bluuebunny/crossref_metadata_2025 huggingface dataset:
An increase in intracellular calcium concentration that is induced by basolateral CO2in rabbit renal proximal tubule
Authors: Bouyer Patrice , Zhou Yuehan , Boron Walter F.
Published: March 2015

Working with isolated perfused S2 proximal tubules, we asked whether the basolateral CO2sensor acts, in part, by raising intracellular Ca2+concentration ([Ca2+]i), monitored with the dye fura 2 (or fura-PE3). In paired experiments, adding 5% CO2/22 mM [Formula: see text] (constant pH 7.40) to the bath (basolateral) solution caused [Ca2+]ito increase from 57 ± 3 to 97 ± 9nM( n = 8, P < 0.002), whereas the same maneuver in the lumen had no effect. Intracellular pH (pHi), measured with the dye BCECF, fell by 0.54 ± 0.08 ( n = 14) when we added [Formula: see text] to the lumen. In 14 tubules in which we added [Formula: see text] to the bath, pHifell by 0.55 ± 0.11 in 9 with a high initial pHi, but rose by 0.28 ± 0.07 in the other 5 with a low initial pHi. Thus it cannot be a pHichange that triggers the [Ca2+]iincrease. Introducing to the bath an out-of-equilibrium (OOE) solution containing 20% CO2/no [Formula: see text] caused [Ca2+]ito rise by 62 ± 17 nM ( n = 10), whereas an OOE solution containing 0% CO2/22 mM [Formula: see text] caused only a trivial increase. Removing Ca2+from the lumen and bath, or adding 10 μM nifedipine (L- and T-type Ca2+-channel blocker) or 2 μM thapsigargin [sarco-(endo) plasmic reticulum Ca2+-ATPase inhibitor] or 4 μM rotenone (mitochondrial inhibitor) to the lumen and bath, failed to reduce the CO2-induced increase in [Ca2+]i. Adding 10 mM caffeine (ryanodine-receptor agonist) had no effect on [Ca2+]i. Thus basolateral CO2, presumably via a basolateral sensor, triggers the release of Ca2+from a nonconventional intracellular pool.
Functional mapping of rbOCT1 and rbOCT2 activity in the S2 segment of rabbit proximal tubule
Authors: Kaewmokul Santi , Chatsudthipong Varanuj , Evans Kristen K. , Dantzler William H. , Wright Stephen H.
Published: March 2015

A strategy was developed to determine the distribution of activity mediated by the organic cation (OC) transporters OCT1 and OCT2 in rabbit renal proximal tubule (RPT). Both transporters displayed similar affinities for tetraethylammonium (TEA; in CHO-K1 cells, TEA concentrations that resulted in half-maximal transport were 19.9 and 34.5 μM for OCT1 and OCT2, respectively). Similarly, some OCs showed little capacity to discriminate between the two processes (IC50values for ephedrine of 13.6 and 24.2 μM for OCT1 and OCT2, respectively). However, OCT2 had a higher affinity for cimetidine and [2-(4-nitro-2,1,3-benzoxadiazol-7-yl) aminoethyl]trimethylammonium (NBD-TMA; 1.3 and 1.4 μM, respectively) than did OCT1 (97.3 and 108 μM, respectively). Conversely, OCT1 had a higher affinity for tyramine and pindolol than did OCT2 (21.2 and 2.4 vs. 361 and 50 μM, respectively). We designated these as “discriminatory inhibitors” and used them to determine the relative contribution of OCT1 and OCT2 for TEA transport in single S2 segments of rabbit RPT. Cimetidine and NBD-TMA were high-affinity inhibitors of TEA transport in S2 segments (median IC50values of 12.3 and 1.4 μM, respectively); in comparison, tyramine and pindolol were low-affinity inhibitors (265 and 69.3 μM, respectively). These IC50values were sufficiently close to those for OCT2 to support the conclusion that TEA transport in the S2 segment of rabbit RPT is dominated by OCT2. However, the profile of inhibition of tyramine (an OCT1-selective substrate) transport in single S2 segments indicated that, despite a comparatively low level of expression, OCT1 can play a dominant role in the uptake of selected OC substrates.
Loop diuretics: from the Na-K-2Cl transporter to clinical use
Authors: Shankar Sudha S. , Brater D. Craig
Published: March 2015

The diuretic response to loop diuretics in various disease states has consistently been found to be subnormal. One of the key determinants of the degree of diuretic response is the functional integrity of the sodium-potassium-chloride transporter in the loop of Henle. Studies in animal models suggest that expression/activity of the transporter may be affected by factors such as altered natural splicing events of NKCC2 (the gene encoding for the renal transporter), renal prostanoids, vasopressin, and other autacoids. We have reviewed the pharmacokinetics and pharmacodynamics of loop diuretics in health and in edematous disorders for which they are used. On the basis of evidence reviewed in this paper, we propose that altered expression or activity of the sodium-potassium-chloride transporter in the loop of Henle, in conjunction with events occurring in other segments of the nephron, possibly accounts for the altered diuretic response to these agents. Thus the modulators of this altered expression/activity could serve as important therapeutic targets for alternative diuretic regimens in these conditions.
Angiotensin II directly stimulates macula densa Na-2Cl-K cotransport via apical AT1receptors
Authors: Kovács Gergely , Peti-Peterdi János , Rosivall László , Bell P. Darwin
Published: March 2015

