bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Ca2+-activated K+channels by multifunctional Ca2+/calmodulin-dependent protein kinase
Authors: Sansom Steven C. , Ma Rong , Carmines Pamela K. , Hall David A.
Published: December 2017

Activation of mesangial cells by ANG II provokes release of intracellular Ca2+stores and subsequent Ca2+influx through voltage-gated channels, events that are reflected by a large transient increase in intracellular concentration [Ca2+]ifollowed by a modest sustained elevation in [Ca2+]i. These ANG II-induced alterations in [Ca2+]ielicit activation of large Ca2+-activated K+channels (BKCa) in a negative-feedback manner. The mechanism of this BKCafeedback response may involve the direct effect of intracellular Ca2+on the channel and/or channel activation by regulatory enzymes. The present study utilized patch-clamp and fura 2 fluorescence techniques to assess the involvement of multifunctional calcium calmodulin kinase II (CAMKII) in the BKCafeedback response. In cell-attached patches, KN62 (specific inhibitor of CAMKII) either abolished or reduced to near zero the ANG II-induced BKCafeedback response. This phenomenon did not reflect direct effects of KN62 on the BKCachannel, because this agent alone did not significantly alter BKCachannel activity in inside-out patches. KN62 also failed to alter either the transient peak or sustained plateau phases of the [Ca2+]iresponse to ANG II. In inside-out patches (1 μM Ca2+in bath), calmodulin plus ATP activated BKCachannels in the presence but not the absence of CAMKII. These observations are consistent with the postulate that CAMKII is involved in the BKCafeedback response of mesangial cells, acting to potentiate the influence of increased [Ca2+]ion the BKCachannel or a closely associated regulator of the channel. An additional effect of CAMKII to activate a voltage-gated Ca2+channel cannot be ruled out by these experiments.
TP receptor-mediated vasoconstriction in microperfused afferent arterioles: roles of O2−and NO
Authors: Schnackenberg Christine G. , Welch William J. , Wilcox Christopher S.
Published: December 2017

Thromboxane A2(TxA2) preferentially constricts the renal afferent arteriole. Nitric oxide (NO) modulates vasoconstriction and is rapidly degraded by superoxide radical (O2−). We investigated the roles of NO and O2−in rabbit isolated, perfused renal afferent arteriole responses to the TxA2/prostaglandin H2(TP) receptor agonist U-46,619. U-46,619 (10−10–10−6M) dose-dependently reduced afferent arteriolar luminal diameter (ED50= 7.5 ± 5.0 nM), which was blocked by the TP receptor antagonist ifetroban (10−6M). Tempol (10−3M) pretreatment, which prevented paraquat-induced vasoconstriction in afferent arterioles, blocked the vasoconstrictor responses to U-46,619. To test whether U-46,619 stimulates NO and whether tempol prevents U-46,619-induced vasoconstriction by enhancing the biological activity of NO, we examined the luminal diameter response to U-46,619 in arterioles pretreated with Nw-nitro-l-arginine methyl ester (l-NAME, 10−4M) or l-NAME + tempol. During l-NAME, the sensitivity and maximal responses of the afferent arteriole to U-46,619 were significantly ( P < 0.05) enhanced. Moreover, l-NAME restored a vasoconstrictor response to U-46,619 in vessels pretreated with tempol. In conclusion, in isolated perfused renal afferent arterioles TP receptor activation stimulates NO production, which buffers the vasoconstriction, and stimulates O2−production, which mediates the vasoconstriction, in part, through interaction with NO.
Selective estrogen receptor modulators suppress mesangial cell collagen synthesis
Authors: Neugarten Joel , Acharya Anjali , Lei Jun , Silbiger Sharon
Published: December 2017

Estrogen receptor modulators (SERMs) are “designer drugs” that exert estrogen-like actions in some cells but not in others. We examined the effects of the SERMs LY-117018 (an analog of raloxifene) and tamoxifen on mesangial cells synthesis of type I and type IV collagen. We found that LY-117018 and tamoxifen suppressed mesangial cell type IV collagen gene transcription and type IV collagen protein synthesis in a dose-dependent manner, with a potency identical to that of estradiol. Type I collagen synthesis was also suppressed by LY-117018 in a dose-dependent manner with a potency identical to that of estradiol but greater than that of tamoxifen. Genistein, which selectively binds to estrogen receptor-β in nanomolar concentrations, suppressed type I and type IV collagen synthesis, suggesting that estrogen receptor-β mediates the effects of estrogen on collagen synthesis. Because matrix accumulation is central to the development of glomerulosclerosis, second-generation SERMs may prove clinically useful in ameliorating progressive renal disease without the adverse effects of estrogen on reproductive tissues.
Urea protects from the proapoptotic effect of NaCl in renal medullary cells
Authors: Zhang Zheng , Tian Wei , Cohen David M.
Published: December 2017

Hypertonic NaCl upregulated two sensitive and specific biochemical indices of apoptosis, caspase-3 activation and annexin V binding, in a time- and dose-dependent fashion in renal medullary mIMCD3 cells. Pretreatment with urea (200 mM for 30 min) protected from the proapoptotic effect of hypertonic stress (200 mosmol/kgH2O) in this model. The protective effect of urea was dose dependent and was effective even when applied a short time (≤1 h) following NaCl exposure; this protective effect was not observed in the nonrenal 3T3 cell line. In both mIMCD3 and 3T3 cells, urea failed to protect from the proapoptotic stressor, ultraviolet (UV)-B irradiation. The ability of urea to protect from hypertonic stress was approximately comparable to the protective effect of peptide mitogens epidermal growth factor and insulin-like growth factor (IGF), but it potentiated the IGF effect. Interestingly, the tyrosine kinase inhibitor, genistein, potentiated the proapoptotic effect of urea yet abrogated the proapoptotic effect of hypertonic stress. In aggregate, these data indicate that urea protects from the proapoptotic effect of hypertonic stress in a potentially cell type-specific and stimulus-specific fashion.
Proximal tubule Na transporter responses are the same during acute and chronic hypertension
Authors: Magyar Clara E. , Zhang Yibin , Holstein-Rathlou Niels-H. , McDonough Alicia A.
Published: December 2017

