bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential expression and acid-base regulation of glutaminase mRNAs in gluconeogenic LLC-PK1-FBPase+cells
Authors: Gstraunthaler Gerhard , Holcomb Thomas , Feifel Elisabeth , Liu Wenlin , Spitaler Nikolaus , Curthoys Norman P.
Published: December 2017

LLC-PK1-FBPase+cells, which are a gluconeogenic substrain of porcine renal LLC-PK1cells, exhibit enhanced oxidative metabolism and increased levels of phosphate-dependent glutaminase (PDG) activity. On adaptation to acidic medium (pH 6.9, 9 mM [Formula: see text]), LLC-PK1-FBPase+cells also exhibit a greater increase in ammonia production and respond with an increase in assayable PDG activity. The changes in PDG mRNA levels were examined by using confluent cells grown on plastic dishes or on permeable membrane inserts. The latter condition increased the state of differentiation of the LLC-PK1-FBPase+cells. The levels of the primary porcine PDG mRNAs were analyzed by using probes that are specific for the 5.0-kb PDG mRNA (p2400) or that react equally with both the 4.5- and 5.0-kb PDG mRNAs (p930 and r1500). In confluent dish- and filter-grown LLC-PK1-FBPase+cells, the predominant 4.5-kb PDG mRNA is increased threefold after 18 h in acidic media. However, in filter-grown epithelia, which sustain an imposed pH and [Formula: see text] gradient, this adaptive increase is observed only when acidic medium is applied to both the apical and the basolateral sides of the epithelia. Half-life experiments established that induction of the 4.5-kb PDG mRNA was due to its stabilization. An identical pattern of adaptive increases was observed for the cytosolic PEPCK mRNA. In contrast, no adaptive changes were observed in the levels of the 5.0-kb PDG mRNA in either cell culture system. Furthermore, cultures were incubated in low-potassium (0.7 mM) media for 24–72 h to decrease intracellular pH while maintaining normal extracellular pH. LLC-PK1-FBPase+cells again responded with increased rates of ammonia production and increased levels of the 4.5-kb PDG and PEPCK mRNAs, suggesting that an intracellular acidosis is the initiator of this adaptive response. Because all of the observed responses closely mimic those characterized in vivo, the LLC-PK1-FBPase+cells represent a valuable tissue culture model to study the molecular mechanisms that regulate renal gene expression in response to changes in acid-base balance.
PTH regulates expression of ClC-5 chloride channel in the kidney
Authors: Silva Ian V. , Blaisdell Carol J. , Guggino Sandra E. , Guggino William B.
Published: December 2017

Mutations in the chloride channel, ClC-5, have been described in several inherited diseases that result in the formation of kidney stones. To determine whether ClC-5 is also involved in calcium homeostasis, we investigated whether ClC-5 mRNA and protein expression are modulated in rats deficient in 1α,25(OH)2vitamin D3with and without thyroparathyroidectomy. Parathyroid hormone (PTH) was replaced in some animals. Vitamin D-deficient, thyroparathyrodectomized rats had lower serum and higher urinary calcium concentrations compared with control animals as well as lower serum PTH and calcitonin concentrations. ClC-5 mRNA and protein levels in the cortex decrease in vitamin D-deficient, thyroparathyroidectomized rats compared with both control and vitamin D-deficient animals. ClC-5 mRNA and protein expression increase near to control levels in vitamin D-deficient, thyroparathyroidectomized rats injected with PTH. No significant changes in ClC-5 mRNA and protein expression in the medulla were detected in any experimental group. Our results suggest that PTH modulates the expression of ClC-5 in the kidney cortex and that neither 1α,25(OH)2vitamin D3nor PTH regulates ClC-5 expression in the medulla. The pattern of expression of ClC-5 varies with urinary calcium. Animals with higher urinary calcium concentrations have lower levels of ClC-5 mRNA and protein expression, suggesting that the ClC-5 chloride channel plays a role in calcium reabsorption.
Decreased vasopressin-mediated renal water reabsorption in rats with chronic aldosterone-receptor blockade
Authors: Jonassen Thomas E. N. , Promeneur Dominique , Christensen Sten , Petersen Jørgen S. , Nielsen Søren
Published: December 2017

Previous studies have suggested that mineralocorticoids are needed for a normal action of vasopressin on collecting duct osmotic water permeability. However, the mechanisms behind this are unknown. To investigate if aldosterone-receptor blockade influences vasopressin type 2 receptor (V2)-mediated renal water reabsorption and the renal expression of the vasopressin-regulated water channel aquaporin-2 (AQP2), rats were treated with the aldosterone-receptor antagonist canrenoate (20 mg/day iv) for 4 wk. Daily urine flow was increased significantly by 44%, and urine osmolality was decreased by 27% in canrenoate-treated rats. Acute V2-receptor blockade (OPC-31260, 800 μg ⋅ kg−1⋅ h−1) was performed under conditions in which volume depletion was prevented. In control rats, OPC-31260 induced a significant increase in urine flow rate (V, +25%) and free water clearance ([Formula: see text], −29%). In canrenoate-treated rats, the effect of OPC-31260 was significantly reduced, and semiquantiative immunoblotting demonstrated a significant reduction (45%) in AQP2 expression. Because rats with common bile duct ligation (CBL) have a reduced vasopressin-mediated water reabsorption compared with normal rats (V: −24%;[Formula: see text]: −28%, and 86% downregulation of AQP2), the effect of canrenoate combined with OPC-31260 was tested. Canrenoate treatment of CBL rats significantly increased daily urine flow, decreased urine osmolality, and impaired the aquaretic response to OPC-31260 (V: −23%;[Formula: see text]: −31%) with maintained suppression of the renal AQP2 expression. Thus canrenoate treatment of normal and CBL rats showed 1) increased urine production, 2) reduced aquaretic effect of acute V2-receptor blockade, and 3) a marked reduction in AQP2 expression. This strongly supports the view that aldosterone plays a significant role for vasopressin-mediated water reabsorption.
Limit-cycle oscillations and tubuloglomerular feedback regulation of distal sodium delivery
Authors: Layton H. E. , Pitman E. Bruce , Moore Leon C.
Published: December 2017