ANG II is a modulator of tubuloglomerular feedback (TGF); however, the site of its action remains unknown. Macula densa (MD) cells sense changes in luminal NaCl concentration ([NaCl]L) via a Na-2Cl-K cotransporter, and these cells do possess ANG II receptors. We tested whether ANG II regulates Na-2Cl-K cotransport in MD cells. MD cell Na+concentration ([Na+]i) was measured using sodium-binding benzofuran isophthalate with fluorescence microscopy. Resting [Na+]iin MD cells was 27.7 ± 1.05 mM ( n = 138) and increased (Δ[Na+]i) by 18.5 ± 1.14 mM ( n = 17) at an initial rate (Δ[Na+]i/Δ t) of 5.54 ± 0.53 × 10−4U/s with an increase in [NaCl]Lfrom 25 to 150 mM. Both Δ[Na+]iand Δ[Na+]i/Δ t were inhibited by 80% with 100 μM luminal furosemide. ANG II (10−9or 10−12M) added to the lumen increased MD resting [Na+]iand [NaCl]L-dependent Δ[Na+]iand caused a twofold increase in Δ[Na+]i/Δ t. Bath (10−9M) ANG II also stimulated cotransport activity, and there was no additive effect of simultaneous addition of ANG II to bath and lumen. The effects of luminal ANG II were furosemide sensitive and abolished by the AT1receptor blocker candesartan. ANG II at 10−6M failed to stimulate the cotransporter, whereas increased cotransport activity could be restored by blocking AT2receptors with PD-123, 319. Thus ANG II may modulate TGF responses via alterations in MD Na-2Cl-K cotransport activity.
Losartan treatment normalizes renal sodium and water handling in rats with mild congestive heart failure
Authors: Staahltoft Dennis , Nielsen Søren , Janjua Nadeem R. , Christensen Sten , Skøtt Ole , Marcussen Niels , Jonassen Thomas E. N.
Published: March 2015

This study was designed to examine the effect of losartan treatment on renal tubular function in rats with mild congestive heart failure (CHF) induced by ligation of the left anterior descending artery. In rats with CHF, there was a significant decrease in daily sodium excretion, which caused sodium retention relative to control rats. Renal function studies revealed that glomerular filtration rate and proximal tubular sodium handling were normal. However, expression of the Na+-K+-2Cl− cotransporter (NKCC2) in the thick ascending limb of Henle's loop was increased. Moreover, vasopressin-mediated renal water reabsorption, as evaluated by the aquaretic response to selective V2-receptor blockade, was significantly increased. Losartan treatment normalized expression of NKCC2 and decreased expression of the vasopressin-regulated water channel aquaporin-2. This was associated with normalization of daily sodium excretion and normalization of the aquaretic response to V2-receptor blockade. Together, these results indicate that, in rats with CHF, losartan treatment inhibits increased sodium reabsorption through NKCC2 in the thick ascending limb of Henle's loop and water reabsorption through aquaporin-2 in the collecting ducts, which may be involved in improving renal function in losartan-treated CHF rats.
Inhibition of heme oxygenase decreases sodium and fluid absorption in the loop of Henle
Authors: Wang Tong , Sterling Hyacinth , Shao Wei A. , Yan QingShang , Bailey Matthew A. , Giebisch Gerhard , Wang Wen-Hui
Published: March 2015

We previously demonstrated that carbon monoxide (CO) stimulates the apical 70-pS K+ channel in the thick ascending limb (TAL) of the rat kidney (Liu HJ, Mount DB, Nasjletti A, and Wang WH. J Clin Invest 103: 963-970, 1999). Because the apical K+ channel plays a key role in K+ recycling, we tested the hypothesis that heme oxygenase (HO)-dependent metabolites of heme may affect Na+ transport in the TAL. We used in vivo microperfusion to study the effect of chromium mesoporphyrin (CrMP), an inhibitor of HO, on fluid absorption ( Jv) and Na+ absorption ( JNa) in the loop of Henle and renal clearance methods to examine the effect of CrMP on renal sodium excretion. Microperfusion experiments demonstrated that addition of CrMP to the loop of Henle decreased Jv by 13% and JNa by 20% in animals on normal rat chow and caused a decrease in Jv (39%) and JNa (40%) in rats on a high-K+ (HK) diet. The effect of CrMP is the result of inhibition of HO because addition of MgPP, an analog of CrMP that does not inhibit HO, had no effect on Jv. Western blot analysis showed that HO-2 is expressed in the kidney and that the level of HO-2 was significantly elevated in animals on a HK diet. Renal clearance studies demonstrated that the infusion of CrMP increased the excretion of urinary Na+ (ENa) and volume (UV) without changes in glomerular filtration rate. The effect of CrMP on ENa and UV was larger in HK rats than those kept on normal chow. We conclude that HK intake increases HO-2 expression in the kidney and that HO-dependent metabolites of heme, presumably CO, play a significant role in the regulation of Na+ transport in the loop of Henle.
Identification of c-kit-positive cells in the mouse ureter: the interstitial cells of Cajal of the urinary tract
Authors: Pezzone Michael A. , Watkins Simon C. , Alber Sean M. , King William E. , de Groat William C. , Chancellor Michael B. , Fraser Matthew O.
Published: March 2015

The existence of a pacemaker system in the urinary tract capable of orchestrating the movement of filtrated urine from the ureteral pelvis to the distal ureter and lower urinary tract seems intuitive. The coordinated activity necessary for such movement or “peristalsis” would likely require an intricate network of cells with pacemaker-like activity, as is the case with the interstitial cells of Cajal (ICC) of the gut. We investigated whether these putative pacemaker cells of the urinary tract are antigenically similar to ICC of the gut by using immunofluorescence staining for c-kit, a cell-surface marker specific for ICC. Ureteral, urinary bladder, and urethral tissues were harvested from female mice of the WBB6F1 strain, and fixed sections were prepared and stained for c-kit. Cell networks composed of stellate-appearing, c-kit-positive, ICC-like cells were found in the lamina propria and at the interface of the inner longitudinal and outer circular muscle layers of the ureteral pelvis but not in the urinary bladder or urethra. Thus, like in the gut, c-kit-positive, ICC-like cells are present in the urinary tract but appear to be restricted to the proximal ureter of this murine species.
Increased renal ENaC subunit and sodium transporter abundances in streptozotocin-induced type 1 diabetes
Authors: Song Jian , Knepper Mark A. , Verbalis Joseph G. , Ecelbarger Carolyn A.
Published: March 2015

