicate that ROMK homozygous loss-of-function mutations cause type II Bartter syndrome, featuring polyuria, renal salt wasting, and hypotension; humans heterozygous for ROMK mutations identified in the Framingham Heart Study have reduced blood pressure. ROMK null mice recapitulate many of the features of type II Bartter syndrome. We have generated an ROMK knockout rat model in Dahl salt-sensitive background by using zinc finger nuclease technology and investigated the effects of knocking out ROMK on systemic and renal hemodynamics and kidney histology in the Dahl salt-sensitive rats. The ROMK(-/-) pups recapitulated features identified in the ROMK null mice. The ROMK(+/-) rats, when challenged with a 4% salt diet, exhibited a reduced blood pressure compared with their ROMK(+/+) littermates. More importantly, when challenged with an 8% salt diet, the Dahl salt-sensitive rats with 50% less ROMK expression showed increased protection from salt-induced blood pressure elevation and signs of protection from renal injury. Our findings in ROMK knockout Dahl salt-sensitive rats, together with the previous reports in humans and mice, underscore a critical role of ROMK in blood pressure regulation.
    Hypertension 06/2013; 62(2). DOI:10.1161/HYPERTENSIONAHA.111.01051 · 7.63 Impact Factor
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    ABSTRACT: To elucidate the role of the insulin receptor (IR) in collecting duct (CD), we bred mice with IR selectively deleted from CD principal cells using an aquaporin-2 promoter to drive Cre-recombinase expression. Young, adult male KO mice had altered plasma and electrolyte homeostasis under high- (HS) and low-sodium (LS) diets, relative to WT littermates. One week of LS feeding led to a significant reduction in urine potassium (K(+)) and sodium (Na(+)) excretion in KO, and a reduction in the ratio of Na(+) to chloride (Cl-) in plasma, relative to WT. HS diet (1-week) increased plasma K+ and reduced urine Na(+) to Cl- ratio, in the KO. Furthermore, KO mice had a significantly (p = 0.025) blunted natriuretic response to benzamil, epithelial sodium channel (ENaC) antagonist. Western blotting of cortex homogenates revealed modestly, but significantly (~15%) lower band density for the β-subunit of ENaC in the KO, versus WT mice, with no differences for the α- or γ-subunits. Moreover, blood pressure (BP), measured by radiotelemetry, was significantly lower in KO versus WT mice under basal conditions (mm Hg): 112 ± 5 (WT), 104 ± 2 (KO), p = 0.023. Chronic insulin infusion reduced heart rate in the WT, but not in the KO, and modestly reduced BP in the WT only. Overall, these results support a fundamental role for insulin through its classic receptor in the modulation of electrolyte homostasis and BP.
    AJP Renal Physiology 11/2012; 304(3). DOI:10.1152/ajprenal.00161.2012 · 4.42 Impact Factor
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    ABSTRACT: STE20/SPS-1-related proline-alanine rich protein kinase (SPAK) and Oxidative Stress Related Kinase (OSR1) activate the potassium-dependent sodium-chloride cotransporter, NKCC2, and thiazide-sensitive sodium-chloride cotransporter, NCC, in-vitro, and both co-localize with a kinase regulatory molecule, Cab39/MO25α, at the apical membrane of the Thick Ascending Limb (TAL) and Distal Convoluted Tubule (DCT). Yet genetic ablation of SPAK in mice causes a selective loss of NCC function while NKCC2 becomes hyperphosphorylated. Here, we explore the underlying mechanisms in wildtype and SPAK-null mice. Unlike in the DCT, OSR1 remains at the TAL apical membrane of KO mice where it is accompanied by an increase in the active, phosphorylated form of AMP-activated Kinase (AMPK). We found an alternative SPAK isoform (putative SPAK2 form), which modestly inhibits co-transporter activity in-vitro, is more abundant in the medulla than the cortex. Thus, enhanced NKCC2 phosphorylation in the SPAK KO may be explained by removal of inhibitory SPAK2, sustained activity of OSR1 and activation of other kinases. By contrast, the OSR1/SPAK/M025α signaling apparatus is disrupted in the DCT. OSR1 becomes largely inactive and displaced from M025α and NCC at the apical membrane, and redistributes to dense punctate structures, containing WNK1, within the cytoplasm. These changes are paralleled by a decrease in NCC phosphorylation and a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the dependent nature of OSR1 on SPAK in the DCT, NCC is unable to be activated. Consequently, SPAK(-/- )mice are highly sensitive to dietary salt-restriction, displaying prolonged negative sodium balance and hypotension.