A mathematical model was used to evaluate the potential effects of limit-cycle oscillations (LCO) on tubuloglomerular feedback (TGF) regulation of fluid and sodium delivery to the distal tubule. In accordance with linear systems theory, simulations of steady-state responses to infinitesimal perturbations in single-nephron glomerular filtration rate (SNGFR) show that TGF regulatory ability (assessed as TGF compensation) increases with TGF gain magnitude γ when γ is less than the critical value γc, the value at which LCO emerge in tubular fluid flow and NaCl concentration at the macula densa. When γ > γc and LCO are present, TGF compensation is reduced for both infinitesimal and finite perturbations in SNGFR, relative to the compensation that could be achieved in the absence of LCO. Maximal TGF compensation occurs when γ ≈ γc. Even in the absence of perturbations, LCO increase time-averaged sodium delivery to the distal tubule, while fluid delivery is little changed. These effects of LCO are consequences of nonlinear elements in the TGF system. Because increased distal sodium delivery may increase the rate of sodium excretion, these simulations suggest that LCO enhance sodium excretion.
Superoxide anion curbs nitric oxide modulation of afferent arteriolar ANG II responsiveness in diabetes mellitus
Authors: Schoonmaker Gwynn C. , Fallet Rachel W. , Carmines Pamela K.
Published: December 2017

Experiments were performed to test the hypothesis that the impact of endogenous nitric oxide (NO) on ANG II-induced renal arteriolar constriction is reduced in rats with insulin-dependent diabetes mellitus (65 mg/kg streptozotocin; STZ). Arteriolar diameter responses to exogenous ANG II were quantified before and during NO synthase inhibition (100 μM Nω-nitro-l-arginine;l-NNA) by using the in vitro blood-perfused juxtamedullary nephron technique. Afferent arteriolar lumen diameter averaged 20.7 ± 2.0 μm in Sham kidneys and 25.9 ± 1.3 μm in STZ kidneys ( P < 0.05). Efferent arteriolar diameter did not differ between Sham and STZ rats. In kidneys from Sham rats, afferent and efferent arteriolar responses to ANG II (0.1–10.0 nM) were exaggerated significantly by l-NNA. l-NNA also augmented efferent arteriolar ANG II responses in kidneys from STZ rats (high-glucose bath) but did not alter ANG II responses in afferent arterioles from STZ rats. l-NNA also accentuated efferent, but not afferent, arteriolar ANG II responses in STZ kidneys during acute restoration of bath glucose to normal levels. Superoxide dismutase (150 U/ml) restored the ability of l-NNA to allow exaggerated afferent arteriolar responses to ANG II in kidneys from STZ rats. These observations indicate that superoxide anion suppresses the modulatory influence of endogenous NO on ANG II-induced afferent arteriolar constriction in diabetes mellitus.
Aquaporin-4 is expressed in basolateral membranes of proximal tubule S3 segments in mouse kidney
Authors: van Hoek Alfred N. , Ma Tonghui , Yang Baoxue , Verkman A. S. , Brown Dennis
Published: December 2017

Because of the availability of knockout mouse models to examine renal transport mechanisms, it has become increasingly important to describe the cellular distribution of major renal transporters in mice. We have used immunocytochemistry and freeze-fracture electron microscopy to compare the renal distribution of aquaporin-4 (AQP4) with that previously described in rat. In rat kidney AQP4 is present exclusively in basolateral membranes of collecting duct principal cells. In mice, however, AQP4 was also detected by immunocytochemistry in basolateral membranes of proximal tubule S3 segments, and not detected in S1 and S2 segments of proximal tubule. Freeze-fracture electron microscopy revealed orthogonal arrays of intramembrane particles (OAPs) on the basolateral membranes of the S3 segment. In AQP4-knockout mice, immunostaining was absent and OAPs were found neither in collecting ducts nor in the S3 segment of the proximal tubule. The urinary concentrating capacity after deletion of both AQP1 and AQP4 was further reduced compared with that of AQP1 or AQP4 null mice, suggesting an additive effect of AQP1 and AQP4 in the concentrating mechanism. The functional significance of the apparent species-dependent expression of AQP4 in proximal tubules is unknown, but may relate to physiological differences between rats and mice.
Prostaglandin E2interaction with AVP: effects on AQP2 phosphorylation and distribution
Authors: Zelenina Marina , Christensen Birgitte Mønster , Palmér Johan , Nairn Angus C. , Nielsen Søren , Aperia Anita
Published: December 2017

Prostaglandin E2(PGE2) antagonizes the action of arginine vasopressin (AVP) on collecting duct water permeability. To investigate the mechanism of this antagonism, rat renal inner medulla (IM) was incubated with the two hormones, and the phosphorylation and subcellular distribution of the water channel, aquaporin-2 (AQP2) were studied. Using a phosphorylation state-specific AQP2 antibody, we demonstrated that AVP stimulates AQP2 phosphorylation at the Ser256protein kinase A consensus site in a time- and dose-dependent manner. In parallel studies using a differential centrifugation technique, we demonstrated that AVP induced translocation of AQP2 from an intracellular vesicle-enriched fraction to a plasma membrane-enriched fraction. PGE2(10−7M) added after AVP (10−8M) did not decrease AQP2 phosphorylation but reversed AVP-induced translocation of AQP2 to the plasma membrane. Preincubation of IM with PGE2did not prevent the effects of AVP on AQP2 phosphorylation and trafficking. PGE2alone did not influence AQP2 phosphorylation and subcellular distribution. Our data indicate that 1) recruitment of AQP2 to the plasma membrane and its retrieval to a pool of intracellular vesicles may be regulated independently, 2) PGE2may counteract AVP action by activation of AQP2 retrieval, 3) dephosphorylation of AQP2 is not a prerequisite for its internalization.
Mutations in sixth transmembrane domain of AQP2 inhibit its translocation induced by vasopression
Authors: Yamashita Yumi , Hirai Keiji , Katayama Yoshifumi , Fushimi Kiyohide , Sasaki Sei , Marumo Fumiaki
Published: December 2017