Uncontrolled diabetes mellitus (DM) is associated with copious water and sodium losses. We hypothesized that the kidney compensates for these losses by increasing the abundances of key sodium and water transporters and channels. Using targeted proteomic analysis via immunoblotting of kidney homogenates, we examined comprehensive regulation of transport proteins. In three studies, streptozotocin (STZ; 65 mg/kg) or vehicle was administered intraperitoneally to male Sprague-Dawley rats. In study 2, to control for potential renal toxicity of STZ, one group of STZ-treated rats was intensively treated with insulin to control diabetes. In another group, the reversibility of DM and related changes was assessed by treating animals with insulin for the final 4 days. In study 3, we correlated blood glucose to transporter changes by treating animals with different doses of insulin. In study 1, STZ treatment resulted in significantly increased band densities for the type 3 sodium/hydrogen exchanger (NHE3), the thiazide-sensitive Na-Cl cotransporter (NCC), and epithelial sodium channel (ENaC) subunits α, β, and γ (85- and 70-kDa bands) to 204, 125, 176, 132, 147, and 241% of vehicle mean, respectively. In study 2, aquaporin-2 (AQP2) and AQP3 were increased with DM, but not AQP1 or AQP4. Neither these changes, nor blood glucose itself, could be returned to normal by short-term intensive insulin treatment. Whole kidney abundance of AQP3, the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), and γ-ENaC (85-kDa band) correlated most strongly with blood glucose in study 3. These comprehensive changes would be expected to decrease volume contraction accompanying large-solute and water losses associated with DM.
ATP masks stretch activation of epithelial sodium channels in A6 distal nephron cells
Authors: Ma He-Ping , Li Li , Zhou Zhen-Hong , Eaton Douglas C. , Warnock David G.
Published: March 2015

The mechanosensitivity of the epithelial sodium channel (ENaC) is controversial. Using cell-attached patch-clamp techniques, we found that mechanical stretch stimulated ENaC in A6 distal nephron cells in only three of nine cell-attached patches. However, stretch consistently activated ENaC after apical ATP was scavenged with apical hexokinase plus glucose or after P2receptors in the patch were blocked. The mean open probability ( P o) of ENaC was increased from 0.31 ± 0.04 to 0.61 ± 0.06 ( P < 0.001; n = 9) when patch pipettes contained hexokinase and glucose, or from 0.24 ± 0.05 to 0.55 ± 0.11 ( P < 0.01; n = 7) when patch pipettes contained suramin, respectively. A poorly hydrolyzable ATP analog, ATPγS, in the patch pipettes inhibited ENaC, reducing the P o from 0.41 ± 0.06 to 0.19 ± 0.05 ( P < 0.01; n = 8). Pretreatment of A6 cells with the phospholipase C (PLC) inhibitor U-73122 abolished the effect of ATP on ENaC activity. These data together suggest that ATP, acting through a PLC-dependent purinergic pathway, masks stretch-induced ENaC activation.
Prostaglandin transporter PGT is expressed in cell types that synthesize and release prostanoids
Authors: Bao Yi , Pucci Michael L. , Chan Brenda S. , Lu Run , Ito Shigekazu , Schuster Victor L.
Published: March 2015

PGT is a broadly expressed transporter of prostaglandins (PGs) and thromboxane that is energetically poised to take up prostanoids across the plasma membrane. To gain insight into the function of PGT, we generated mouse monoclonal antibody 20 against a portion of putative extracellular loop 5 of rat PGT. Immunoblots of endogenous PGT in rat kidney revealed a 65-kDa protein in a zonal pattern corresponding to PG synthesis rates (papilla ≅ medulla > cortex). Immunocytochemically, PGT in rat kidneys was expressed in glomerular endothelial and mesangial cells, arteriolar endothelial and muscularis cells, principal cells of the collecting duct, medullary interstitial cells, medullary vasa rectae endothelia, and papillary surface epithelium. Proximal tubules, which are known to take up and metabolize PGs, were negative. Immunoblotting and immunocytochemistry revealed that rat platelets also express abundant PGT. Coexpression of the PG synthesis apparatus (cyclooxygenase) and PGT by the same cell suggests that prostanoids may undergo release and reuptake.
NF-κB inhibits transcription of the H+-K+-ATPase α2-subunit gene: role of histone deacetylases
Authors: Zhang Wenzheng , Kone Bruce C.
Published: March 2015

The H+-K+-ATPase α2(HKα2) gene plays a central role in potassium homeostasis, yet little is known about its transcriptional control. We recently demonstrated that the proximal promoter confers basal transcriptional activity in mouse inner medullary collecting duct 3 cells. We sought to determine whether the κB DNA binding element at −104 to −94 influences basal HKα2gene transcription in these cells. Recombinant NF-κB p50 footprinted the region −116/−94 in vitro. Gel shift and supershift analysis revealed NF-κB p50- and p65-containing DNA-protein complexes in nuclear extracts of mouse inner medullary collecting duct 3 cells. A promoter-luciferase construct with a mutated −104/−94 NF-κB element exhibited higher activity than the wild-type promoter in transfection assays. Overexpression of NF-κB p50, p65, or their combination trans-repressed the HKα2promoter. The histone deacetylase (HDAC) inhibitor trichostatin A partially reversed NF-κB-mediated trans-repression of the HKα2promoter. HDAC6 overexpression inhibited HKα2promoter activity, and HDAC6 coimmunoprecipitated with NF-κB p50 and p65. These results suggest that HDAC6, recruited to the DNA protein complex, acts with NF-κB to suppress HKα2transcription and identify NF-κB p50 and p65 as novel binding partners for HDAC6.
Altered expression of major renal Na transporters in rats with bilateral ureteral obstruction and release of obstruction
Authors: Li Chunling , Wang Weidong , Kwon Tae-Hwan , Knepper Mark A. , Nielsen Søren , Frøkiær Jørgen
Published: March 2015

