Kidney Outer Medulla Research Articles

Intra-renal pressures during warm arterial and venous ischemia-reperfusion in rats

Red blood cell (RBC) trapping presents as the expansion- and obstruction of-, the outer-medullary capillaries of the kidney with tightly packed RBCs. RBC trapping has been shown to contribute to kidney injury and glomerular filtration rate decline, however, the mechanisms mediating RBC trapping remain unclear. It has previously been proposed that swelling of tubular cells during warm-ischemia initiates RBC trapping by increasing intra-renal pressure. This increase in pressure is thought to compresses the vasa recta capillaries that drain the renal medulla, initiating RBC trapping in the medullary circulation during reperfusion. In the current study we tested the hypothesis that ‘Intra-renal pressure is increased during warm ischemia-reperfusion’. We tested our hypothesis using young male Wistar-Kyoto rats (n=6). We examined pressure changes in the left kidney during ischemia-reperfusion from warm arterial (n=3) and venous (n=3) clamping using micro-serrefines. Rats were anesthetized with isoflurane (2-5%), a midline abdominal incision performed and the left artery and vein isolated. The left kidney was then cleared of connective tissue and placed in a kidney-cup to prevent movement. Intra-renal pressure was measured using a Millar micro-tip catheter pressure transducer which was inserted into the kidney outer-medulla. Intra-renal pressure was recorded during baseline and following 45 minutes of ischemia and 1 hour of reperfusion. Body temperature was maintained throughout the study using a servo-controlled heating table and heat lamp. At the end of the study, the left kidney was harvested and correct catheter placement confirmed. The kidneys were then fixed for later histological analysis. Data were compared using 2-way RM ANOVA (Graphpad Prism).Arterial clamping significantly increased intra-renal pressure (p<0.001). Following arterial clamping, intra-renal pressure rapidly increased reaching a peak of ~30mmHg above baseline after ~6 minutes. Intra-renal pressure then fell slightly averaging 17±3mmHg above baseline over the clamp period. Upon release of the arterial clamp, intrarenal pressure fell to below baseline levels within 270 seconds. Following clamping of the renal vein, intrarenal pressure increased, reaching a peak of 28mmHg above baseline. The increase in average intrarenal pressure across the clamp period was significantly greater with venous compared to arterial clamping (p<0.001), averaging 25±1mmHg. Similar to arterial clamping, intra-renal pressure rapidly fell to baseline levels following removal of the venous clamp.Our data indicate that both arterial and venous clamping increase intra-renal pressure during the clamp/ischemic period. As intra-renal pressure rapidly returns to baseline following removal of the clamp, however, compression of the venous vessels that drain the renal medulla by high intra-renal pressures is unlikely to mediate RBC trapping during reperfusion. NIH NIDDK ISAC 21AU4231, NIH NHLBI P01 HL134604 and an American Heart Association Transformational Project Award 970585 to P.O. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Reverse Electron Transfer is a More Dominant Source of Mitochondrial ROS Production in the Heart and Kidney Outer Medulla than in the Kidney Cortex

RationaleReactive oxygen species (ROS; e.g. O2·– and H2O2) play important roles in both physiological and pathophysiological processes. ROS in low concentrations contribute to physiological processes, such as cellular redox signaling and phagocytosis, whereas ROS in high concentrations are toxic to the cell causing tissue injury contributing to the pathogenesis of cardiovascular and chronic renal diseases, including salt‐sensitive hypertension. Mitochondria, which produce ROS as byproducts of aerobic respiration via both forward electron transfer (FET) and reverse electron transfer (RET) are known to be one of the major cellular sources of ROS. Although it is recognized that the RET mechanism in which electrons flow back from complex II to complex I contribute significantly to ROS production in cardiac mitochondria, the mechanisms of ROS production and the role of RET in kidney mitochondria has remained poorly understood.MethodWe evaluated the relative contributions of FET and RET towards overall ROS production in mitochondria isolated from the heart and kidney cortex and outer medulla (OM) of adult Sprague‐Dawley rats. H2O2 emission was measured by a spectrofluorometer in isolated mitochondria in the presence of either Succinate (Suc) simulating RET or succinate+rotenone (Suc+Rot) simulating FET. Furthermore, we measured mitochondrial rates of H2O2 production along with respiration and membrane potential under three respiratory states namely (i) leak state (state 2; after substrate addition), (ii) oxidative phosphorylation (OxPhos) state (state 3; after ADP addition), and (iii) maximum respiratory state (state 5; after the addition of the uncoupler FCCP).ResultsIt was found that mitochondria isolated from the heart and kidney cortex produced the least and the most ROS, respectively. The rate of ROS production in the presence of Suc+Rot compared to Suc alone decreased significantly in the heart and to a lesser extent in OM, indicating significant contribution of RET to overall ROS production in the heart and slightly in the OM. In contrast, there was not significant difference in ROS production rates in the presence of Suc and Suc +Rot in mitochondria from kidney cortex, showing that RET is not predominant in the kidney cortex. Also, we observed significant reduction in the ROS production rate in state 4 compared to state 2 in the heart mitochondria compared to kidney cortex and OM. A possible explanation for these differential results is that oxaloacetate (OAA), produced by the tricarboxylic acid cycle, accumulates resulting in succinate dehydrogenase (SDH) inhibition more rapidly in the heart than in the kidney affecting mitochondrial ROS production, respiration, and bioenergetics.ConclusionRET mechanism contributes to mitochondrial ROS production significantly in the heart and slightly in the kidney OM, but not in the kidney cortex. OAA accumulation contributes to SDH inhibition significantly in the heart than in the kidney.