Vasopression-induced phosphorylation of serine 256 of the aquaporin-2 (AQP2) water channel triggers translocation of the protein from cystolic reservoir vesicles to the apical membrane of collecting duct principal cells. Dileucine motifs are located in the sixth transmembrane domain (6TM) of AQP2 and are known as the signal sequence for internalization, sorting from the trans-Golgi network to endosomes/lysosomes, and basolateral sorting. In this study, involvement of 6TM in vasopressin-induced translocation of the protein was investigated. A series of mutations in 6TM of AQP2 was introduced to rat cDNA and expressed in LLC-PK1cells. Immunofluorescence microscopy indicated that the mutant AQP2 proteins were retained in the cytoplasm after vasopressin stimulation, which actually promoted the plasma membrane expression of wild-type protein. Immunoelectron microscopy showed that the mutant AQP2 proteins reached the endosomes but did not reach the plasma membrane. These results demonstrate that 6TM has essential domains for vasopressin-induced translocation from endosomes to the plasma membrane.
Role of AP2 consensus sites in regulation of rat Npt2 (sodium-phosphate cotransporter) promoter
Authors: Shachaf C. , Skorecki K. L. , Tzukerman M.
Published: December 2017

Expression of the Npt2 gene, encoding the type II sodium-dependent phosphate cotransporter, is restricted to renal proximal tubule epithelium. We have isolated a 4,740-bp fragment of the 5′-flanking sequence of the rat Npt2 gene, identified the transcription initiation site, and demonstrated that this 5′-flanking sequence drives luciferase-reporter gene expression, following transfection in the proximal tubule cell-derived opossum kidney (OK) cell line but not in unrelated cell lines. Analysis of the promoter sequence revealed the presence of 10 consensus binding motifs for the AP2 transcription factor. Transient transfection assays revealed an important effect of the number of tandemly repeated AP2 sites in enhancing promoter activity. The promoter sequence also revealed a pair of inverted repeats enclosing 1,324 bp of intervening sequence and containing 8 of the total 10 AP2 consensus sites in the promoter sequence. Deletion or reversal of orientation of the distal inverted repeat resulted in marked enhancement of promoter activity. Electrophoretic mobility shift analysis revealed a distinct pattern of transcription factor binding to oligonucleotides containing AP2 sites, using nuclear extracts from OK cells, compared with unrelated cell lines. Taken together, these results suggest an important role for AP2 consensus binding sites in regulating Npt2 gene expression and suggest a mechanism of regulation mediated by the interaction of inverted repeats enclosing these sites.
Enhanced renal function in bradykinin B2 receptor transgenic mice
Authors: Wang Danzhao , Yoshida Hideaki , Song Qing , Chao Lee , Chao Julie
Published: December 2017

The tissue kallikrein-kinin system has been recognized as a paracrine and/or autocrine hormonal system that regulates arterial pressure, renal hemodynamics, and electrolyte excretion. We have created a transgenic mouse model overexpressing human bradykinin B2receptor, and the mice developed lifetime hypotension. With this animal model, we further analyzed the potential role of B2receptors in regulation of renal function. Baseline urinary excretion, urinary potassium excretion, and pH were significantly increased in transgenic mice, whereas urinary sodium excretion and serum sodium concentration were unaltered. Transgenic mice exhibited increased renal blood flow, glomerular filtration rate, and urine flow. Enhanced renal function was accompanied by significant increases in urinary nitrate/nitrite, cGMP, and cAMP levels with unaltered urinary kinin levels in transgenic mice compared with control siblings. Renal cGMP and cAMP content was also significantly increased in transgenic mice. Because the renin-angiotensin system exerts vasoconstriction buffering vasodilation of the kallikrein-kinin system, expression of renin-angiotensin components was examined by Northern blot analysis. We found a significant increase in hepatic angiotensinogen expression with no changes in renal renin and pulmonary angiotensin-converting enzyme mRNA levels in B2 receptor transgenic mice. These studies showed that overexpression of B2 receptors in transgenic mice resulted in hypotension and enhanced renal function through activation of nitric oxide-cGMP and cAMP signal transduction pathways.
Citrate and succinate transport in proximal tubule cells
Authors: Hering-Smith Kathleen S. , Gambala Cecilia T. , Hamm L. Lee
Published: December 2017

Urinary citrate, which inhibits calcium nephrolithiasis, is determined by proximal reabsorption via an apical dicarboxylate transporter. Citrate is predominantly trivalent at physiological pH, but citrate− 2 is transported at the apical membrane. We now demonstrate that low-Ca solutions induce transport of citrate− 2 and succinate in opossum kidney cells. With 1.2 mM extracellular Ca, citrate uptake was pH insensitive and not competed by succinate− 2. In contrast, with low extracellular Ca, citrate uptake increased twofold, was inhibited by succinate (and other dicarboxylates), was stimulated by lowering extracellular pH (consistent with citrate− 2 transport), and increased further by lowering extracellular Mg. The effect of Ca was incrementally concentration dependent, between 0 and 1.2 mM. The effect of Ca was not simply complexation with citrate because succinate (which is complexed significantly less) was affected by Ca similarly. Incubation of cells for 48 h in a low-pH media increased citrate transport (studied at control pH) more than twofold, suggesting induction of transporters.
Variable imprinting of the heterotrimeric G protein Gsα-subunit within different segments of the nephron
Authors: Weinstein Lee S. , Yu Shuhua , Ecelbarger Carolyn A.
Published: December 2017