    Journal of Biological Chemistry 09/2012; DOI:10.1074/jbc.M112.402800 · 4.60 Impact Factor
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    ABSTRACT: Apical SK/ROMK and BK channels mediate baseline and flow-induced K secretion (FIKS), respectively, in the cortical collecting duct (CCD). BK channels are detected in acid-base transporting intercalated (IC) and Na-absorbing principal (PC) cells. Although the density of BK channels is greater in IC than PC, Na-K-ATPase activity in IC is considered inadequate to sustain high rates of urinary K secretion. To test the hypothesis that basolateral NKCC in the CCD contributes to BK channel-mediated FIKS, we measured net K secretion (J(K)) and Na absorption (J(Na)) at slow (∼1) and fast (∼5 nl·min(-1)·mm(-1)) flow rates in rabbit CCDs microperfused in vitro in the absence and presence of bumetanide, an inhibitor of NKCC, added to the bath. Bumetanide inhibited FIKS but not basal J(K), J(Na), or the flow-induced [Ca(2+)](i) transient necessary for BK channel activation. Addition of luminal iberiotoxin, a BK channel inhibitor, to bumetanide-treated CCDs did not further reduce J(K). Basolateral Cl removal reversibly inhibited FIKS but not basal J(K) or J(Na). Quantitative PCR performed on single CCD samples using NKCC1- and 18S-specific primers and probes and the TaqMan assay confirmed the presence of the transcript in this nephron segment. To identify the specific cell type to which basolateral NKCC is localized, we exploited the ability of NKCC to accept NH(4)(+) at its K-binding site to monitor the rate of bumetanide-sensitive cytosolic acidification after NH(4)(+) addition to the bath in CCDs loaded with the pH indicator dye BCECF. Both IC and PC were found to have a basolateral bumetanide-sensitive NH(4)(+) entry step and NKCC1-specific antibodies labeled the basolateral surfaces of both cell types in CCDs. These results suggest that BK channel-mediated FIKS is dependent on a basolateral bumetanide-sensitive, Cl-dependent transport pathway, proposed to be NKCC1, in both IC and PC in the CCD.
    AJP Renal Physiology 08/2011; 301(5):F1088-97. DOI:10.1152/ajprenal.00347.2011 · 4.42 Impact Factor
  • James B Wade
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    ABSTRACT: Editorial Focus- F001-2011.
    AJP Renal Physiology 05/2011; 301(2):F308. DOI:10.1152/ajprenal.00248.2011 · 4.42 Impact Factor
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    ABSTRACT: ROMK channels are well-known to play a central role in renal K secretion, but the absence of highly specific and avid-ROMK antibodies has presented significant roadblocks toward mapping the extent of expression along the entire distal nephron and determining whether surface density of these channels is regulated in response to physiological stimuli. Here, we prepared new ROMK antibodies verified to be highly specific, using ROMK knockout mice as a control. Characterization with segmental markers revealed a more extensive pattern of ROMK expression along the entire distal nephron than previously thought, localizing to distal convoluted tubule regions, DCT1 and DCT2; the connecting tubule (CNT); and cortical collecting duct (CD). ROMK was diffusely distributed in intracellular compartments and at the apical membrane of each tubular region. Apical labeling was significantly increased by high-K diet in DCT2, CNT1, CNT2, and CD (P < 0.05) but not in DCT1. Consistent with the large increase in apical ROMK, dramatically increased mature glycosylation was observed following dietary potassium augmentation. We conclude 1) our new antibody provides a unique tool to characterize ROMK channel localization and expression and 2) high-K diet causes a large increase in apical expression of ROMK in DCT2, CNT, and CD but not in DCT1, indicating that different regulatory mechanisms are involved in K diet-regulated ROMK channel functions in the distal nephron.