Urinary tract obstruction impairs urinary concentrating capacity and reabsorption of sodium. To clarify the molecular mechanisms of these defects, expression levels of renal sodium transporters were examined in rats with 24-h bilateral ureteral obstruction (BUO) or at day 3 or 14 after release of BUO (BUO-R). BUO resulted in downregulation of type 3 Na+/H+exchanger (NHE3) to 41 ± 14%, type 2 Na-Pi cotransporter (NaPi-2) to 26 ± 6%, Na-K-ATPase to 67 ± 8%, type 1 bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1) to 20 ± 7%, and thiazide-sensitive cotransporter (TSC) to 37 ± 9%. Immunocytochemistry confirmed downregulation of NHE3, NaPi-2, Na-K-ATPase, BSC-1, and TSC. Consistent with this downregulation, BUO-R was associated with polyuria, reduced urinary osmolality, and increased urinary sodium and phosphate excretion. BUO-R for 3 days caused a persistant downregulation of NHE3 to 53 ± 10%, NaPi-2 to 57 ± 9%, Na-K-ATPase to 62 ± 8%, BSC-1 to 50 ± 12%, and TSC to 56 ± 16%, which was associated with a marked reduction in the net renal reabsorption of sodium (616 ± 54 vs. 944 ± 24 μmol · min-1· kg-1; P < 0.05) and phosphate (6.3 ± 0.9 vs. 13.1 ± 0.4 μmol · min-1· kg-1; P < 0.05) demonstrating a defect in renal sodium and phosphate reabsorption capacity. Moreover, downregulation of Na-K-ATPase and TSC persisted in BUO-R for 14 days, whereas NHE3, NaPi-2, and BSC-1 were normalized to control levels. In conclusion, downregulation of renal Na transporters in rats with BUO and release of BUO are likely to contribute to the associated urinary concentrating defect, increased urinary sodium excretion, and postobstructive polyuria.
Formate-stimulated NaCl absorption in the proximal tubule is independent of the pendrin protein
Authors: Karniski Lawrence P. , Wang Tong , Everett Lorraine A. , Green Eric D. , Giebisch Gerhard , Aronson Peter S.
Published: March 2015

A significant fraction of active chloride reabsorption across the apical membrane of the proximal tubule is mediated by a chloride/formate exchange process, whereby intracellular formate drives the transport of chloride into the cell. When chloride/formate exchange operates in parallel with Na+/H+exchange and H+-coupled recycling of formate, the net result is electroneutral NaCl reabsorption. Pendrin is the protein product of the PDS gene (SLC26A4) and functions in several different anion exchange modes, including chloride/formate exchange. Pendrin is expressed in the kidney and may serve as the transporter responsible for formate-dependent NaCl reabsorption. In the present study, Pds-knockout mice were used to determine the role of pendrin in proximal tubule chloride reabsorption. We show that formate-dependent NaCl absorption in microperfused proximal tubules is similar between wild-type and pendrin-deficient mice. In addition, there is no difference in the rate of formate-mediated chloride transport in brush-border membrane vesicles isolated from wild-type and pendrin-deficient mice. These studies demonstrate that pendrin is not responsible for formate-dependent NaCl reabsorption in the proximal tubule.
Proximal tubular cholesterol loading after mitochondrial, but not glycolytic, blockade
Authors: Zager Richard A. , Johnson Ali C. M. , Hanson Sherry Y.
Published: March 2015

Diverse forms of injury cause proximal tubular cholesterol accumulation. However, underlying mechanisms in general, and those involved with ATP depletion injury in particular, remain poorly defined. To help elucidate this issue, cholesterol homeostasis and its determinants were assessed after partial ATP depletion states. Serum-exposed HK-2 cells were subjected to mild ATP depletion, induced by mitochondrial inhibition (antimycin A; AA) or glycolytic blockade (2-deoxyglucose; DG). Four or 18 h later, cell cholesterol levels, hydroxymethylglutaryl (HMG)-CoA reductase (HMGCR), the LDL receptor (LDL-R), and ABCA1/SR-B1 cholesterol transporters were assessed. AA and DG each induced mild, largely sublethal ATP depletion injury. Each also caused significant HMGCR increments and SR-B1 decrements and left ABCA1 intact. In contrast, only AA increased the LDL-R, and only AA evoked a cholesterol-loading state (∼25% ↑). One-half of this increase was statin inhibitable, and one-half could be blocked by serum deletion, implying that both synthetic and nonsynthetic (e.g., LDL-R transport) pathways were involved. The AA-induced HMGCR and LDL-R protein changes were paralleled by their mRNAs, suggesting the presence of altered transcriptional events. We conclude that 1) sublethal ATP depletion, whether induced by mitochondrial or glycolytic blockade, can upregulate HMGCR and decrease SR-B1, and these changes represent a previously unrecognized ATP depletion “phenotype”; 2) mitochondrial blockade can also upregulate the LDL-R and evoke a cholesterol-loading state; 3) the latter likely occurs via synthetic and transport pathways; and 4) the mitochondrion may be a critical, and previously unrecognized, determinant of postinjury cell cholesterol homeostasis, potentially by impacting the LDL-R.
Upregulation of type I collagen by TGF-β in mesangial cells is blocked by PPARγ activation
Authors: Zheng Feng , Fornoni Alessia , Elliot Sharon J. , Guan Youfei , Breyer Matthew D. , Striker Liliane J. , Striker Gary E.
Published: March 2015