The heterotrimeric G protein Gsis required for hormone-stimulated intracellular cAMP generation because it couples hormone receptors to the enzyme adenylyl cyclase. Hormones that activate Gsin the kidney include parathyroid hormone, glucagon, calcitonin, and vasopressin. Recently, it has been demonstrated that the Gsα gene is imprinted in a tissue-specific manner, leading to preferential expression of Gsα from the maternal allele in some tissues. In the kidney, Gsα is imprinted in the proximal tubule but not in more distal nephron segments, such as the thick ascending limb or collecting duct. This most likely explains why in both humans and mice heterozygous mutations in the maternal allele lead to parathyroid hormone resistance in the proximal tubule whereas mutations in the paternal allele do not. In contrast, heterozygous mutations have little effect on vasopressin action in the collecting ducts. In mice with heterozygous null Gsα mutations (both those with mutations on the maternal or paternal allele), expression of the Na-K-2Cl cotransporter was decreased in the thick ascending limb, suggesting that its expression is regulated by cAMP. The Gsα genes also generate alternative, oppositely imprinted transcripts encoding XLαs, a Gsα isoform with a long NH2-terminal extension, and NESP55, a chromogranin-like neurosecretory protein. The role, if any, of these proteins in renal physiology is unknown.
IP3, IP3receptor, and cellular senescence
Authors: Huang Ming-Shyan , Adebanjo Olugbenga A. , Awumey Emmanuel , Biswas Gopa , Koval Antoliy , Sodam Bali R. , Sun Li , Moonga Baljit S. , Epstein Joshua , Goldstein Samuel , Lai F. Anthony , Lipschitz David , Zaidi Mone
Published: December 2017

Herein we demonstrate that replicative cellular senescence in vitro results in sharply reduced inositol 1,4,5-trisphosphate (IP3) receptor levels, reduced mitogen-evoked IP3formation and Ca2+release, and Ca2+store depletion. Human diploid fibroblasts (HDFs) underwent either 30 mean population doublings [mean population doublings (MPDs) thymidine labeling index (TI) >92% (“young”) or between 53 and 58 MPDs (TI < 28%; “senescent”)]. We found that the cytosolic Ca2+release triggered by either ionomycin or by several IP3-generating mitogens, namely bradykinin, thrombin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), was attenuated markedly in senescent HDFs. Notably, the triggered cytosolic Ca2+transients were of a smaller magnitude in senescent HDFs. However, the response latency seen with both PDGF and EGF was greater for senescent cells. Finally, a smaller proportion of senescent HDFs showed oscillations. In parallel, IP3formation in response to bradykinin or EGF was also attenuated in senescent HDFs. Furthermore, senescent HDFs displayed a sharply diminished Ca2+release response to intracellularly applied IP3. Finally, to compare IP3receptor protein levels directly in young and senescent HDFs, their microsomal membranes were probed in Western blots with a highly specific anti-IP3receptor antiserum, Ab40. A ∼260-kDa band corresponding to the IP3receptor protein was noted; its intensity was reduced by ∼50% in senescent cells. Thus, we suggest that reduced IP3receptor expression, lowered IP3formation, and Ca2+release, as well as Ca2+store depletion, all contribute to the deficient Ca2+signaling seen in HDFs undergoing replicative senescence.
Renal angiotensin II receptors and protein kinase C in diabetic rats: effects of insulin and ACE inhibition
Authors: Amiri Farhad , Garcia Raul
Published: December 2017

It has been shown that glomerular ANG II receptors are downregulated and protein kinase C (PKC) activity is enhanced in diabetes mellitus. Therefore, we investigated glomerular and preglomerular vascular ANG II receptors and PKC isoform regulation in streptozotocin (STZ)-diabetic rats treated with insulin and/or captopril. Diabetic rats were prepared by injecting STZ (60 mg/kg). Those that developed diabetes after 48 h were treated with low or high doses of insulin, or with a low dose of insulin as well as captopril, and killed 14 days later. Their glomeruli and preglomerular vessels were purified, competitive binding studies were performed by using the ANG II antagonists losartan and PD-123319, and PKC analysis was carried out by Western blotting. Competitive binding studies showed that the AT1receptor was the only ANG II receptor detected on both glomeruli and preglomerular vessels of all groups. Preglomerular vascular AT1receptor density (Bmax) was significantly upregulated in low insulin-treated STZ rats, whereas glomerular AT1Bmaxwas downregulated. Furthermore, both the captopril- and high insulin-treated groups had less glomerulosclerosis and vascular damage than the low insulin-treated group. PKCα, PKCδ, PKCε, and PKCμ isoforms found in preglomerular vessels were upregulated by captopril and high insulin doses, respectively, whereas no such regulation occurred in glomeruli. We conclude that in STZ-diabetic rats ANG II receptors and PKC isoforms on preglomerular vessels and glomeruli are differentially regulated by treatment with insulin and/or captopril.
Heme oxygenase-1 gene ablation or expression modulates cisplatin-induced renal tubular apoptosis
Authors: Shiraishi Fumie , Curtis Lisa M. , Truong Leigh , Poss Kenneth , Visner Gary A. , Madsen Kirsten , Nick Harry S. , Agarwal Anupam
Published: December 2017

