The Molecular Physiology of the Urinary Concentrating Mechanism

Mark A . Knepper 대한신장학회 1998 춘계학술대회 초록집 Vol.1998 No.-

Aquaporins in Health and Disease

Mark A . knepper 대한신장학회 1998 춘계학술대회 초록집 Vol.1998 No.-

Molecular physiology of water balance.

Knepper, Mark A,Kwon, Tae-Hwan,Nielsen, Soren Massachusetts Medical Society 2015 The New England journal of medicine Vol.372 No.14

Identification of β-catenin-interacting proteins in nuclear fractions of native rat collecting duct cells

Hwang, Jacqueline R.,Chou, Chung-Lin,Medvar, Barbara,Knepper, Mark A.,Jung, Hyun Jun American Physiological Society 2017 American Journal of Physiology Vol.313 No.1

<P>The gene encoding the aquaporin-2 water channel is regulated transcriptionally in response to vasopressin. In the renal collecting duct, vasopressin stimulates the nuclear translocation and phosphorylation (at Ser<SUP>552</SUP>) of β-catenin, a multifunctional protein that acts as a transcriptional coregulator in the nucleus. The purpose of this study was to identify β-catenin-interacting proteins that might be involved in transcriptional regulation in rat inner medullary collecting duct (IMCD) cells, using experimental and computational approaches. We used a standard chromatin immunoprecipitation procedure coupled to mass spectrometry (ChIP-MS) in a nuclear fraction isolated from rat IMCD suspensions. Over four biological replicates, we reproducibly identified 43 β-catenin-binding proteins, including several known β-catenin-binding partners as well as novel interacting proteins. Multiple proteins involved in transcriptional regulation were identified (Taf1, Jup, Tdrd3, Cdh1, Cenpj, and several histones). Many of the identified β-catenin-binding partners were found in prior studies to translocate to the nucleus in response to vasopressin. There was only one DNA-binding transcription factor (TF), specifically Taf1, part of the RNA-polymerase II preinitiation complex. To identify sequence-specific TFs that might interact with β-catenin, Bayes’ theorem was used to integrate data from several information sources. The analysis identified several TFs with potential binding sites in the <I>Aqp2</I> gene promoter that could interact with β-catenin in the regulation of <I>Aqp2</I> gene transcription, specifically Jun, Junb, Jund, Atf1, Atf2, Mef2d, Usf1, Max, Pou2f1, and Rxra. The findings provide information necessary for modeling the transcriptional response to vasopressin.</P>

Biphasic effects of ANP infusion in conscious, euvolumic rats: roles of AQP2 and ENaC trafficking.

Wang, Weidong,Li, Chunling,Nejsum, Lene N,Li, Hongyan,Kim, Soo Wan,Kwon, Tae-Hwan,Jonassen, Thomas E N,Knepper, Mark A,Thomsen, Klaus,Frokiaer, Jorgen,Nielsen, Soren American Physiological Society 2006 American journal of physiology. Renal physiology Vol.290 No.2

<P>Atrial natriuretic peptide (ANP) acutely promotes water and sodium excretion, whereas subchronic effects involve water retention. Renal hemodynamics, water and sodium excretion, and aquaporin-2 (AQP2) and epithelial Na channel (ENaC) subcellular trafficking were determined in response to continuous ANP infusion in conscious rats, where body sodium and fluid balance was constantly maintained. ANP (0.5 microg x kg(-1) x min(-1)) evoked a transient (peak at 10 min) fivefold diuresis followed by reduced urine production to control levels (30- to 90-min period). The fractional distal water excretion was significantly increased initially and then decreased in response to ANP. There was no change in the subcellular localization of AQP2 and AQP2 phosphorylated in PKA consensus site S256 (p-AQP2) 10 min after ANP infusion. In contrast, after 90 min a marked increase in apical labeling of AQP2 and p-AQP2 was observed in the inner and outer medullary collecting ducts but not in cortical collecting ducts. In support of this, ANP induced plasma membrane targeting of AQP2 in transiently AQP2-transfected cells. ANP infusion evoked an instant increase in renal sodium excretion, which persisted for 90 min. Ten minutes of ANP infusion induced no changes in the subcellular localization of ENaC subunits, whereas a marked increase in apical targeting of alpha- and gamma-subunits was observed after 90 min. In conclusion, 1) ANP infusion induced a sustained natriuresis and transient diuresis; 2) there were no changes in the subcellular localization of AQP2 and ENaC subunits after 10 min of ANP infusion; and 3) there was a marked increase in apical targeting of AQP2, p-AQP2, and alpha- and gamma-ENaC after 90 min of ANP infusion. The increased targeting of ENaC and AQP2 likely represents direct or compensatory effects to increase sodium and water reabsorption and to prevent volume depletion in response to prolonged ANP infusion.</P>