We found that peroxisome proliferator-activated receptor-γ (PPARγ) mRNA was reduced by 77% in glomeruli of diabetic mice. Because mesangial cells play an important role in diabetic nephropathy, we examined regulation of type I collagen expression by PPARγ and transforming growth factor-β1(TGF-β1) in mouse mesangial cells in the presence of 6 and 25 mM glucose. Mesangial cells contained functionally active PPARγ. Exposure to 25 mM glucose resulted in reduced PPARγ expression and transcriptional activity, accompanied by increased type I collagen expression. Restoration of PPARγ activity to normal levels in cells cultured in 25 mM glucose, by transfection with a PPARγ expression construct and treatment with the PPARγ agonist troglitazone, returned type I collagen levels toward normal values. Activation of PPARγ by troglitazone also decreased type I collagen mRNA and blocked TGF-β1-mediated upregulation of type I collagen mRNA and protein. Moreover, PPARγ activation suppressed basal and activated TGF-β1responses in mesangial cells. This action was blocked by transfection of cells with a dominant-negative PPARγ construct. In summary, PPARγ suppresses the increased type I collagen mRNA and protein expression mediated by TGF-β1in mesangial cells.
α-Melanocyte-simulating hormone and interleukin-10 do not protect the kidney against mercuric chloride-induced injury
Authors: Miyaji Takehiko , Hu Xuzhen , Star Robert A.
Published: March 2015

The anti-inflammatory cytokines α-melanocyte-stimulating hormone (MSH) and interleukin (IL)-10 inhibit acute renal failure (ARF) after ischemia or cisplatin administration; however, these agents have not been tested in a pure nephrotoxic model of ARF. Therefore, we examined the effects of α-MSH and IL-10 in HgCl2-induced ARF. Mice were injected subcutaneously with HgCl2and then given vehicle, α-MSH, or IL-10 by intravenous injection. Animals were killed to study serum creatinine, histology, and myeloperoxidase activity. Treatment with either α-MSH or IL-10 did not alter the increase in serum creatinine, tubular damage, or leukocyte accumulation at 48 h after HgCl2injection. Because α-MSH and IL-10 are active in other injury models that involve leukocytes, we studied the time course of tubular damage and leukocyte accumulation to investigate whether leukocytes caused the tubular damage or accumulated in response to the tubular damage. Tubular damage was present in the outer stripe 12 h after HgCl2injection. In contrast, the number of leukocytes and renal myleoperoxidase activity were normal at 12 h but were significantly increased at 24 and 48 h after injection. We conclude that neither α-MSH nor IL-10 altered the course of HgCl2-induced renal injury. Because the tubular damage preceded leukocyte infiltration, the delayed leukocyte accumulation may play a role in the removal of necrotic tissue and/or tissue repair in HgCl2-induced ARF.
Untraditional methods for targeting the kidney in transgenic mice
Authors: Bianco Robert A. , Keen Henry L. , Lavoie Julie L. , Sigmund Curt D.
Published: March 2015

With the completion of the human genome project and the sequencing of many genomes of experimental models, there is a pressing need to determine the physiological relevance of newly identified genes. Gene-targeting approaches have become an important tool in our arsenal to dissect the significance of genes expressed in many tissues. A wealth of experimental models has been made to assess the role of gene expression in renal function and development. The development of new and informative models is presently limited by the anatomic complexity of the kidney and the lack of cell-specific promoters to target the numerous diverse cell types in that organ. Because of this, new approaches may have to be developed. In this review, we will discuss several untraditional methods to target gene expression to the kidney. These approaches should provide some additional tricks and tools to help in developing additional informative models for studying renal physiology.
Role of renal nerves in stimulation of renin, COX-2, and nNOS in rat renal cortex during salt deficiency
Authors: Höcherl K. , Kammerl M. , Kees F. , Krämer B. K. , Grobecker H. F. , Kurtz A.
Published: March 2015

We investigated a possible involvement of the sympathetic nervous system in the parallel increase of renin, cyclooxygenase-2 (COX-2), and neuronal nitric oxide synthase (nNOS) gene expression in the juxtaglomerular apparatus of rat kidneys induced by salt deficiency. Therefore, we determined the effects of renal denervation and the β-adrenoreceptor antagonist metoprolol (50 mg/kg body wt po, twice a day) on renocortical expression of renin, COX-2, and nNOS in rats fed a low-salt (0.02% wt/wt) diet or treated for 1 wk with ramipril (10 mg/kg body wt) in combination with a low-salt diet. We found that a low-salt diet in combination with ramipril strongly increased renocortical mRNA levels of renin, COX-2, and nNOS 9-, 7-, and 2.5-fold, respectively. Treatment with metoprolol did not change basal expression of the three genes or induction of renin and COX-2 gene expression, while induction of nNOS expression was clearly attenuated. Similarly, unilateral renal denervation attenuated induction of nNOS expression but had no effect on all other parameters. These findings suggest that β-adrenergic stimulation is not required for stimulation of renin and COX-2 gene expression in the juxtaglomerular apparatus during salt deficiency. However, β-adrenoreceptor activity or renal nerve activity appears to be required for the full stimulation of nNOS expression by low salt intake or combined with angiotensin-converting enzyme inhibition.
IGF-1 inhibits the mitochondrial apoptosis program in mesangial cells exposed to high glucose
Authors: Kang Barinder P. S. , Urbonas Arunas , Baddoo Andrew , Baskin Stuart , Malhotra Ashwani , Meggs Leonard G.
Published: March 2015