Heme oxygenase-1 (HO-1) is a 32-kDa microsomal enzyme that catalyzes the conversion of heme to biliverdin, releasing iron and carbon monoxide. Induction of HO-1 occurs as a protective response in cells/tissues exposed to a wide variety of oxidant stimuli. The chemotherapeutic effects of cis-diamminedichloroplatinum(II) (cisplatin), a commonly used anticancer drug, are limited by significant nephrotoxicity, which is characterized by varying degrees of renal tubular apoptosis and necrosis. The purpose of this study was to evaluate the functional significance of HO-1 expression in cisplatin-induced renal injury. Our studies demonstrate that transgenic mice deficient in HO-1 (−/−), develop more severe renal failure and have significantly greater renal injury compared with wild-type (+/+) mice treated with cisplatin. In vitro studies in human renal proximal tubule cells demonstrate that hemin, an inducer of HO-1, significantly attenuated cisplatin-induced apoptosis and necrosis, whereas inhibition of HO-1 enzyme activity reversed the cytoprotective effect. Overexpression of HO-1 resulted in a significant reduction in cisplatin-induced cytotoxicity. These studies provide a basis for future studies using targeted gene expression of HO-1 as a therapeutic and preventive modality in high-risk settings of acute renal failure.
Similarities and differences in the subcellular localization of hamartin and tuberin in the kidney
Authors: Murthy Vanishree , Haddad Luciana A. , Smith Nicole , Pinney Denise , Tyszkowski Robert , Brown Dennis , Ramesh Vijaya
Published: December 2017

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomas in multiple organs, notably the brain and kidneys. The disease is caused by mutations in TSC1or TSC2 genes, coding hamartin and tuberin, respectively. Immunofluorescence analysis of tuberin and hamartin performed here demonstrates that both proteins are specifically expressed in the distal urinary tubule, comprising the distal tubules, connecting segment, and collecting ducts. Hamartin, distinct from tuberin, is expressed in the thick ascending limbs of Henle and in juxtaglomerular cells, where it colocalizes with renin. In positive epithelial cells, tuberin localizes to the cytoplasm as well as the apical membrane. Hamartin, however, preferentially localizes to the apical membrane. The two proteins colocalize at the apical membrane of type A intercalated cells and connecting tubule cells, whereas in type B intercalated cells they reveal a variable pattern of expression. The cell-specific expression of tuberin and hamartin described here will provide critical insight into the cell types that give rise to kidney lesions, and the tumor suppressor role of these proteins in TSC.
Chlorpropamide upregulates antidiuretic hormone receptors and unmasks constitutive receptor signaling
Authors: Durr Jacques A. , Hensen Johannes , Ehnis Tobias , Blankenship Mary S. ,   (With the Technical Assistance of C. Klein)
Published: December 2017

The mechanism by which chlorpropamide (CP) treatment promotes antidiuresis is unknown. CP competitively inhibited antidiuretic hormone (ADH) binding and adenylyl cyclase (AC) stimulation (inhibition constants Kiand K′iof 2.8 mM and 250 μM, respectively) in the LLC-PK1cell line. CP (333 μM) increased the apparent Kaof ADH for AC activation (0.31 vs. 0.08 nM) without affecting a maximal response, suggesting competitive antagonism. Because CP lowers “basal” AC activity and the AC activation-ADH receptor occupancy relationship (A-O plots), it is an ADH inverse agonist. Twenty-four-hour CP exposure (100 μM) upregulated the ADH receptors without affecting affinity. This lowered Kaand increased basal AC activity and maximal response (1.86 vs. 1.35 and 14.9 vs. 10.6 fmol cAMP ⋅ min−1⋅ 103cells−1, n = 6, P < 0.05). NaCl, which potentiates ADH stimulation, also increased basal AC activity. This, together with the CP-ADH inverse agonism and increased basal AC activity at higher receptor density, unmasks constitutive receptor signaling. The CP-ADH inverse agonism explains receptor upregulation and predicts the need for residual ADH with functional isoreceptors for CP-mediated antidiuresis. This could be why CP ameliorates partial central diabetes insipidus but not nephrogenic diabetes insipidus.
V1 receptors in luminal action of vasopressin on distal K+ secretion
Authors: Amorim José B. O. , Malnic Gerhard
Published: December 2017

Luminal perfusion with collected proximal fluid increases distal K+ secretion compared with artificial solutions. Arginine vasopressin (AVP), present in luminal fluid, might be responsible for this observation. K+ secretion rate ( J K) was measured by K+-sensitive microelectrodes during paired luminal stationary microperfusion with control and AVP-containing 0.5 mM K+ solutions. J K was 1.34 ± 0.35 ( n = 24 tubules) nmol ⋅ cm− 2 ⋅ s− 1during perfusion with 10− 9 M AVP, against 0.90 ± 0.12 nmol ⋅ cm−2 ⋅ s−1( n = 21) in control ( P < 0.02). With 10− 9 M AVP+10− 6 M β-mercapto-β-β-cyclopenta-methylenepropionyl1, O-Me-Tyr2-Arg8 vasopressin (MCMV), a specific peptide V1-receptor antagonist, J K was 0.36 ± 0.067 against 0.77 ± 0.10 (control; n = 9) nmol ⋅ cm− 2 ⋅ s− 1( P < 0.01). With 10− 6 M MCMV alone, J K was 0.37 ± 0.04 against a control of 0.62 ± 0.06 ( n = 19) nmol ⋅ cm− 2 ⋅ s− 1( P < 0.01). A peptide V2 antagonist had no such effect. In Brattleboro rats, which do not produce endogenous AVP, MCMV had no effect when given alone, although AVP still stimulated J K. In conclusion, luminal AVP stimulates distal J K significantly. The V1 antagonist MCMV inhibits the effect of AVP but also reduces J Kwhen given alone. This suggests that AVP acts luminally via V1 receptors but also that there appears to be a background effect of endogenous AVP blocked by the antagonist.
Bidirectional regulation of tonicity-responsive enhancer binding protein in response to changes in tonicity
Authors: Woo Seung Kyoon , Dahl Stephen C. , Handler Joseph S. , Kwon H. Moo
Published: December 2017