    AJP Renal Physiology 03/2011; 300(6):F1385-93. DOI:10.1152/ajprenal.00592.2010 · 4.42 Impact Factor
  • James B. Wade, Bruce A. Stanton, Dennis Brown
    Comprehensive Physiology, 12/2010; , ISBN: 9780470650714
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    ABSTRACT: Evidence is mounting that a multi-gene kinase network is central to the regulation of renal Na(+) and K(+) excretion and that aberrant signaling through the pathway can result in renal sodium retention and hypertension (HTN). The kinase network minimally includes the Ste20-related proline-alanine-rich kinase (SPAK), the with-no-lysine kinases (WNKs), WNK4 and WNK1, and their effectors, the thiazide-sensitive NaCl cotransporter and the potassium secretory channel, ROMK. Available evidence indicates that the kinase network normally functions as a switch to change the mineralocorticoid hormone response of the kidney to either conserve sodium or excrete potassium, depending on whether aldosterone is induced by a change in dietary sodium or potassium. Recently, common genetic variants in the SPAK gene have been identified as HTN susceptibility factors in the general population, suggesting that altered WNK-SPAK signaling plays an important role in essential HTN. Here, we highlight recent breakthroughs in this emerging field and discuss areas of consensus and uncertainty.
    Kidney International 04/2010; 77(12):1063-9. DOI:10.1038/ki.2010.103 · 8.52 Impact Factor
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    ABSTRACT: Renal outer medullary potassium (ROMK) channels are exquisitely regulated to adjust renal potassium excretion and maintain potassium balance. Clathrin-dependent endocytosis plays a critical role, limiting urinary potassium loss in potassium deficiency. In renal disease, aberrant ROMK endocytosis may contribute to potassium retention and hyperkalemia. Previous work has indicated that ROMK endocytosis is stimulated by with-no-lysine (WNK) kinases, but the endocytotic signal and the internalization machinery have not been defined. Here, we found that ROMK bound directly to the clathrin adaptor molecule autosomal recessive hypercholesterolemia (ARH), and this interaction was mediated by what we believe to be a novel variant of the canonical "NPXY" endocytotic signal, YxNPxFV. ARH recruits ROMK to clathrin-coated pits for constitutive and WNK1-stimuated endocytosis, and ARH knockdown decreased basal rates of ROMK endocytosis, in a heterologous expression system, COS-7 cells. We found that ARH was predominantly expressed in the distal nephron where it coimmunoprecipitated and colocalized with ROMK. In mice, the abundance of kidney ARH protein was modulated by dietary potassium and inversely correlated with changes in ROMK. Furthermore, ARH-knockout mice exhibited an altered ROMK response to potassium intake. These data suggest that ARH marks ROMK for clathrin-dependent endocytosis, in concert with the demands of potassium homeostasis.
    The Journal of clinical investigation 11/2009; 119(11):3278-89. DOI:10.1172/JCI37950 · 13.77 Impact Factor
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    ABSTRACT: With-no-lysine kinase 4 (WNK4) inhibits electroneutral sodium chloride reabsorption by attenuating the cell surface expression of the thiazide-sensitive NaCl cotransporter (NCC). The underlying mechanism for this effect remains poorly understood. Here, we explore how WNK4 affects the trafficking of NCC through its interactions with intracellular sorting machinery. An analysis of NCC cell surface lifetime showed that WNK4 did not alter the net rate of cotransporter internalization. In contrast, direct measurements of forward trafficking revealed that WNK4 attenuated the rate of NCC surface delivery, inhibiting the anterograde movement of cotransporters traveling to the plasma membrane from the trans-Golgi network. The response was paralleled by a dramatic reduction in NCC protein abundance, an effect that was sensitive to the lysosomal protease inhibitor leupeptin, insensitive to proteasome inhibition, and attenuated by endogenous WNK4 knockdown. Subcellular localization studies performed in the presence of leupeptin revealed that WNK4 enhanced the accumulation of NCC in lysosomes. Moreover, NCC immunoprecipitated with endogenous AP-3 complexes, and WNK4 increased the fraction of cotransporters that associate with this adaptor, which facilitates cargo transport to lysosomes. WNK4 expression also increased LAMP-2-positive lysosomal content, indicating that the kinase may act by a general AP-3-dependent mechanism to promote cargo delivery into the lysosomal pathway. Taken together, these findings indicate that WNK4 inhibits NCC activity by diverting the cotransporter to the lysosome for degradation by way of an AP-3 transport carrier.