The activated insulin-like growth factor-1 receptor (IGF-1R) protects cells from a wide range of apoptotic stimuli. Hyperglycemia promotes the intracellular generation of superoxide anion and hydrogen peroxide, both of which have been linked to the activation of the mitochondrial apoptosis program. Here, we report for the first time that ligand activation of the IGF-1R protects normal human mesangial cells and SV40 murine mesangial cells from the glycol-oxidant-induced apoptosis program. The IGF-1R antiapoptosis program was dependent on the recruitment of both Akt/PKB and the ERK subfamily of mitogen-activated protein kinases. IGF-1 treatment also protected the redox potential of mesangial cells maintained at high ambient glucose concentration, by inhibiting the generation of reactive oxygen intermediates and preserving mitochondrial transmembrane potential. IGF-1R survival signals targeted the Bcl-2 family of proteins to protect against glucose-induced apoptosis and oxidative stress. IGF-1-treated cells exhibited a decrease in the Bax/Bcl-2 ratio; increased phosphorylation/inactivation of Bad at Ser112 and Ser136; inhibition of cytochrome c release; perturbations directionally opposed to the initiation of the apoptosis program. In addition, we demonstrate IGF-1R-activated ERK signaling modules phosphorylate Ser112 of the mitochondrial Bad protein, establishing a direct link between surface IGF-1R and the survival program in mitochondria. Our findings indicate that in mesangial cells maintained at high ambient glucose concentration, IGF-1 activates a survival program that maintains the integrity of mitochondria and prevents the expression of the genetic program for apoptosis.
Angiotensin II stimulates basolateral K channels in rat cortical collecting ducts
Authors: Wei Yuan , Wang Wenhui
Published: March 2015

We used the patch-clamp technique to study the effects of angiotensin II (ANG II) on basolateral K channels in cortical collecting ducts (CCDs). Application of ANG II (100 pM–100 nM) increased the activity of basolateral 18-pS K channels. This effect of ANG II was completely abolished by losartan, which is an antagonist of type 1 angiotensin (AT1) receptors. In contrast, inhibition of type 2 angiotensin (AT2) receptors did not block the stimulatory effect of ANG II. Also, application of ANG II significantly increased intracellular Ca2+concentrations, which were measured with fura 2 dye. To explore the role of Ca2+-dependent pathways in the regulation of basolateral K channels, the effects of ANG II on channel activity were examined in the presence of arachidonyltrifluoromethyl ketone to inhibit phospholipase A2(PLA2), GF-109203X [a protein kinase C (PKC) inhibitor], and NG-nitro-l-arginine methyl ester (l-NAME) to inhibit nitric oxide synthase. Inhibition of either PLA2or PKC did not block the effect of ANG II on basolateral K-channel activity. However, the stimulatory effect of ANG II was absent in the CCDs treated with l-NAME. Moreover, addition of the membrane-permeant 8-bromo-guanosine 3′,5′-cyclic monophosphate (8-bromo-cGMP) not only increased channel activity but also abolished the stimulatory effect of ANG II on channel activity. We conclude that ANG II increases basolateral K-channel activity via the stimulation of AT1receptors, and the stimulatory effect of ANG II is mediated by a nitric oxide-dependent cGMP pathway.
Apical heterodimeric cystine and cationic amino acid transporter expressed in MDCK cells
Authors: Bauch Christian , Verrey François
Published: March 2015

The luminal uptake ofl-cystine and cationic amino acids by (re)absorptive epithelia, as found in the small intestine and the proximal kidney tubule, is mediated by the transport system b0,+, which is defective in cystinuria. Expression studies in Xenopus laevis oocytes and other nonepithelial cells as well as genetic studies on cystinuria patients have demonstrated that two gene products, the glycoprotein rBAT and the multitransmembrane-domain protein b0,+AT, are required for system b0,+function. To study the biosynthesis, surface expression, polarity, and function of this heterodimer in an epithelial context, we established stable Madin-Darby canine kidney (MDCK) cell lines expressing rBAT and/or b0,+AT. Confocal immunofluorescence microscopy shows that both subunits depend on each other for apical surface expression. Immunoprecipitation of biosynthetically labeled proteins indicates that b0,+AT is stable in the absence of rBAT, whereas rBAT is rapidly degraded in the absence of b0,+AT. When both are coexpressed, they associate covalently and rBAT becomes fully glycosylated and more stable. Functional experiments show that the expressed transport is of the high-affinity b0,+-type and is restricted to the apical side of the epithelia. In conclusion, coexpression experiments in MDCK cell epithelia strongly suggest that the intracellular association of rBAT and b0,+AT is required for the surface expression of either subunit, which together form a functional heterocomplex at the apical cell membrane.
Sodium and calcium transport pathways along the mammalian distal nephron: from rabbit to human
Authors: Loffing Johannes , Kaissling Brigitte
Published: March 2015

The final adjustment of renal sodium and calcium excretion is achieved by the distal nephron, in which transepithelial ion transport is under control of various hormones, tubular fluid composition, and flow rate. Acquired or inherited diseases leading to deranged renal sodium and calcium balance have been linked to dysfunction of the distal nephron. Diuretic drugs elicit their effects on sodium balance by specifically inhibiting sodium transport proteins in the apical plasma membrane of distal nephron segments. The identification of the major apical sodium transport proteins allows study of their precise distribution pattern along the distal nephron and helps address their cellular and molecular regulation under various physiological and pathophysiological settings. This review focuses on the topological arrangement of sodium and calcium transport proteins along the cortical distal nephron and on some aspects of their functional regulation. The availability of data on the distribution of transporters in various species points to the strengths, as well as to the limitations, of animal models for the extrapolation to humans.
Upregulation of Na+transporter abundances in response to chronic thiazide or loop diuretic treatment in rats
Authors: Na Ki Young , Oh Yoon Kyu , Han Jin Suk , Joo Kwon Wook , Lee Jung Sang , Earm Jae-Ho , Knepper Mark A. , Kim Gheun-Ho
Published: March 2015