Tonicity-responsive enhancer binding protein (TonEBP) regulates transcription of tonicity responsive genes such as the sodium- myo-inositol cotransporter (SMIT), the sodium-chloride-betaine cotransporter (BGT1), and aldose reductase (AR). To characterize signals that activate TonEBP in Madin-Darby canine kidney (MDCK) cells, the abundance and nuclear distribution of TonEBP were studied after the osmolality of the culture medium was changed. Hypertonicity but not hyperosmolality is effective in activation of TonEBP as expected. Surprisingly, exposure to hypotonic medium leads to a dramatic downregulation of TonEBP both in abundance and nuclear distribution, indicating that under isotonic conditions, TonEBP is at a low-level activated state and can respond to both increase and decrease in tonicity. Additional experiments suggest that cellular ionic strength is the signal that initiates regulation of TonEBP. The increase in abundance of TonEBP is mediated by an increase in mRNA abundance and a parallel increase in synthesis of TonEBP. The stability of TonEBP mRNA is not affected by hypertonicity indicating that transcription plays a major role in the induction of TonEBP by hypertonicity.
Inhibition of type I and III IP3Rs by TGF-β is associated with impaired calcium release in mesangial cells
Authors: Sharma Kumar , Mc Gowan Tracy A. , Wang Lewei , Madesh Muniswamy , Kaspar Vince , Szalai Gabor , Thomas Andrew P. , Hajnóczky György
Published: December 2017

Inositol 1,4,5-trisphosphate receptors (IP3Rs) mediate cytosolic free calcium concentration ([Ca2+]c) signals in response to a variety of agonists that stimulate mesangial cell contraction and proliferation. In the present study, we demonstrate that mesangial cells express both type I and III IP3Rs and that these receptors occupy different cellular locations. Chronic treatment with transforming growth factor-β1 (TGF-β1; 10 ng/ml, 24 h) leads to downregulation of both type I and III IP3Rs as measured by immunoblot and confocal analysis. TGF-β1 treatment does not affect IP3levels, and downregulation of type I IP3R is not due to enhanced degradation of the protein, as the half-life of type I IP3R is unchanged in the presence or absence of TGF-β1. Functional effects of TGF-β1-induced downregulation of the IP3Rs were evaluated by measuring [Ca2+]cchanges in response to epidermal growth factor (EGF) in intact cells and sensitivity of [Ca2+]crelease to IP3in permeabilized cells. TGF-β1 pretreatment led to a significant decrease of [Ca2+]crelease induced by EGF in intact cells and by submaximal IP3(400 nm) in permeabilized cells. Total IP3-sensitive [Ca2+]cstores were not changed, as assessed by stimulation with maximal doses of IP3(10.5 μm) and thapsigargin-mediated calcium release in permeabilized cells. We conclude that prolonged exposure to TGF-β1 leads to downregulation of both type I and III IP3Rs in mesangial cells and this is associated with impaired sensitivity to IP3.
Amino acid transport in podocytes
Authors: Gloy Joachim , Reitinger Steffen , Fischer Karl-Georg , Schreiber Rainer , Boucherot Anissa , Kunzelmann Karl , Mundel Peter , Pavenstädt Hermann
Published: December 2017

It has recently been shown that formation of podocyte foot processes is dependent on a constant source of lipids and proteins (Simons M, Saffrich R, Reiser J, and Mundel P. J Am Soc Nephrol 10: 1633–1639, 1999). Here we characterize amino acid transport mechanisms in differentiated cultured podocytes and investigate whether it may be disturbed during podocyte injury. RT-PCR studies detected mRNA for transporters of neutral amino acids (ASCT1, ASCT2, and B0/+), cationic AA (CAT1 and CAT3), and anionic AA (EAAT2 and EAAT3). Alanine (Ala), asparagine, cysteine (Cys), glutamine (Gln), glycine (Gly), leucine (Leu), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), glutamic acid (Glu), arginine (Arg), and histidine (His) depolarized podocytes and increased their whole cell conductances. Depletion of extracellular Na+completely inhibited the depolarization induced by Ala, Gln, Glu, Gly, Leu, and Pro and decreased the depolarization induced by Arg and His, indicating the presence of Na+-dependent amino acid transport. Incubation of podocytes with 100 μg/ml puromycin aminonucleoside for 24 h significantly attenuated the effects induced by the various amino acids by ∼70%. The data indicate the existence of different amino acid transporter systems in podocytes. Alteration of amino acid transport may participate in podocyte injury and disturbed foot process formation.
Increased renal medullary endothelin-1 synthesis in prehypertensive DOCA- and salt-treated rats
Authors: Hsieh Tusty-Jiuan , Lin Shiu-Ru , Lee Yau-Jiunn , Shin Shyi-Jang , Lai Yung-Hsiung , Hsu Chin-Hsun , Tsai Juei-Hsiung
Published: December 2017

To investigate the role of renal endothelin-1 (ET-1) synthesis in water-sodium homeostasis, we measured mRNA expressions, protein levels, enzyme activity, and receptor binding of the renal ET-1 system in a DOCA- and salt-treated rat model. Male Wistar rats were divided into control and DOCA- and salt-treated (DOCA-Salt) groups. The DOCA-Salt group received 25 mg/kg body wt DOCA and was maintained on 1% NaCl drinking water. Rats were killed on days 1, 2, 4, and 10 of the experiment. Urinary ET-1-like immunoreactivity significantly increased from the second day in the DOCA-Salt group and correlated well with the urinary sodium excretion rate ( r = 0.81, P < 0.001). Renal endothelin-converting enzyme (ECE) activity, ET-1, and ECE-1 mRNA expressions were significantly increased in the renal medullary area of DOCA-Salt rats. In situ hybridization and immunohistochemical studies showed that the increase in ET-1 synthesis was mainly localized in the inner medullary collecting ducts. The maximum binding of endothelin B receptor also increased from the second day in the renal medulla of the DOCA-Salt group. Our results suggest that renal medullary synthesized ET-1 may be a natriuretic factor and may participate in the intrarenal regulation of water and salt homeostasis in prehypertensive DOCA-and salt-treated rats.
Cloning of mesangial cell Na+/Ca2+exchangers from Dahl/Rapp salt-sensitive/resistant rats
Authors: Unlap M. Tino , Peti-Peterdi Janos , Bell P. Darwin
Published: December 2017