Acute hypertension in Sprague-Dawley rats (SD) provokes a decrease in renal proximal tubule (PT) salt and fluid reabsorption, redistribution of apical Na/H exchanger isoform 3 (NHE3) and Na-Picotransporter type 2 (NaPi2) out of the brush border into higher density membranes, and inhibition of renal cortical Na-K-ATPase (NKA) activity (41). The aims of this study were to determine 1) whether an increase in arterial pressure affects distribution or activity of Na transporters in the spontaneously hypertensive rat (SHR) and 2) whether development of chronic hypertension in SHR leads to persistent adaptive changes in NHE3 and NaPi2 distribution and/or NKA activity. Renal cortex Na transporter protein density distributions and activities were compared by subcellular fractionation in 1) adult SHR with an acute increase or decrease in arterial pressure and 2) young SD (YSD) and young SHR (YSHR) vs. adult SD and SHR. In adult hypertensive SHR NHE3 was shifted to membranes of higher densities, analogous to SD with acute hypertension, and there were no further changes with a further increase or decrease in arterial pressure. There was no change in total pool size of NHE3 in cortex in YSHR vs. SHR. NHE3, NaPi2, megalin , NKA α-/β-subunit, dipeptidyl peptidase IV (DPPIV), and villin distributions were the same in YSHR vs. YSD. NHE3, NaPi2, and megalin shifted to higher densities in adult SHR, but not SD, with age. Basolateral NKA and apical alkaline phosphatase activities were 40% greater in YSHR than YSD and decreased to SD levels in adults. We conclude that there are persistent changes in Na+transporter distributions and activity in response to chronic hypertension in SHR that mimic the responses to acute hypertension seen in SD rats and that elevated sodium pump activity per transporter in YSHR may contribute to the generation of hypertension.
Signal complex regulation of renal transport proteins: NHERF and regulation of NHE3 by PKA
Authors: Weinman Edward J. , Minkoff Charles , Shenolikar Shirish
Published: December 2017

The activity of the sodium/hydrogen exchanger 3 (NHE3) isoform of the sodium/hydrogen exchanger in the brush-border membrane of the renal proximal tubule is tightly regulated. Recent biochemical and cellular experiments have established the essential requirement for a new class of regulatory factors, sodium/hydrogen exchanger regulatory factor (NHERF) and NHERF-like proteins, in cAMP-mediated inhibition of NHE3 activity. NHERF is the first PSD-95/Dlg/ZO-1 (PDZ) motif-containing protein localized to apical membranes and appears to facilitate cAMP-dependent protein kinase A (PKA) phosphorylation of NHE3 by interacting with the cytoskeleton to target a multiprotein complex to the brush-border membrane. Other recent experiments have indicated that NHERF also regulates the activity of other renal transport proteins, suggesting that the signal complex model of signal transduction in the kidney may be more common than presently appreciated. This article reviews studies on the regulation of NHE3 by NHERF, PKA, and ezrin and introduces the concept of regulation of renal transporters by signal complexes. Although not the primary focus of this review, recent studies have indicated a role for NHERF in membrane targeting, trafficking, and sorting of transporters, receptors, and signaling proteins. Thus NHERF and related PDZ-containing proteins appear to be essential adapters for regulation of renal transporters in the mammalian kidney that maintain salt and water balance.
Differential localization of human nongastric H+-K+-ATPase ATP1AL1 in polarized renal epithelial cells
Authors: Reinhardt Jürgen , Grishin Alexander V. , Oberleithner Hans , Caplan Michael J.
Published: December 2017

The human H+-K+-ATPase, ATP1AL1, belongs to the subgroup of nongastric, K+-transporting ATPases. In concert with the structurally related gastric H+-K+-ATPase, it plays a major role in K+reabsorption in various tissues, including colon and kidney. Physiological and immunocytochemical data suggest that the functional heteromeric ion pumps are usually found in the apical plasma membranes of renal epithelial cells. However, the low expression levels of characteristic nongastric ion pumps makes it difficult to verify their spatial distribution in vivo. To investigate the sorting behavior of ATP1AL1, we expressed this pump by stable transfection in MDCK and LLC-PK1renal epithelial cell lines. Stable interaction of ATP1AL1 with either the endogenous Na+-K+-ATPase β-subunit or the gastric H+-K+-ATPase β-subunit was tested by confocal immunofluorescence microscopy and surface biotinylation. In cells transfected with ATP1AL1 alone, the α-subunit accumulated intracellularly, consistent with its inability to assemble and travel to the plasma membrane with the endogenous Na+-K+-ATPase β-subunit. Cotransfection of ATP1AL1 with the gastric H+-K+-ATPase β-subunit resulted in plasma membrane localization of both pump subunits. In cotransfected MDCK cells the heteromeric ion pump was predominantly polarized to the apical plasma membrane. Functional expression of ATP1AL1 was confirmed by86Rb+uptake measurements. In contrast, cotransfected LLC-PK1cells accumulate ATP1AL1 at the lateral membrane. The distinct polarization of ATP1AL1 indicates that the α-subunit encodes sorting information that is differently interpreted by cell type-specific sorting mechanisms.
Identification of a novel kidney-specific gene downregulated in acute ischemic renal failure
Authors: Hu Erding , Chen Zunxuan , Fredrickson Todd , Gellai Miklos , Jugus Malcolm , Contino Lisa , Spurr Nigel , Sims Matthew , Halsey Wendy , Van Horn Stephanie , Mao Joyce , Sathe Ganesh , Brooks David
Published: December 2017