    Journal of Biological Chemistry 05/2009; 284(27):18471-80. DOI:10.1074/jbc.M109.008185 · 4.60 Impact Factor
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    ABSTRACT: Hypertension places a major burden on individual and public health, but the genetic basis of this complex disorder is poorly understood. We conducted a genome-wide association study of systolic and diastolic blood pressure (SBP and DBP) in Amish subjects and found strong association signals with common variants in a serine/threonine kinase gene, STK39. We confirmed this association in an independent Amish and 4 non-Amish Caucasian samples including the Diabetes Genetics Initiative, Framingham Heart Study, GenNet, and Hutterites (meta-analysis combining all studies: n = 7,125, P < 10(-6)). The higher BP-associated alleles have frequencies > 0.09 and were associated with increases of 3.3/1.3 mm Hg in SBP/DBP, respectively, in the Amish subjects and with smaller but consistent effects across the non-Amish studies. Cell-based functional studies showed that STK39 interacts with WNK kinases and cation-chloride cotransporters, mutations in which cause monogenic forms of BP dysregulation. We demonstrate that in vivo, STK39 is expressed in the distal nephron, where it may interact with these proteins. Although none of the associated SNPs alter protein structure, we identified and experimentally confirmed a highly conserved intronic element with allele-specific in vitro transcription activity as a functional candidate for this association. Thus, variants in STK39 may influence BP by increasing STK39 expression and consequently altering renal Na(+) excretion, thus unifying rare and common BP-regulating alleles in the same physiological pathway.
    Proceedings of the National Academy of Sciences 01/2009; 106(1):226-31. DOI:10.1073/pnas.0808358106 · 9.81 Impact Factor
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    ABSTRACT: There is increasing evidence that mammalian urinary tract epithelial cells utilize membrane channels and transporters to transport solutes across their apical (luminal) and basalateral membranes to modify solute concentrations in both cell and urine. This study investigates the expression, localization, and regulation of the ROMK (K(ir) 1.1) potassium channels in rat and dog ureter and bladder tissues. Immunoblots of homogenates of whole ureter, whole bladder, bladder epithelial cells, and bladder smooth muscle tissues in both rat and dog identified approximately 45- to 50-kDa bands characteristic of ROMK in all tissues. RT-PCR identified ROMK mRNA in these same tissues in both animal species. ROMK protein localized by immunocytochemistry was strongly expressed in the apical membranes of the large umbrella cells lining the bladder lumen and to a lesser extent in the cytoplasm of epithelial cells and smooth muscle cells in the rat bladder. ROMK protein and mRNA were also discovered in cardiac, striated, and smooth muscle in diverse organs. There was no difference in immunoblot expression of ROMK abundance in bladder homogenates (whole bladder, epithelial cell, or muscle cell) or ureteral homogenates between groups of rats fed high- or low-potassium diets. Although the functional role of ROMK in urinary tract epithelia and smooth muscle is unknown, ROMK may participate in the regulation of epithelial and smooth muscle cell volume and osmolality, in the dissipation of potassium leaked or diffused from urine across the epithelial cell apical membranes or tight junctions, and in net or bidirectional potassium transport across urinary tract epithelia.
    American journal of physiology. Renal physiology 10/2008; 295(6):F1658-65. DOI:10.1152/ajprenal.00022.2008 · 3.30 Impact Factor
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    ABSTRACT: Renal tubule epithelial cells express the insulin receptor (IR); however, their value has not been firmly established. We generated mice with renal epithelial cell-specific knockout of the IR by Cre-recombinase-loxP recombination using a kidney-specific (Ksp) cadherin promoter. KO mice expressed significantly lower levels of IR mRNA and protein in kidney cortex (49-56% of the WT) and medulla (32-47%) homogenates. Immunofluorescence showed the greatest relative reduction in the thick ascending limb and collecting duct cell types. Body weight, kidney weight, and food and water intakes were not different from WT littermates. However, KO mice had significantly increased basal systolic blood pressure (BP, 15 mm Hg higher) as measured by radiotelemetry. In response to a volume load by gavage (20 ml/kg of body weight, 0.9% NaCl, 15% dextrose), KO mice had impaired natriuresis (37 +/- 10 versus 99 +/- 9 mmol of Na(+) per 2 h in WT). Furthermore, volume load led to a sustained increase in BP in KO mice only. In contrast, insulin administration i.p. (0.5 units/kg of body weight) resulted in a significant fall in BP in WT, but not in KO mice. To test the role of reduced renal nitric oxide (NO) production in these responses, basal urinary nitrates plus nitrites excretion (UNOx) was measured and found to be 61% lower in KO vs. WT mice. Furthermore, acute insulin increased UNOx by 202% in the WT, relative to a significantly blunted rise (67%) in KO animals. These results illuminate a previously uncharacterized role for renal IR to reduce BP and facilitate sodium and water excretion, possibly via NO production.