Furosemide and hydrochlorothiazide (HCTZ) exert their diuretic actions by binding to apical Na+transporters, viz., the Na+-K+-2Cl−cotransporter in the thick ascending limb and the Na+-Cl−cotransporter in the distal convoluted tubule, respectively. We carried out semiquantitative immunoblotting and immunohistochemistry of rat kidneys to investigate whether chronic administration of furosemide or HCTZ is associated with compensatory changes in the abundance of Na+transporters downstream from the primary site of action. Osmotic minipumps were implanted into Sprague-Dawley rats to deliver furosemide (12 mg/day) or HCTZ (3.75 mg/day) for 7 days. To prevent volume depletion, all animals were offered tap water and a solution containing 0.8% NaCl and 0.1% KCl as drinking fluid. The diuretic/natriuretic response was quantified in response to both agents by using quantitative urine collections. Semiquantitative immunoblotting revealed that the abundances of thick ascending limb Na+-K+-2Cl−cotransporter and all three subunits of the epithelial Na+channel (ENaC) were increased by furosemide infusion. HCTZ infusion increased the abundances of thiazide-sensitive Na+-Cl−cotransporter and β-ENaC in the cortex and β- and γ-ENaC in the outer medulla. Consistent with these results, β-ENaC immunohistochemistry showed a remarkable increase in immunoreactivity in the principal cells of collecting ducts with either diuretic treatment. These increases in the abundance of Na+transporters in response to chronic diuretic treatment may account for the generation of diuretic tolerance associated with long-term diuretic use.
Role of blood cells in leucine kinetics across the human kidney
Authors: Garibotto Giacomo , Russo Rodolfo , Sofia Antonella , Vettore Monica , Dertenois Laura , Robaudo Cristina , Deferrari Giacomo , Zanetti Michela , Tessari Paolo
Published: March 2015

To evaluate the role of blood cells in interorgan amino acid transport and in the estimates of regional protein turnover, we studied the effects of plasma vs. whole blood sampling on regional leucine kinetics in postabsorptive humans. Studies were carried out by combining the arteriovenous difference technique with the measurement of [14C]- and [15N]leucine isotope exchange across the human kidney, the splanchnic area, and the leg. In the kidney, whole blood-derived rates of leucine-carbon appearance, disappearance, and net balance (NB) were greater (by 3–15 times; P < 0.035) than those calculated in plasma. In addition, the net leucine-carbon (i.e., protein) balance across the kidney was negative in whole blood (−5.6 ± 1.3 μmol/min × 1.73 m2, P < 0.01 vs. 0) but neutral in plasma [−0.24 ± 1.33, P = not significant from 0; P < 0.01 vs. whole blood]. A net leucine transport out of renal cells was shown in blood but not in plasma. In contrast, rates of leucine-carbon appearance, disappearance, NB, and net transport, in both the splanchnic area and the leg, were similar in whole blood and plasma. These data suggest that blood cells play a key role in leucine transport out of the kidney and, consequently, in the leucine-derived estimates of renal protein degradation and NB, which is at variance with what is observed across the splanchnic organs or the leg. These data also emphasize the need for complete whole blood arteriovenous measurements to accurately estimate protein turnover across the kidney.
Involvement of ERK pathway in albumin-induced MCP-1 expression in mouse proximal tubular cells
Authors: Takaya Kiho , Koya Daisuke , Isono Motohide , Sugimoto Toshiro , Sugaya Takeshi , Kashiwagi Atsunori , Haneda Masakazu
Published: March 2015

Persistent proteinuria has been indicated to be a major risk factor for the development of tubulointerstitial damage through a process of proinflammatory molecule expression. Monocyte chemoattractant protein-1 (MCP-1) was shown to contribute to recruitment of immune cells into the renal interstitium in acute and chronic renal diseases. However, the molecular mechanisms by which proteinuria causes MCP-1 expression in proximal tubular cells have not been fully clarified. In this study, we examined whether albumin overload-induced MCP-1 expression was regulated by mitogen-activated protein kinase (MAPK) in mouse proximal tubular (mProx) cells. Exposure of mProx cells to delipidated bovine serum albumin (BSA) induced mRNA and protein expression of MCP-1 in a time- and dose-dependent manner. BSA activated extracellular signal-regulated kinase (ERK1/2) and p38 MAPK. The MEK inhibitor U-0126 partially suppressed BSA-induced MCP-1 expression and MCP-1 promoter/luciferase reporter activity. U-0126 also inhibited an increase in nuclear factor-κB and activator protein-1 DNA-binding activity of MCP-1 promoter by protein overload in mProx cells. In addition, we found that U-0126 inhibited BSA-induced nuclear factor-κB reporter activity and inhibitory protein degradation in mProx cells. In conclusion, these findings indicate that ERK signaling is involved in BSA-induced MCP-1 expression in mProx cells.
Mineralocorticoid regulation of cyclooxygenase-2 expression in rat renal medulla
Authors: Zhang Ming-Zhi , Hao Chuan-Ming , Breyer Matthew D. , Harris Raymond C. , McKanna James A.
Published: March 2015