Modulation of Cl⁻ signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT

Vachel, Laura,Shcheynikov, Nikolay,Yamazaki, Osamu,Fremder, Moran,Ohana, Ehud,Son, Aran,Shin, Dong Min,Yamazaki-Nakazawa, Ai,Yang, Chin-Rang,Knepper, Mark A.,Muallem, Shmuel AAAS 2018 Science signaling Vol.11 No.554

<P><B>Combinations to regulate Cl<SUP>−</SUP> signaling</B></P><P>The secretion of HCO<SUB>3</SUB><SUP>−</SUP>-containing fluids is vital to the function of all epithelia and is enabled in part by the activity of the Na<SUP>+</SUP>-coupled HCO<SUB>3</SUB><SUP>−</SUP> transporter NBCe1-B. Vachel <I>et al</I>. identified five serine residues in NBCe1-B whose phosphorylation status was controlled by the regulatory protein IRBIT. The phosphorylation status of Ser<SUP>12</SUP> and Ser<SUP>65</SUP> affected the Cl<SUP>−</SUP> sensitivity of two intracellular Cl<SUP>−</SUP>-sensing motifs. Moreover, IRBIT recruited a distinct kinase/phosphatase pair for each serine residue. The three remaining phosphorylation sites were phosphorylated in distinct combinations that determined the relative basal activity level of NBCe1-B and the potential for further activation by IRBIT. These results demonstrate how distinct phosphorylation patterns may enable epithelial cells to fine-tune the HCO<SUB>3</SUB><SUP>−</SUP> transport activity of NBCe1-B in response to varying conditions in different parts of the organ.</P><P>IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl<SUP>−</SUP>-sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl<SUP>−</SUP> (Cl<SUP>−</SUP><SUB>in</SUB>). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl<SUP>−</SUP><SUB>in</SUB>. Mutational analysis suggested that the phosphorylation status of Ser<SUP>232</SUP>, Ser<SUP>233</SUP>, and Ser<SUP>235</SUP> was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser<SUP>232</SUP>, Ser<SUP>233</SUP>, or Ser<SUP>235</SUP> produced NBCe1-B in the conformations pSer<SUP>233</SUP>/pSer<SUP>235</SUP>, pSer<SUP>232</SUP>/pSer<SUP>235</SUP>, or pSer<SUP>232</SUP>/pSer<SUP>233</SUP>, respectively. The activity of the pSer<SUP>233</SUP>/pSer<SUP>235</SUP> form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl<SUP>−</SUP><SUB>in</SUB>. The properties of the pSer<SUP>232</SUP>/pSer<SUP>235</SUP> form were similar to those of wild-type NBCe1-B, whereas the pSer<SUP>232</SUP>/pSer<SUP>233</SUP> form was partially active, further activated by IRBIT, but retained inhibition by Cl<SUP>−</SUP><SUB>in</SUB>. Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser<SUP>65</SUP>, which affected Cl<SUP>−</SUP><SUB>in</SUB> sensing by the <SUP>32</SUP>GXXXP<SUP>36</SUP> motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser<SUP>12</SUP>, which affected Cl<SUP>−</SUP><SUB>in</SUB> sensing by the <SUP>194</SUP>GXXXP<SUP>198</SUP> motif. Ser<SUP>232</SUP>, Ser<SUP>233</SUP>, and Ser<SUP>235</SUP> are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO<SUB>3</SUB><SUP>−</SUP> secretion.</P>

Treating lithium-induced nephrogenic diabetes insipidus with a COX-2 inhibitor improves polyuria via upregulation of AQP2 and NKCC2.

Kim, Gheun-Ho,Choi, Nak Won,Jung, Ju-Young,Song, Ji-Hyun,Lee, Chang Hwa,Kang, Chong Myung,Knepper, Mark A American Physiological Society 2008 American Journal of Physiology Vol.294 No.4

<P>Prostaglandin E(2) may antagonize vasopressin-stimulated salt absorption in the thick ascending limb and water absorption in the collecting duct. Blockade of prostaglandin E(2) synthesis by nonsteroidal anti-inflammatory drugs (NSAIDs) enhances urinary concentration, and these agents have antidiuretic effects in patients with nephrogenic diabetes insipidus (NDI) of different etiologies. Because renal prostaglandins are derived largely from cyclooxygenase-2 (COX-2), we hypothesized that treatment of NDI with a COX-2 inhibitor may relieve polyuria through increased expression of Na-K-2Cl cotransporter type 2 (NKCC2) in the thick ascending limb and aquaporin-2 (AQP2) in the collecting duct. To test this hypothesis, semiquantitative immunoblotting and immunohistochemistry were carried out from the kidneys of lithium-induced NDI rats with and without COX-2 inhibition. After male Sprague-Dawley rats were fed an LiCl-containing rat diet for 3 wk, the rats were randomly divided into control and experimental groups. The COX-2 inhibitor DFU (40 mg.kg(-1).day(-1)) was orally administered to the experimental rats for an additional week. Treatment with the COX-2 inhibitor significantly relieved polyuria and raised urine osmolality. Semiquantitative immunoblotting using whole-kidney homogenates revealed that COX-2 inhibition caused significant increases in the abundance of AQP2 and NKCC2. Immunohistochemistry for AQP2 and NKCC2 confirmed the effects of COX-2 inhibition in lithium-induced NDI rats. The upregulation of AQP2 and NKCC2 in response to the COX-2 inhibitor may underlie the therapeutic mechanisms by which NSAIDs enhance antidiuresis in patients with NDI.</P>