To gain further insights into the molecular mechanisms involved in acute renal failure, we have isolated a new gene from rat and human, named KSP32 (kidney-specific protein with a molecular mass of 32 kDa). KSP32 encodes a novel gene that shows little homology to other mammalian proteins. It, however, shares extensive homology with several proteins found in the nematode Caenorhabditis elegans and plants. The expression of KSP32 mRNA is highly restricted to kidney. In situ hybidization analysis revealed that the expression of KSP32 mRNA was prominent in the boundary of kidney cortex and outer medulla, exhibiting a raylike formation extending from the medulla into the cortex. Finally, KSP32 mRNA was dramatically downregulated in rat following induction of acute ischemic renal failure. Rapid loss of KSP32 mRNA expression was observed beginning at ∼5 h following renal injury and mRNA levels remained depressed for at least 96 h. Both KSP32 mRNA levels as well as renal function recovered 14 days after injury. Administration of an endothelin receptor antagonist (SB-209670), known to restore renal function, significantly increased KSP32 expression.
Long-term regulation of renal Na-dependent cotransporters and ENaC: response to altered acid-base intake
Authors: Kim Gheun-Ho , Martin Stephen W. , Fernández-Llama Patricia , Masilamani Shyama , Packer Randall K. , Knepper Mark A.
Published: December 2017

Increased systemic acid intake is associated with an increase in apical Na/H exchange in the renal proximal tubule mediated by the type 3 Na/H exchanger (NHE3). Because NHE3 mediates both proton secretion and Na absorption, increased NHE3 activity could inappropriately perturb Na balance unless there are compensatory changes in Na handling. In this study, we use semiquantitative immunoblotting of rat kidneys to investigate whether acid loading is associated with compensatory decreases in the abundance of renal tubule Na transporters other than NHE3. Long-term (i.e., 7-day) acid loading with NH4Cl produced large decreases in the abundances of the thiazide-sensitive Na-Cl cotransporter (TSC/NCC) of the distal convoluted tubule and both the β- and γ-subunits of the amiloride-sensitive epithelial Na channel (ENaC) of the collecting duct. In addition, the renal cortical abundance of the proximal type 2 Na-dependent phosphate transporter (NaPi-2) was markedly decreased. In contrast, abundances of the bumetanide-sensitive Na-K-2Cl cotransporter of the thick ascending limb and the α-subunit of ENaC were unchanged. A similar profile of changes was seen with short-term (16-h) acid loading. Long-term (7-day) base loading with NaHCO3resulted in the opposite pattern of response with marked increases in the abundances of the β- and γ-subunits of ENaC and NaPi-2. These adaptations may play critical roles in the maintenance in Na balance when changes in acid-base balance occur.
Molecular cloning, chromosomal organization, and functional characterization of a sodium-dicarboxylate cotransporter from mouse kidney
Authors: Pajor Ana M. , Sun Nina N.
Published: December 2017

The sodium-dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs filtered Krebs cycle intermediates and plays an important role in the regulation of urinary citrate concentrations.1Low urinary citrate is a risk factor for the development of kidney stones. As an initial step in the characterization of NaDC-1 regulation, the genomic structure and functional properties of the mouse Na+-dicarboxylate cotransporter (mNaDC-1) were determined. The gene coding for mNaDC-1, Slc13a2, is found on chromosome 11. The gene is ∼24.9 kb in length and contains 12 exons. The mRNA coding for mNaDC-1 is found in kidney and small intestine. Expression of mNaDC-1 in Xenopus laevis oocytes results in increased transport of di- and tricarboxylates. The Michaelis-Menten constant ( Km) for succinate was 0.35 mM, and the Kmfor citrate was 0.6 mM. The transport of citrate was stimulated by acidic pH, whereas the transport of succinate was insensitive to pH changes. Transport by mNaDC-1 is electrogenic, and substrates produced inward currents in the presence of sodium. The sodium affinity was relatively high in mNaDC-1, with half-saturation constants for sodium of 10 mM (radiotracer experiments) and 28 mM at −50 mV (2-electrode voltage clamp experiments). Lithium acts as a potent inhibitor of transport, but it can also partially substitute for sodium. In conclusion, the mNaDC-1 is related in sequence and function to the other NaDC-1 orthologs. However, its function more closely resembles the rabbit and human orthologs rather than the rat NaDC-1, with which it shares higher sequence similarity.
Chaotic behavior of renal sympathetic nerve activity: effect of baroreceptor denervation and cardiac failure
Authors: DiBona Gerald F. , Jones Susan Y. , Sawin Linda L.
Published: December 2017

Nonlinear dynamic analysis was used to examine the chaotic behavior of renal sympathetic nerve activity in conscious rats subjected to either complete baroreceptor denervation (sinoaortic and cardiac baroreceptor denervation) or induction of congestive heart failure (CHF). The peak interval sequence of synchronized renal sympathetic nerve discharge was extracted and used for analysis. In control rats, this yielded a system whose correlation dimension converged to a low value over the embedding dimension range of 10–15 and whose greatest Lyapunov exponent was positive. Complete baroreceptor denervation was associated with a decrease in the correlation dimension of the system (before 2.65 ± 0.27, after 1.64 ± 0.17; P < 0.01) and a reduction in chaotic behavior (greatest Lyapunov exponent: 0.201 ± 0.008 bits/data point before, 0.177 ± 0.004 bits/data point after, P < 0.02). CHF, a state characterized by impaired sinoaortic and cardiac baroreceptor regulation of renal sympathetic nerve activity, was associated with a similar decrease in the correlation dimension (control 3.41 ± 0.23, CHF 2.62 ± 0.26; P < 0.01) and a reduction in chaotic behavior (greatest Lyapunov exponent: 0.205 ± 0.048 bits/data point control, 0.136 ± 0.033 bits/data point CHF, P < 0.02). These results indicate that removal of sinoaortic and cardiac baroreceptor regulation of renal sympathetic nerve activity, occurring either physiologically or pathophysiologically, is associated with a decrease in the correlation dimensions of the system and a reduction in chaotic behavior.
Downregulation of the calpain inhibitor protein calpastatin by caspases during renal ischemia-reperfusion
Authors: Shi Yuexian , Melnikov Vyacheslav Y. , Schrier Robert W. , Edelstein Charles L.
Published: December 2017