    Proceedings of the National Academy of Sciences 05/2008; 105(17):6469-74. DOI:10.1073/pnas.0711283105 · 9.81 Impact Factor
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    ABSTRACT: The adaptor proteins sodium/hydrogen exchanger regulatory factor (NHERF)-1 and NHERF-2 have overlapping tissue distribution in renal cells and overlapping specificity in their binding to renal transporters and other proteins. To compare the kidney-specific differences in the function of these adaptor proteins, NHERF-1 and NHERF-2 null mice were compared with wild-type control mice. In NHERF-2 null mice, the renal proximal tubule abundance and distribution of NHERF-1 and NHERF-3 were not different from those in wild-type animals. The glomerular expression of podocalyxin and ZO-1 also did not differ. NHERF-1 null mice had increased urinary excretion of phosphate, calcium, and uric acid compared with wild-type control and NHERF-2 null mice. Because of the association between NHERF-2 and podocalyxin in glomeruli and ClC-5 in the renal proximal tubule, the urinary excretion of protein was determined. There were no differences in the urinary excretion of protein or low-molecular-weight proteins between wild-type control, NHERF-1(-/-), and NHERF-2(-/-) mice. These studies indicate that the increased urinary excretion of phosphate and uric acid are specific to NHERF-1 null mice and highlight the fact that predictions about the role of adaptor proteins such as the NHERF proteins obtained from studies of model cell systems must be confirmed in whole animals.
    American journal of physiology. Renal physiology 05/2008; 294(4):F1001-7. DOI:10.1152/ajprenal.00504.2007 · 3.30 Impact Factor
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    ABSTRACT: Sodium-hydrogen exchanger regulatory factor-1-deficient (NHERF-1(-/-)) mice demonstrate increases in the urinary excretion of phosphate, calcium, and uric acid associated with interstitial deposition of calcium in the papilla of the kidney. These studies examine the role of NHERF-1 in the tubular reabsorption of uric acid and regulation of mouse urate transporter 1 (mURAT1), a newly described transporter that is responsible for the renal tubular reabsorption of uric acid. In primary cultures of mouse renal proximal tubule cells, uric acid uptake was significantly lower in NHERF-1(-/-) cells compared with wild-type cells over a large range of uric acid concentrations in the media. Western immunoblotting revealed a 56 +/- 6% decrease in the brush border membrane (BBM) expression of mURAT1 in NHERF-1(-/-) compared with wild-type control kidneys (P < 0.05). Confocal microscopy confirmed the reduced apical membrane expression of mURAT1 in NHERF-1(-/-) kidneys and demonstrated mislocalization of mURAT1 to intracellular vesicular structures. Para-aminohippurate significantly inhibited uric acid uptake in wild-type cells (41 +/- 2%) compared with NHERF-1(-/-) cells (8.2 +/- 3%). Infection of NHERF-1(-/-) cells with adenovirus-green fluorescence protein-NHERF-1 resulted in significantly higher rates of uric acid transport (15.4 +/- 1.1 pmol/microg protein per 30 min) compared with null cells that were infected with control adenovirus-green fluorescence protein (7.9 +/- 0.3) and restoration of the inhibitory effect of para-aminohippurate (% inhibition 34 +/- 4%). These findings indicate that NHERF-1 exerts a significant effect on the renal tubular reabsorption of uric acid in the mouse by modulating the BBM abundance of mURAT1 and possibly other BBM uric acid transporters.