The Dahl/Rapp rat model of hypertension is characterized by a marked increase in blood pressure and a progressive fall in glomerular filtration rate when salt-sensitive (S) rats are placed on an 8% NaCl diet. On the same diet, the salt-resistant (R) rat does not exhibit these changes. In previous studies we found that protein kinase C (PKC) upregulates Na+/Ca2+exchanger activity in afferent arterioles and mesangial cells from R but not S rats. One possible reason for the difference in PKC sensitivity may be due to differences in the S and R Na+/Ca2+exchanger protein. We now report the cloning of Na+/Ca2+exchangers from R (RNCX1) and S (SNCX1) mesangial cells. At the amino acid level, SNCX1 differs from RNCX1 at position 218 in the NH2-terminal domain where it is isoleucine in RNCX1 but phenylalanine in SNCX1. These two exchangers also differ by 23 amino acids at the alternative splice site within the cytosolic domain. RNCX1 and SNCX1 were expressed in OK-PTH cells and45Ca2+-uptake studies were performed. Acute phorbol 12-myristate 13-acetate (PMA) treatment (300 nM, 20 min) upregulated exchanger activity in cells expressing RNCX1 but failed to stimulate exchanger activity in SNCX1 expressing cells. Upregulation of RNCX1 could be prevented by prior 24-h pretreatment with PMA, which downregulates PKC. These results demonstrate a difference in PKC-Na+/Ca2+exchange activity between the isoform of the exchanger cloned from the R vs. the S rat. Lack of PKC activation of SNCX1 may contribute to a dysregulation of intracellular Ca2+concentration and enhanced renal vasoreactivity in this model of hypertension.
Cellular localization of type 5 and type 6 ACs in collecting duct and regulation of cAMP synthesis
Authors: Héliès-Toussaint Cécile , Aarab Lotfi , Gasc Jean-Marie , Verbavatz Jean-Marc , Chabardès Danielle
Published: December 2017

The cellular distribution of Ca2+-inhibitable adenylyl cyclase (AC) type 5 and type 6 mRNAs in rat outer medullary collecting duct (OMCD) was performed by in situ hybridization. Kidney sections were also stained with specific antibodies against either collecting duct intercalated cells or principal cells. The localization of type 5 AC in H+-ATPase-, but not aquaporin-3-, positive cells demonstrated that type 5 AC mRNA is expressed only in intercalated cells. In contrast, type 6 AC mRNA was observed in both intercalated and principal cells. In microdissected OMCDs, the simultaneous superfusion of carbachol and PGE2elicited an additive increase in the intracellular Ca2+concentration, suggesting that the Ca2+-dependent regulation of these agents occurs in different cell types. Glucagon-dependent cAMP synthesis was inhibited by both a pertussis toxin-sensitive PGE2pathway (63.7 ± 4.6% inhibition, n = 5) and a Ca2+-dependent carbachol pathway (48.6 ± 3.3%, n = 5). The simultaneous addition of both agents induced a cumulative inhibition of glucagon-dependent cAMP synthesis (78.2 ± 3.3%, n = 5). The results demonstrate a distinct cellular localization of type 5 and type 6 AC mRNAs in OMCD and the functional expression of these Ca2+-inhibitable enzymes in intercalated cells.
Metanephrogenic mesenchyme-to-epithelium transition induces profound expression changes of ion channels
Authors: Huber Stephan M. , Braun Gerald S. , Segerer Stephan , Veh Rüdiger W. , Horster Michael F.
Published: December 2017

The expression patterns of plasma membrane transporters that specify the epithelial cell type are acquired with ontogeny. To study this process during metanephrogenic mesenchyme-to-epithelium transition, branching ureteric buds with their adjacent mesenchymal blastema (mouse embryonic day E14) were dissected and explanted on a collagen matrix. In culture, induced mesenchymal cells condensed, aggregated, and converted to the comma- and S-shaped body. During in vitro condensation and aggregation, transcription factor Pax-2 protein was downregulated while the epithelial markers E-cadherin and β-catenin proteins were upregulated. In addition, Wilms' tumor suppressor protein WT-1 was detectable upon condensation and downregulated in the S stage, where expression persisted in the long arm of the S. Patch-clamp, whole cell conductance ( G, in nS/10 pF) of pre-epithelial condensed mesenchymal cells ( n = 7) was compared with that of tubular proximal S-shaped-body epithelium ( n = 6). Both stages expressed E-cadherin and WT-1 mRNA, as demonstrated by single-cell RT-PCR, testifying further to the epithelial as well as the nephrogenic commitment of the recorded cells. Mesenchymal cells exhibited whole cell currents ( G = 6.7 ± 1.3) with reversal potentials ( Vrev, in mV) near equilibrium potential for Cl−( ECl) ( Vrev= −40 ± 7) suggestive of a high fractional Cl−conductance. Currents of the S-shaped-body cells ( G = 4.0 ± 1.1), in sharp contrast, had a Vrevat EK( Vrev= −82 ± 6) indicating a high fractional K+conductance. Further, analysis of K+-selective whole cell tail currents and single-channel recording revealed a change in K+channel expression. Also, Kir6.1 K+channel mRNA and protein were downregulated between both stages, whereas KvLQT K+channel mRNA was abundant throughout. In conclusion, metanephrogenic mesenchyme-to-epithelium transition is accompanied by a profound reorganization of plasma membrane ion channel conductance.
Modeling exchange of plasma proteins between microcirculation and interstitium of the renal medulla
Authors: Wang W. , Michel C. C.
Published: December 2017