The interaction between the cysteine proteases calpain and caspases during renal ischemia-reperfusion (I/R) was investigated. An increase in the activity of calpain, as determined by 1) the appearance of calpain-mediated spectrin breakdown products and 2) the conversion of procalpain to active calpain, was demonstrated. Because intracellular calpain activity is regulated by calpastatin, the effect of I/R on calpastatin was determined. On immunoblot of renal cortex, there was a 50–100% decrease of a low molecular weight (LMW) form of calpastatin (41 kDa) after I/R. Calpastatin activity was also significantly decreased after I/R compared with sham-operated rats, indicating that the decreased protein expression had functional significance. In rats treated with the caspase inhibitor, z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-d-DCB), the decrease in both calpastatin activity and protein expression was normalized, suggesting that caspases may be proteolyzing calpastatin. Caspase 3 activity increased significantly after I/R and was attenuated in ischemic kidneys from rats treated with the caspase inhibitor. In summary, during renal I/R injury, there is 1) calpain activation associated with downregulation of calpastatin protein and decreased calpastatin activity and 2) activation of caspase 3. In addition, in vivo caspase inhibition reverses the decrease in calpastatin activity. In conclusion, proteolysis of calpastatin by caspase 3 may regulate calpain activity during I/R injury. Although the protective effect of cysteine protease inhibition against hypoxic necrosis of proximal tubules has previously been demonstrated, the functional significance in ischemic acute renal failure in vivo merits further study.
Defective fluid and HCO3−absorption in proximal tubule of neuronal nitric oxide synthase-knockout mice
Authors: Wang Tong , Inglis Fiona M. , Kalb Robert G.
Published: December 2017

Using renal clearance techniques and in situ microperfusion of proximal tubules, we examined the effects of NG-monomethyl-l-arginine methyl ester (l-NAME) on fluid and HCO3−transport in wild-type mice and also investigated proximal tubule transport in neuronal nitric oxide synthase (nNOS)-knockout mice. In wild-type mice, administration of l-NAME (3 mg/kg bolus iv) significantly increased mean blood pressure, urine volume, and urinary Na+excretion. l-NAME, given by intravenous bolus and added to the luminal perfusion solution, decreased absorption of fluid (60%) and HCO3−(49%) in the proximal tubule. In nNOS-knockout mice, the urinary excretion of HCO3−was significantly higher than in the wild-type mice (3.12 ± 0.52 vs. 1.40 ± 0.33 mM) and the rates of HCO3−and fluid absorption were 62 and 72% lower, respectively. Both arterial blood HCO3−concentration (20.7 vs. 25.7 mM) and blood pH (7.27 vs. 7.34) were lower, indicating a significant metabolic acidosis in nNOS-knockout mice. Blood pressure was lower in nNOS-knockout mice (76.2 ± 4.6 mmHg) than in wild-type control animals (102.9 ± 8.4 mmHg); however, it increased in response to l-NAME (125.5 ± 5.07 mmHg). Plasma Na+and K+were not significantly different from control values. Our data show that a large component of HCO3−and fluid absorption in the proximal tubule is controlled by nNOS. Mice without this isozyme are defective in absorption of fluid and HCO3−in the proximal tubule and develop metabolic acidosis, suggesting that nNOS plays an important role in the regulation of acid-base balance.
Potassium deprivation upregulates expression of renal basolateral Na+-HCO3−cotransporter (NBC-1)
Authors: Amlal Hassane , Habo Khalid , Soleimani Manoocher
Published: December 2017

The purpose of the present experiments was to examine the effect of potassium deprivation on the expression of the renal basolateral Na+-HCO3−cotransporter (NBC-1). Rats were placed on a K+-free diet for various time intervals and examined. NBC-1 mRNA levels increased by about threefold in the cortex ( P < 0.04) at 72 h of K+deprivation and remained elevated at 21 days. NBC activity increased by ∼110% in proximal tubule suspensions, with the activity increasing from 0.091 in control to 0.205 pH/min in the K+-deprived group ( P < 0.005). The inner stripe of outer medulla and cells of medullary thick ascending limb of Henle (mTAL) showed induction of NBC-1 mRNA and activity in K+-deprived rats, with the activity in mTAL increasing from 0.010 in control to 0.133 pH/min in the K+-deprived group ( P < 0.004). K+deprivation also increased NBC-1 mRNA levels in the renal papilla ( P < 0.02). We conclude that 1) K+deprivation increases NBC-1 expression and activity in proximal tubule and 2) K+deprivation causes induction of NBC-1 expression and activity in mTAL tubule and inner medulla. We propose that NBC-1 likely mediates enhanced HCO3−reabsorption in proximal tubule, mTAL, and inner medullary collecting duct in K+deprivation and contributes to the maintenance of metabolic alkalosis in this condition.
Angiotensin II releases 20-HETE from rat renal microvessels
Authors: Croft Kevin D. , McGiff John C. , Sanchez-Mendoza Alicia , Carroll Mairead A.
Published: December 2017