Maintained ENaC trafficking in aldosterone-infused rats during mineralocorticoid and glucocorticoid receptor blockade.

Nielsen, Jakob,Kwon, Tae-Hwan,Frokiaer, Jorgen,Knepper, Mark A,Nielsen, Soren American Physiological Society 2007 American Journal of Physiology Vol.292 No.1

<P>Aldosterone induces redistribution of epithelial sodium channel (ENaC) to the apical plasma membrane from intracellular vesicles in renal connecting tubule (CNT) and cortical collecting duct (CCD). The role of the classical mineralocorticoid receptor (MR) in ENaC trafficking is still debated. We examined whether the MR antagonist spironolactone affects ENaC regulation in the kidney cortex of aldosterone-infused rats. Aldosterone infusion for 7 days resulted in a plasma aldosterone concentration in the high physiological range (3 to 4 nM). Aldosterone infusion decreased plasma K(+) concentration compared with untreated control rats. Cotreatment with spironolactone completely blocked the aldosterone-induced decrease in plasma K(+). Immunoblotting and immunohistochemistry showed increased protein abundance of Na-K-ATPase alpha(1)-subunit and NCC in the kidney cortex, in response to aldosterone infusion that was blocked by spironolactone. In contrast, aldosterone-induced redistribution of ENaC subunits from the cytoplasm to the apical plasma membrane domain in CNT and CCD was unaffected by spironolactone. Immunoblotting of alphaENaC showed increased protein abundance in aldosterone-infused rats that was not blocked by spironolactone treatment. To exclude possible glucocorticoid receptor (GR)-mediated effects of aldosterone, we treated aldosterone-infused rats with both spironolactone and the GR antagonist RU486. Combined MR and GR blockade prevented neither ENaC trafficking nor the upregulation of alphaENaC protein abundance in aldosterone-infused rats. We provide new evidence for ENaC trafficking occurring independent of MR and GR activation in aldosterone-infused rats.</P>

포스터 발표 : 포스터 연제 ; 발생중인 흰쥐콩팥에서 요소운반체 A 발현에 대한 면역조직화학적 연구

김동언 ( Kim Dong Eon ),김영희 ( Kim Yeong Hui ),한기환 ( Han Gi Hwan ),차정호 ( Cha Jeong Ho ),김진 ( Kim Jin ),( Mark A. Knepper ),( Kirsten M. Madsen ) 대한신장학회 1999 춘계학술대회 초록집 Vol.18 No.2

Altered Renal Sodium Transporter Expression in an Animal Model of Type 2 Diabetes Mellitus

주권욱,Jay Wook Lee,Un Sil Jeon,임춘수,Jin Suk Han,Mark A. Knepper,나기영,오윤규 대한의학회 2007 Journal of Korean medical science Vol.22 No.6

Hemodynamic factors play an important role in the development and/or progression of diabetic nephropathy. We hypothesized that renal sodium transporter dysregulation might contribute to the hemodynamic alterations in diabetic nephropathy. Otsuka Long Evans Tokushima Fatty (OLETF) rats were used as an animal model for type 2 diabetes. Long Evans Tokushima (LETO) rats were used as controls. Renal sodium transporter regulation was investigated by semiquantitative immunoblotting and immunohistochemistry of the kidneys of 40-week-old animals. The mean serum glucose level in OLETF rats was increased to 235±25 mg/dL at 25 weeks, and the hyperglycemia continued up to the end of 40 weeks. Urine protein/ creatinine ratios were 10 times higher in OLETF rats than in LETO rats. At 40th week, the abundance of the epithelial sodium channel (ENaC) -subunit was increased in OLETF rats, but the abundance of the ENaC -subunit was decreased. No significant differences were observed in the ENaC -subunit or other major sodium transporters. Immunohistochemistry for the ENaC -subunit showed increased immunoreactivity in OLETF rats, whereas the ENaC -subunit showed reduced immunoreactivity in these rats. In OLETF rats, ENaC -subunit upregulation and ENaC -subunit downregulation after the development of diabetic nephropathy may reflect an abnormal sodium balance.