    Journal of the American Society of Nephrology 05/2007; 18(5):1419-25. DOI:10.1681/ASN.2006090980 · 9.47 Impact Factor
  • David A Spector, Qing Yang, James B Wade
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    ABSTRACT: Although the mammalian urinary tract is generally held to be solely a transit and storage vehicle for urine made by the kidney, in vivo data suggest reabsorption of urea and other urine constituents across urinary tract epithelia. To determine whether urinary tract tissue concentrations are increased as a result of such reabsorption, we measured urea nitrogen and creatinine concentrations and determined whether urea transporter B (UT-B) was present in bladder, ureter, and other tissues from dogs and rats. Mean urea nitrogen and creatinine concentrations in dogs and rats were three- to sevenfold higher in urinary tract tissues than in serum and were comparable to those in renal cortex. In water-restricted or water-loaded rats, urea nitrogen concentrations in bladder tissues fell inversely with the state of hydration, were proportional to urine urea nitrogen concentrations, and were greater than the corresponding serum urea nitrogen concentration in every animal. Immunoblots of rat and dog urinary tract tissues demonstrated the presence of UT-B in homogenates of bladder and ureter, and immunocytochemical analysis localized UT-B to epithelial cell membranes. These findings are consistent with the notion that urea and creatinine are continuously reabsorbed from the urine across the urothelium, urea in part via UT-B, and that urine is thus altered in its passage through the urinary tract. Urea reabsorption across urinary tract epithelia may be important during conditions requiring nitrogen conservation and may contribute to pathophysiological states characterized by high blood urea nitrogen, such as prerenal azotemia and obstructive uropathy.
    American journal of physiology. Renal physiology 02/2007; 292(1):F467-74. DOI:10.1152/ajprenal.00181.2006 · 3.30 Impact Factor
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    ABSTRACT: PDZ proteins usually contain multiple protein-protein interaction domains and act as molecular scaffolds that are important for the generation and maintenance of cell polarity and cell signaling. Here, we identify and characterize TIP-1 as an atypical PDZ protein that is composed almost entirely of a single PDZ domain and functions as a negative regulator of PDZ-based scaffolding. We found that TIP-1 competes with the basolateral membrane mLin-7/CASK complex for interaction with the potassium channel Kir 2.3 in model renal epithelia. Consequently, polarized plasma membrane expression of Kir 2.3 is disrupted resulting in pronounced endosomal targeting of the channel, similar to the phenotype observed for mutant Kir 2.3 channels lacking the PDZ-binding motif. TIP-1 is ubiquitously expressed, raising the possibility that TIP-1 may play a similar role in regulating the expression of other membrane proteins containing a type I PDZ ligand.
    Molecular Biology of the Cell 11/2006; 17(10):4200-11. DOI:10.1091/mbc.E06-02-0129 · 4.55 Impact Factor
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    Richard A Coleman, Jie Liu, James B Wade
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    ABSTRACT: We have discovered that the immunoreactivity of the fluorophore Alexa Fluor 488 survives glutaraldehyde and osmium tetroxide fixation and epoxy resin embedding and etching. We have developed new localization methods that for the first time take advantage of this property. The antigen is localized in cryosections using suitable primary antibody and an Alexa Fluor 488-conjugated secondary antibody. Cryosection fluorescence can be photographed for later correlation with electron microscopy (EM) findings. The sections are then further fixed with glutaraldehyde and OsO4, if desired and flat-embedded in epoxy resin. Semi-thin sections are etched completely with sodium ethoxide, whereas thin sections are partially etched. Alexa Fluor 488 is then localized with rabbit anti-Alexa Fluor 488 and goat anti-rabbit conjugated to Alexa Fluor 488 [light microscopy (LM)] or to colloidal gold (EM). A second antigen may also be localized using Alexa Fluor 568. When used without postfixation, these methods produce high-resolution semi-thin, or even thin, sections that retain a high level of fluorescence for LM observations. These methods allow highly sensitive immunolocalizations in tissue while preserving cell fine structure through traditional fixation and epoxy embedding. In demonstration of the methods, we describe the localization of the thiazide-sensitive sodium/chloride cotransporter and the epithelial sodium channel in rat kidney.
    Journal of Histochemistry and Cytochemistry 08/2006; 54(7):817-27. DOI:10.1369/jhc.6A6929.2006 · 2.40 Impact Factor

Publication Stats

3k Citations
384.19 Total Impact Points


  • 1988–2013
    • University of Maryland, Baltimore
      • • Department of Physiology
      • • Department of Medicine
      Baltimore, Maryland, United States
  • 2007
    • Johns Hopkins University
      Baltimore, Maryland, United States
  • 2005
    • University of California, San Francisco
      San Francisco, California, United States
  • 2003–2004
    • Duke University
      Durham, North Carolina, United States
  • 2001
    • Georgetown University
      • Division of Endocrinology
      Washington, D. C., DC, United States
  • 1988–1999
    • National Heart, Lung, and Blood Institute
      • Systems Biology Center
      Maryland, United States