We studied hydroxyeicosatetraenoic acid (HETE) release in response to ANG II from preglomerular microvessels (PGMVs), the vascular segment governing changes in renal vascular resistance. PGMVs were isolated from Sprague-Dawley rats and incubated with NADPH and hormones at 37°C. Eicosanoids were extracted, and cytochrome P-450 (CYP)-derived HETEs were purified and quantitated by negative chemical ionization gas chromatography-mass spectroscopy. PGMVs produced primarily 20- and 19-HETEs, namely, 7.9 ± 1.7 and 2.2 ± 0.5 ng/mg protein, respectively. ANG II (5 nM) increased CYP-HETE release by two- to threefold; bradykinin, phenylephrine, and Ca2+ionophore were without effect. [Sar1]ANG II (0.1–100 μM) dose dependently stimulated 19- and 20-HETEs, an effect blocked by the AT2-receptor antagonist PD-123319 as well as by U-73122, a phospholipase C inhibitor. Microvascular 20-HETE release was increased more than twofold by the third day in response to ANG II (120 ng · kg−1· min−1) infused subcutaneously for 2 wk; it was not further enhanced after 14 days, although blood pressure continued to rise. Thus an AT2-phospholipse C effector unit is associated with synthesis of a vasoconstrictor product, 20-HETE, in a key renovascular segment.
Functional and pharmacological characterization of human Na+-carnitine cotransporter hOCTN2
Authors: Wagner Carsten A. , Lükewille Ulrike , Kaltenbach Simone , Moschen Ivano , Bröer Angelika , Risler Teut , Bröer Stefan , Lang Florian
Published: December 2017

l-Carnitine is essential for the translocation of acyl-carnitine into the mitochondria for β-oxidation of long-chain fatty acids. It is taken up into the cells by the recently cloned Na+-driven carnitine organic cation transporter OCTN2. Here we expressed hOCTN2 in Xenopus laevis oocytes and investigated with two-electrode voltage- clamp and flux measurements its functional and pharmacological properties as a Na+-carnitine cotransporter. l-carnitine transport was electrogenic. The l-carnitine-induced currents were voltage and Na+ dependent, with half-maximal currents at 0.3 ± 0.1 mM Na+ at −60 mV. Furthermore,l-carnitine-induced currents were pH dependent, decreasing with acidification. In contrast to other members of the organic cation transporter family, hOCTN2 functions as a Na+-coupled carnitine transporter. Carnitine transport was stereoselective, with an apparent Michaelis-Menten constant ( K m) of 4.8 ± 0.3 μM for l-carnitine and 98.3 ± 38.0 μM for d-carnitine. The substrate specificity of hOCTN2 differs from rOCT-1 and hOCT-2 as hOCTN2 showed only small currents with classic OCT substrates such as choline or tetraethylammonium; by contrast hOCTN2 mediated transport of betaine. hOCTN2 was inhibited by several drugs known to induce secondary carnitine deficiency. Most potent blockers were the antibiotic emetine and the ion channel blockers quinidine and verapamil. The apparent IC50 for emetine was 4.2 ± 1.2 μM. The anticonvulsant valproic acid did not induce a significant inhibition of carnitine transport, pointing to a different mode of action. In summary, hOCTN2 mediates electrogenic Na+-dependent stereoselective high-affinity transport ofl-carnitine and Na+. hOCTN2 displays transport properties distinct from other members of the OCT family and is directly inhibited by several substances known to induce systemic carnitine deficiency.
Differential actions of renal ischemic injury on the intrarenal angiotensin system
Authors: Allred Alicia J. , Chappell Mark C. , Ferrario Carlos M. , Diz Debra I.
Published: December 2017

The present study determined the effect of either occlusion of the left renal artery for 60 min (ischemia) or sham operation on angiotensin (ANG) receptors and tissue and urinary levels of ANG peptides between 24 and 72 h recovery in male Sprague-Dawley rats. At 24 h postischemia, urinary concentrations of ANG I and ANG-(1–7) rose by an average of 83 and 64%, respectively ( P < 0.05) but had declined to control levels by 72 h. Tissue ANG II rose at 24 h in postischemic kidneys by an average of 63% compared with the contralateral nonischemic kidney ( P < 0.05). Whereas the enzymatic activity of angiotensin-converting enzyme and neprilysin was reduced after ischemia, renal renin activity in ischemic kidneys rose by 74% compared with sham-operated kidneys. Receptor autoradiography using125I-labeled [Sar1,Thr8]ANG II (125I-Sarthran) (0.8 nM) revealed a decreased apparent density of ANG receptors (>80% AT1) in ischemic kidneys with a trend for a decrease in the contralateral nonischemic kidneys compared with the kidneys from sham-operated rats. Twenty-four hours after ischemia, ANG II receptors decreased by 68% in glomeruli ( P < 0.05), 49% in the outer cortical tubulointerstitial area ( P < 0.05), and 48% in the inner cortical-outer medullary area of the vasa recta ( P < 0.05). Medullary binding decreased ∼50% in both the ischemic kidney and the contralateral nonischemic kidney compared with sham. In all regions of the ischemic kidney, receptors recovered by 72 h to levels not different from sham control rats. The marked change in urinary ANG I and ANG-(1–7) at 24 h following occlusion indicates these peptides may be potential urinary markers for acute renal ischemia. The reduction of receptors in vascular and tubular regions of the ischemic kidney provides a mechanism for the loss of vasoconstrictor responses to ANG II following ischemia previously reported by others.
Hexosamine regulation of glucose-mediated laminin synthesis in mesangial cells involves protein kinases A and C
Authors: Singh Lalit P. , Crook Errol D.
Published: December 2017