The renal inner medulla and its distal one-third, the papilla, are major sites of prostanoid synthesis involved in water and electrolyte homeostasis. These sites contain variable levels of cyclooxygenase (COX)-2, a key prostaglandin synthase enzyme that is sensitive to adrenal steroids. Immunoreactive renal medullary COX-2, restricted to interstitial cells in control adult rats, shows a gradient of intense staining at the tip of the papilla that gradually diminishes to undetectable levels in the proximal inner medulla. We used adrenalectomy (ADX) and steroid replacement to investigate the effects of steroids on papillary COX-2. Immunoblots demonstrate that papillary COX-2 was reduced by one-half after 2 wk ADX; glucocorticoid replacement ameliorated the decline but not to control levels. Mineralocorticoid (deoxycorticosterone acetate; DOCA) replacement stimulated papillary COX-2 more than fivefold over control; both the intensity of immunostaining and the numbers of COX-2-positive cells in the inner medulla increased. Similar stimulation of papillary COX-2 resulted from DOCA treatment of normal control rats, but the response was blunted in rats fed a low-salt diet and absent in Brattleboro rats. DOCA treatment of mouse renal medullary interstitial cells in culture had no effect, but increased tonicity of the culture medium with NaCl caused strong upregulation of COX-2. Urea, a permeant molecule, had no effect. Together, these results suggest that mineralocorticoids lead to upregulation of COX-2 in rat renal medulla by indirect pathways, probably involving induced electrolyte hypertonicity in the interstitial fluid.
Proteolysis, the ubiquitin-proteasome system, and renal diseases
Authors: Debigaré Richard , Price S. Russ
Published: March 2015

Protein degradation is a critical process for the growth and function of cells. Proteolysis eliminates abnormal proteins, controls many cellular regulatory processes, and supplies amino acids for cellular remodeling. When substrates of proteolytic pathways are poorly recognized or there is mistiming of proteolysis, profound changes in cell function can occur. Based on these potential problems, it is not surprising that alterations in proteolytic enzymes/cofactors or in the structure of protein substrates that render them more or less susceptible to degradation are responsible for disorders associated with kidney cell malfunctions. Multiple pathways exist for protein degradation. The best-described proteolytic system is the ubiquitin-proteasome pathway, which requires ATP and degrades the bulk of cellular and some membrane proteins. This review will survey examples of renal abnormalities that are associated with defective protein degradation involving the ubiquitin-proteasome pathway. Loss of muscle mass associated with chronic renal failure, von Hippel-Lindau disease, Liddle syndrome, and ischemic acute renal failure will be discussed. These examples are indicative of the diverse roles of the ubiquitin-proteasome system in renal-associated pathological conditions.
Expression of urea transporters in the developing rat kidney
Authors: Kim Young-Hee , Kim Dong-Un , Han Ki-Hwan , Jung Ju-Young , Sands Jeff M. , Knepper Mark A. , Madsen Kirsten M. , Kim Jin
Published: March 2015

Urea transport in the kidney is mediated by a family of transporter proteins that includes renal urea transporters (UT-A) and erythrocyte urea transporters (UT-B). Because newborn rats are not capable of producing concentrated urine, we examined the time of expression and the distribution of UT-A and UT-B in the developing rat kidney by light and electron microscopic immunocytochemistry. Kidneys from 16-, 18-, and 20-day-old fetuses, 1-, 4-, 7-, 14-, and 21-day-old pups, and adult animals were studied. In the adult kidney, UT-A was expressed intensely in the inner medullary collecting duct (IMCD) and terminal portion of the short-loop descending thin limb (DTL) and weakly in long-loop DTL in the outer part of the inner medulla. UT-A immunoreactivity was not present in the fetal kidney but was observed in the IMCD and DTL in 1-day-old pups. The intensity of UT-A immunostaining in the IMCD gradually increased during postnatal development. In 4- and 7-day-old pups, UT-A immunoreactivity was present in the DTL at the border between the outer and inner medulla. In 14- and 21-day-old pups, strong UT-A immunostaining was observed in the terminal part of short-loop DTL in the outer medulla, and weak labeling remained in long-loop DTL descending into the outer part of the inner medulla. In the adult kidney, there was intense staining for UT-B in descending vasa recta (DVR) and weak labeling of glomeruli. In the developing kidney, UT-B was first observed in the DVR of a 20-day-old fetus. After birth there was a striking increase in the number of UT-B-positive DVR, in association with the formation of vascular bundles. The intensity of immunostaining remained strong in the outer medulla but gradually decreased in the inner medulla. We conclude that the expression of urea transporters in short-loop DTL and DVR coincides with the development of the ability to produce a concentrated urine.
Transfection of CYP4A1 cDNA increases vascular reactivity in renal interlobar arteries
Authors: Kaide Jun-Ichi , Wang Mong-Heng , Wang Ji-Shi , Zhang Fan , Gopal V.Raj , Falck John R. , Nasjletti Alberto , Laniado-Schwartzman Michal
Published: March 2015

20-HETE, a cytochrome P-450 4A (CYP4A1)-derived arachidonic acid metabolite, is a major eicosanoid formed in renal and extrarenal microcirculation. 20-HETE inhibits Ca2+-activated K+ channels in vascular smooth muscle cells and thereby may modulate vascular reactivity. We transfected renal interlobar arteries with an expression plasmid containing the cDNA of CYP4A1, the low- K m arachidonic acid ω-hydroxylase, and examined the consequences of increasing 20-HETE synthesis on constrictor responses to phenylephrine. CYP4A1-transfected interlobar arteries demonstrated a twofold increase in CYP4A protein levels and 20-HETE production compared with arteries transfected with the empty plasmid; they also showed increased sensitivity to phenylephrine, as evidenced by a decrease in EC50 from 0.37 ± 0.04 μM in plasmid-transfected arteries to 0.07 ± 0.01 μM in CYP4A1-transfected arteries. The increased sensitivity to phenylephrine was greatly attenuated by N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), a selective inhibitor of 20-HETE synthesis, and by 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid, a specific 20-HETE antagonist. This effect of DDMS was reversed by addition of 20-HETE, further substantiating the notion that increased levels of 20-HETE contribute to the increased sensitivity to phenylephrine in vessels overexpressing CYP4A1. These data suggest that 20-HETE of vascular origin sensitizes renal vascular smooth muscle to phenylephrine.