In the absence of evidence for lymphatics in the inner medulla of the kidney, it has been proposed that plasma proteins are cleared by convection out of the medullary interstitial fluid (ISF) directly into the ascending vasa recta (AVR). To clarify this hypothesis we have developed a mathematical model of the microvascular exchange of fluid, plasma proteins, and small solutes among the descending vasa recta (DVR), the AVR, and the ISF. The model represents the DVR and AVR as limbs of a countercurrent exchange loop separated and surrounded by the ISF. Steady-state exchange of fluid and solute are considered by using conservation and exchange equations. We have used values for parameters based on experimental measurements and investigated the effects of the properties of the vasa recta, the flow, and the gradient of small solutes on the distribution of plasma proteins. Results from the model agree reasonably well with experimental measurements, suggesting that convection may account for the clearance of plasma proteins from the renal medulla maintaining their concentration below that of the AVR.
Pathophysiological role of T lymphocytes in renal ischemia-reperfusion injury in mice
Authors: Rabb Hamid , Daniels Frank , O'Donnell Michael , Haq Mahmud , Saba Sabiha R. , Keane William , Tang Winson W.
Published: December 2017

Mononuclear cell infiltrates are found in human renal ischemia-reperfusion injury (IRI), and peritubular T lymphocytes have been identified in experimental IRI. However, the role of T cells in the pathogenesis of renal IRI is unknown. We hypothesized that T cells are one of the important mediators of renal IRI. To test this hypothesis, we used an established mouse model of renal IRI, and evaluated mice with genetically engineered deficiency of both CD4+ and CD8+ T cells. At 48 h postischemia, CD4/CD8-knockout (KO) mice had marked improvement in renal function compared with control C57BL/6 mice (serum creatinine: 0.7 ± 0.4 vs. 2.5 ± 0.3 mg/dl, respectively; P < 0.05). Neutrophil infiltration into postischemic kidney was reduced in CD4/CD8 KO mice, compared with control mice, at both 24 h [polymorphonuclear neutrophils (PMNs)/10 high power fields: 714 ± 354 vs. 3,514 ± 660, respectively; P < 0.05] and 48 h (88 ± 32 vs. 1,979 ± 209, respectively; P< 0.05). Tubular necrosis score in CD4/CD8 KO mice, compared with control mice, was significantly less at 48 h (0.4 ± 0.1 vs. 2.4 ± 0.2, respectively; P < 0.05). Because adhesion between T cells and renal tubular epithelial cells (RTECs) may underlie the pathophysiological role of T cells in renal IRI, we also measured T cell adhesion to primary murine RTECs in vitro. Exposure of RTECs to 2 h of hypoxia followed by 1 h of reoxygenation increased T cell adhesion more than twofold. Phorbol ester treatment, which activates integrins, increased T cell adhesion threefold. These data suggest that T lymphocytes can mediate experimental renal IRI. Moreover, adhesion of infiltrating T cells to renal tubular cells may provide a potential mechanism underlying postischemic tubular dysfunction.
Altered expression of renal AQPs and Na+transporters in rats with Lithium-induced NDI
Authors: Kwon Tae-Hwan , Laursen Ulla H. , Marples David , Maunsbach Arvid B. , Knepper Mark A. , Frøkiær Jørgen , Nielsen Søren
Published: December 2017

Lithium (Li) treatment is often associated with nephrogenic diabetes insipidus (NDI). The changes in whole kidney expression of aquaporin-1 (AQP1), -2, and -3 as well as Na-K-ATPase, type 3 Na/H exchanger (NHE3), type 2 Na-Pi cotransporter (NaPi-2), type 1 bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1), and thiazide-sensitive Na-Cl cotransporter (TSC) were examined in rats treated with Li orally for 4 wk: protocol 1, high doses of Li (high Na+intake), and protocol 2, low doses of Li (identical food and normal Na+intake in Li-treated and control rats). Both protocols resulted in severe polyuria. Semiquantitative immunoblotting revealed that whole kidney abundance of AQP2 was dramatically reduced to 6% ( protocol 1) and 27% ( protocol 2) of control levels. In contrast, the abundance of AQP1 was not decreased. Immunoelectron microscopy confirmed the dramatic downregulation of AQP2 and AQP3, whereas AQP4 labeling was not reduced. Li-treated rats had a marked increase in urinary Na+excretion in both protocols. However, the expression of several major Na+transporters in the proximal tubule, loop of Henle, and distal convoluted tubule was unchanged in protocol 2, whereas in protocol 1significantly increased NHE3 and BSC-1 expression or reduced NaPi-2 expression was associated with chronic Li treatment. In conclusion, severe downregulation of AQP2 and AQP3 appears to be important for the development of Li-induced polyuria. In contrast, the increased or unchanged expression of NHE3, BSC-1, Na-K-ATPase, and TSC indicates that these Na+transporters do not participate in the development of Li-induced polyuria.
MAPK signaling and the kidney
Authors: Tian Wei , Zhang Zheng , Cohen David M.
Published: December 2017

Following an overview of the biochemistry of mitogen-activated protein kinase (MAPK) pathways, the relevance of these signaling events to specific models of renal cell function and pathophysiology, both in vitro and in vivo, will be emphasized. In in vitro model systems, events activating the principal MAPK families [extracellular signal-regulated and c-Jun NH2-terminal kinase and p38] have been best characterized in mesangial and tubular epithelial cell culture systems and include peptide mitogens, cytokines, lipid mediators, and physical stressors. Several in vivo models of proliferative or toxic renal injury are also associated with aberrant MAPK regulation. It is anticipated that elucidation of downstream effector signaling mechanisms and a clearer understanding of the immediate and remote upstream activating pathways, when applied to these highly clinically relevant model systems, will ultimately provide much greater insight into the basis for specificity now seemingly absent from these signaling events.