Hyperglycemia leads to alterations in mesangial cell function and extracellular matrix (ECM) protein accumulation. These adverse effects of glucose may be mediated by glucose metabolism through the hexosamine biosynthesis pathway (HBP). The HBP converts fructose-6-phosphate to glucosamine-6-phosphate via the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFA). We have investigated the effects of high glucose (HG, 25 mM) and glucosamine (GlcN, 1.5 mM) on the synthesis of the ECM protein laminin in a SV-40-transformed rat kidney mesangial (MES) cell line. The roles of protein kinases C (PKC) and A (PKA) in mediating laminin accumulation were also investigated. Treatment of MES cells with HG or GlcN for 48 h increased laminin levels in cellular extracts more than twofold compared with 5 mM glucose (low glucose; LG). The presence of the GFA inhibitor diazo-oxo-norleucine (DON, 10 μM) blocked HG but not GlcN-induced laminin synthesis. HG resulted in a time-dependent increase in total PKC and PKA activities, 57±11.3 ( P< 0.01 vs. LG) and 85±17.4% ( P < 0.01 vs. LG), respectively. GlcN had no effect on the total PKC activity; however, both glucose and glucosamine increased membrane-associated PKC activity by twofold compared with LG. GlcN stimulated total PKA activity by 47±8.4% ( P < 0.01 vs. LG). Similarly, membrane- associated PKA activity was also increased by HG and GlcN ∼1.8 and 1.5-fold, respectively. HG and GlcN increased cellular cAMP levels 2.2- and 3.4-fold, respectively. Pharmacological downregulation of PKC by long-term incubation of MES cells with 0.5 μM phorbol 12-myristate 13-acetate (PMA) or inhibition of PKA activity by 2 μM H-8 blocked the effects of HG and GlcN on laminin synthesis. These results demonstrate that glucose-induced laminin synthesis in MES cells is mediated by flux through the HBP and that this stimulation involves PKC and PKA signaling pathways.
Early polyuria and urinary concentrating defect in potassium deprivation
Authors: Amlal Hassane , Krane Carissa M. , Chen Qian , Soleimani Manoocher
Published: December 2017

The time course of the onset of nephrogenic diabetes insipidus and its relationship to aquaporin-2 (AQP2) expression in K+ deprivation (KD) remains unknown. Rats were fed a K+-free diet and killed after 12 h, 1, 2, 3, 6, or 21 days. Serum K+ concentration was decreased only after, but not before, 3 days of a K+-free diet. Urine osmolality, however, decreased as early as 12 h of KD (1,061 ± 26 vs. 1,487 ± 102 mosmol/kgH2O in control, P < 0.01). It decreased further at 24 h (to 858 ± 162 mosmol/kgH2O in KD, P < 0.004) and remained low at 21 days of KD (436 ± 58 mosmol/kgH2O, P < 0.0001 compared with baseline). Water intake decreased at 12 h ( P < 0.002) but increased at 24 h ( P < 0.05) and remained elevated at 21 days of KD. Urine volume increased at 24 h of KD (8 ± 2 to 15 ± 2 ml/24 h, P < 0.05) and remained elevated at 21 days. Immunoblot analysis demonstrated that AQP2 protein abundance in the outer medulla remained unchanged at 12 h ( P > 0.05), decreased at 24 h (∼44%, P < 0.001), and remained suppressed (∼52%, P < 0.03) at 21 days of KD. In the inner medulla the AQP2 protein abundance remained unchanged at both 12 and 24 h of KD. AQP2 protein abundance in the cortex, however, decreased at 12 h (∼47%, P < 0.01) and remained suppressed at 24 h (∼77%, P < 0.001) of KD. Northern blot analysis showed that AQP2 mRNA decreased as early as 12 h of KD in both cortex ( P < 0.02) and outer medulla ( P < 0.01) and remained suppressed afterward. In conclusion, the urinary concentrating defect in KD is an early event and precedes the onset of hypokalemia. These studies further suggest that the very early urinary concentrating defect in KD (after 12 but before 24 h) results primarily from the suppression of cortical AQP2, whereas the later onset of a urinary concentrating defect (after 24 h) also involves a downregulation of medullary AQP2.
Overexpression of GFAT activates PAI-1 promoter in mesangial cells
Authors: James Leighton R. , Fantus I. George , Goldberg Howard , Ly Hao , Scholey James W.
Published: December 2017

Effects of hyperglycemia on glomerular cells may be mediated by glucose entry into the hexosamine pathway, and mesangial cell (MC) expression of the hexosamine pathway rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) is increased in diabetic glomerulosclerosis. We hypothesized that GFAT activity would be an important determinant of gene expression in glomerular MC. When overexpressed in primary MC, GFAT produced a two- to threefold increase in the activity of plasminogen activator inhibitor-1 (PAI-1) promoter. There was a 1.4-fold increase in PAI-1 promoter activity in cells exposed to high glucose (20 mM), whereas in MC overexpressing GFAT, exposure to high glucose caused a 3.5- to 4-fold increase in promoter activity. PAI-1 promoter activation was dependent on GFAT enzyme activity because o-diazoacetyly-l-serine and 6-diazo-5-oxonorleucine, inhibitors of GFAT enzyme activity, abrogated the activation of PAI-1 promoter in MC overexpressing GFAT. Glucosamine, which is downstream of GFAT in the hexosamine pathway, produced a 2.5-fold increase in the PAI-1 promoter activity. In addition to increasing the mRNA levels for transforming growth factor-β1 (TGF-β1), GFAT overexpression also increased mRNA levels for the TGF-β type I and type II receptors. TGF-β-neutralizing antibody did not normalize PAI-1 promoter activity in MC exposed to glucosamine or those overexpressing GFAT. We conclude that GFAT expression and activity are important determinants of gene expression in MC and that flux through the hexosamine pathway activates expression of genes implicated in vascular injury pathways.
CD2AP is expressed with nephrin in developing podocytes and is found widely in mature kidney and elsewhere
Authors: Li Cong , Ruotsalainen Vesa , Tryggvason Karl , Shaw Andrey S. , Miner Jeffrey H.
Published: December 2017

CD2-associated protein (CD2AP) is an adapter molecule that can bind to the cytoplasmic domain of nephrin, a component of the glomerular slit diaphragm. Mice lacking CD2AP exhibit a congenital nephrotic syndrome characterized by extensive foot process effacement, suggesting that CD2AP-nephrin interactions are critical to maintaining slit diaphragm function. We have examined the patterns of expression of both CD2AP and nephrin in developing mouse and human kidney. Both proteins were first detected in developing podocytes at the capillary loop stage of glomerulogenesis and eventually became concentrated near the glomerular basement membrane. CD2AP was also observed diffusely in collecting duct and apically in many cells of proximal and distal tubule. Kidneys from Cd2ap −/− mice initially exhibited normal nephrin localization, but as the mice aged and foot processes became effaced, nephrin disappeared. In laminin-β2 mutant mice exhibiting nephrotic syndrome, CD2AP in glomeruli was aberrantly localized in a primarily punctate pattern. Extensive extrarenal expression of CD2AP was observed in endothelial and epithelial cells, in many cases with a specific subcellular localization. Together, these results suggest that CD2AP is not only involved in maintaining the slit diaphragm but may also have a general role in maintaining specialized subcellular architecture. The severity of kidney disease in Cd2ap mutant mice may have eclipsed manifestation of defects in other tissues.
Angiotensin II increases vasopressin-stimulated facilitated urea permeability in rat terminal IMCDs
Authors: Kato Akihiko , Klein Janet D. , Zhang Chi , Sands Jeff M.
Published: December 2017

Angiotensin II receptors are present along the rat inner medullary collecting duct (IMCD), although their physiological role is unknown. Because urea is one of the major solutes transported across the terminal IMCD, we measured angiotensin II's effect on urea permeability. In the perfused rat terminal IMCD, angiotensin II had no effect on basal urea permeability but significantly increased vasopressin-stimulated urea permeability by 55%. Angiotensin II, both without and with vasopressin, also increased the amount of32P incorporated into urea transporter (UT)-A1 in inner medullary tissue exposed to these hormones ex vivo. Because angiotensin II activates protein kinase C, we tested the effect of staurosporine (SSP). In the absence of angiotensin II, SSP had no effect on vasopressin-stimulated urea permeability in the perfused terminal IMCD. However, SSP completely and reversibly blocked the angiotensin II-mediated increase in vasopressin-stimulated urea permeability. SSP and chelerythrine reduced the angiotensin II-stimulated32P incorporation into UT-A1 in inner medullary tissue exposed ex vivo. We conclude that angiotensin II increases vasopressin-stimulated facilitated urea permeability and32P incorporation into the 97- and 117-kDa UT-A1 proteins via a protein kinase C-mediated signaling pathway. These data suggest that angiotensin II augments vasopressin-stimulated facilitated urea transport in the rat terminal IMCD and may play a physiological role in the urinary concentrating mechanism by augmenting the maximal response to vasopressin.
Dynamic interaction between myogenic and TGF mechanisms in afferent arteriolar blood flow autoregulation
Authors: Walker Matthew , Harrison-Bernard Lisa M. , Cook Anthony K. , Navar L. Gabriel
Published: December 2017

The dynamic activity of afferent arteriolar diameter (AAD) and blood flow (AABF) responses to a rapid step increase in renal arterial pressure (100–148 mmHg) was examined in the kidneys of normal Sprague-Dawley rats ( n = 11) before [tubuloglomerular feedback (TGF)-intact] and after interruption of distal tubular flow (TGF-independent). Utilizing the in vitro blood-perfused juxtamedullary nephron preparation, fluctuations in AAD and erythrocyte velocity were sampled by using analog-to-digital computerized conversion, video microscopy, image shearing, and fast-frame, slow-frame techniques. These assessments enabled dynamic characterization of the autonomous actions and collective interactions between the myogenic and TGF mechanisms at the level of the afferent arteriole. The TGF-intact and TGF-independent systems exhibited common initial (0–24 vs. 0–13 s, respectively) response slope kinetics (−0.53 vs. −0.47% ΔAAD/s; respectively) yet different maximum vasoconstrictive magnitude (−11.28 ± 0.1 vs. −7.02 ± 0.9% ΔAAD; P < 0.05, respectively). The initial AABF responses similarly exhibited similar kinetics but differing magnitudes. In contrast, during the sustained pressure input (13–97 s), the maximum vasoconstrictor magnitude (−7.02 ± 0.9% ΔAAD) and kinetics (−0.01% ΔAAD/s) of the TGF-independent system were markedly blunted whereas the TGF-intact system exhibited continued vasoconstriction with slower kinetics (−0.20% ΔAAD/s) until a steady-state plateau was reached (−25.9 ± 0.4% ΔAAD). Thus the TGF mechanism plays a role in both direct mediation of vasoconstriction and in modulation of the myogenic response.
Mapping and functional analysis of an instability element in phosphoenolpyruvate carboxykinase mRNA
Authors: Laterza Omar F. , Taylor Lynn , Unnithan Shashikala , Nguyen Ly , Curthoys Norman P.
Published: December 2017

Phosph oenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of renal gluconeogenesis. The 3′-nontranslated region of the PEPCK mRNA contains an instability element that facilitates its rapid turnover and contributes to the regulation of PEPCK gene expression. Such processes are mediated by specific protein-binding elements. Thus RNA gel shift analysis was used to identify proteins in rat renal cortical cytosolic extracts that bind to the 3′-nontranslated region of the PEPCK mRNA. Deletion constructs were then used to map the binding interactions to two adjacent RNA segments (PEPCK-6 and PEPCK-7). However, competition experiments established that only the binding to PEPCK-7 was specific. Functional studies were performed by cloning similar segments in a luciferase reporter construct, pLuc/Zeo. This analysis indicated that both PEPCK-6 and PEPCK-7 segments were necessary to produce a decrease in luciferase activity equivalent to that observed with the full-length 3′-nontranslated region. Thus the PEPCK-7 segment binds a specific protein that may recruit one or more proteins to form a complex that mediates the rapid decay of the PEPCK mRNA.
Method for measuring luminal efflux of fluorescent organic compounds in isolated, perfused renal tubules
Authors: Shuprisha Apichai , Wright Stephen H. , Dantzler William H.
Published: December 2017

To examine directly in real time the efflux of organic compounds [e.g., organic anions (OAs) such as fluorescein (FL)] across the luminal membrane of isolated, perfused renal tubules during net secretion, we devised an approach utilizing a recently developed epifluorescence microscopy system for continuous monitoring of fluorescence in the collected perfusate. To illustrate this approach, we measured the luminal efflux rate of FL in mineral oil-covered, isolated, perfused S2 segments of rabbit renal proximal tubules. The washout profile of FL showed a deviation from linearity at time 0 when plotted on a semilog scale, indicating that the luminal efflux of FL was a saturable process. We were able for the first time to determine the kinetic parameters of luminal efflux [FL concentration at one-half maximal FL efflux ( Ktlumen) of ∼560 μM and maximal rate of FL efflux across the luminal membrane ( Jmaxlumen) of ∼635 fmol · min−1· mm−1]. From the present study, we conclude that the transport step for OAs across the luminal membrane of OAs is a carrier-mediated process. This approach will work to measure luminal transport in real time for any secreted organic compound that is sufficiently fluorescent to be measured with commonly available, highly sensitive optical equipment.
Production of gastric intrinsic factor, transcobalamin, and haptocorrin in opossum kidney cells
Authors: Brada N. , Gordon M. M. , Shao J.-S. , Wen J. , Alpers D. H.
Published: December 2017

Opossum kidney epithelial cells were shown previously to synthesize and secrete two cobalamin (Cbl)-binding proteins, presumed to be haptocorrin (Hc) and transcobalamin II (TCII). The present study examines the hypothesis that renal tubular cells also produce intrinsic factor (IF), and this production provides an explanation for the presence of IF in urine. By using antisera raised against human IF and against TCII, the presence of TCII was confirmed, and that of IF discovered in the media of opossum kidney (OK) cells in culture. The apparent molecular weight of IF and TCII was 68 and 43 kDa, respectively. Immunoreactivity on Western blot of the putative IF protein was blocked by recombinant human IF. When proteins secreted into the media were separated electrophoretically under nondenaturing conditions after binding with [57Co]Cbl, a broad major band migrated at a relative front independently of recombinant IF or TCII, and probably represents Hc, as the Cbl binding is blocked by cobinamide. Small amounts of bound [57Co]Cbl migrated in the position of both IF and TCII, when cobinamide was present. The presence of IF and TCII in OK cells was confirmed by immunohistology. Specific reactivity for IF (blocked by recombinant IF) was found in proximal tubules of opossum kidney, but not in other portions of the nephron, confirming the ability of anti-human IF antiserum to detect opossum IF. A 732-bp fragment of IF, nearly identical in sequence to rat IF, was isolated by RT-PCR from opossum kidney mRNA, and Western blot confirmed the presence of IF protein. The presence of IF was also documented in rat kidney by isolation of an RT-PCR fragment, immunocytochemistry, and Western blot. IF should be added to the list of renal (proximal) tubular antigens that are shared by other epithelia.
P2Y2receptor of MDCK cells: cloning, expression, and cell-specific signaling
Authors: Zambon Alexander C. , Hughes Richard J. , Meszaros J. Gary , Wu J. Julie , Torres Brian , Brunton Laurence L. , Insel Paul A.
Published: December 2017

Madin-Darby canine kidney (MDCK)-D1 cells, a canine renal epithelial cell line, co-express at least three different P2Y receptor subtypes: P2Y1, P2Y2, and P2Y11(24). Stimulation of P2Y receptors in these cells results in the release of arachidonic acid (AA) and metabolites and the elevation of intracellular cAMP. To define in more precise terms the signaling contributed by the MDCK-D1 P2Y2(cP2Y2) receptor, we have cloned and heterologously expressed it in CF2Th (canine thymocyte) cells, a P2Y2-null cell. Analysis by RT-PCR indicated that canine P2Y2receptors are expressed in skeletal muscle, spleen, kidney, lung, and liver. When expressed in CF2Th cells, cP2Y2receptors promoted phospholipase C-mediated phosphatidylinositol (PI) hydrolysis [uridine 5′-triphosphate ≥ ATP > adenosine 5′-diphosphate > 2MT-ATP] and mobilization of intracellular Ca2+. In contrast to their actions in MDCK-D1 cells, cP2Y2receptors did not stimulate formation of cAMP or AA release when expressed in CF2Th cells. The data indicate that cell setting plays an essential role in the ability of P2Y receptors to regulate AA release and cAMP formation. In particular, renal epithelial cells preferentially express components critical for cP2Y2-induced cAMP formation, including the expression of enzymes involved in the generation and metabolism of AA and receptors that respond to PGE2.
Mechanisms of albuminuria in the chronic nitric oxide inhibition model
Authors: Arcos Michelle Indira , Fujihara Clarice Kazue , Sesso Antônio , Brandão De Almeida Prado Euthymia , De Almeida Prado Maria JoséBrandão , De Nucci Gilberto , Zatz Roberto
Published: December 2017

Chronic nitric oxide (NO) inhibition causes hypertension and renal injury. Concomitant salt overload promotes massive albuminuria. We investigated the mechanisms whereby these treatments impair glomerular permselectivity. Adult male Munich-Wistar rats received either a standard-salt (SS; 0.5% Na) or high-salt (HS; 3.1% Na) diet and either no treatment or the NO inhibitor Nω-nitro-l-arginine methyl ester (l-NAME). At 30 days, albuminuria was moderate, the density of fixed anionic sites at the glomerular basement membrane (GBM), estimated by cationic ferritin binding, declined by ∼35%, and the fractional clearance of 70-kDa neutral dextran (ϕ) rose moderately in rats receiving l-NAME and SS. Rats given l-NAME and HS exhibited massive albuminuria, whereas ϕ was nearly tripled. Depletion of GBM anionic sites was also seen in these rats. The GBM was thickened in bothl-NAME-treated groups. These abnormalities were largely reversed after cessation of treatments. These results indicate that chronic l-NAME treatment promotes reversible albuminuria by impairing both glomerular size and charge selectivity. These effects likely reflect functional rather than structural disruption of the glomerular wall.