Substrate- and Calcium-Dependent Differential Regulation of Mitochondrial Oxidative Phosphorylation and Energy Production in the Heart and Kidney.

Mitochondrial dehydrogenases are differentially stimulated by Ca2+. Ca2+ has also diverse regulatory effects on mitochondrial transporters and other enzymes. However, the consequences of these regulatory effects on mitochondrial oxidative phosphorylation (OxPhos) and ATP production, and the dependencies of these consequences on respiratory substrates, have not been investigated between the kidney and heart despite the fact that kidney energy requirements are second only to those of the heart. Our objective was, therefore, to elucidate these relationships in isolated mitochondria from the kidney outer medulla (OM) and heart. ADP-induced mitochondrial respiration was measured at different CaCl2 concentrations in the presence of various respiratory substrates, including pyruvate + malate (PM), glutamate + malate (GM), alpha-ketoglutarate + malate (AM), palmitoyl-carnitine + malate (PCM), and succinate + rotenone (SUC + ROT). The results showed that, in both heart and OM mitochondria, and for most complex I substrates, Ca2+ effects are biphasic: small increases in Ca2+ concentration stimulated, while large increases inhibited mitochondrial respiration. Furthermore, significant differences in substrate- and Ca2+-dependent O2 utilization towards ATP production between heart and OM mitochondria were observed. With PM and PCM substrates, Ca2+ showed more prominent stimulatory effects in OM than in heart mitochondria, while with GM and AM substrates, Ca2+ had similar biphasic regulatory effects in both OM and heart mitochondria. In contrast, with complex II substrate SUC + ROT, only inhibitory effects on mitochondrial respiration was observed in both the heart and the OM. We conclude that the regulatory effects of Ca2+ on mitochondrial OxPhos and ATP synthesis are biphasic, substrate-dependent, and tissue-specific.

Characterizing Substrate Dependent Differential Regulation of Mitochondrial Respiration in the Heart and Kidney Using Computational Modeling

It is recognized that the efficiency of mitochondrial ATP synthesis depends on the choice of respiratory substrates, which differentially generate reducing equivalents NADH and FADH2 through tricarboxylic acid (TCA) cycle that feed electrons to electron transport chain (ETC) for oxidative phosphorylation (OxPhos). Specifically, various substrate combinations represent differential contributions from different substrate oxidation pathways in mitochondria towards OxPhos and ATP synthesis. However, the relevance of these substrates and their combinations has not been quantitatively or systematically characterized in neither the heart nor the kidney. We determined the respiratory responses of mitochondria isolated from the heart ventricle and kidney outer medulla (OM) and cortex to different substrate combinations and different ADP concentrations. State 3 O2 consumption rates (OCR) were determined comparing different substrates for complexes I & II (pyruvate+malate, glutamate+malate, α‐ketoglutarate+malate, succinate, succinate+rotenone) in response to increasing concentrations of ADP. Comparisons of the state 3 OCR of the heart and kidney (OM & cortex) mitochondria yielded dramatically different values, which were also distinct for different substrates. State 3 OCR of the heart mitochondria was significantly greater than that found in the kidney mitochondria (OM & cortex). Heart mitochondria energized with succinate without rotenone (blocker of complex I) and saturating concentrations of ADP failed to stimulate state 3 respiration. This was in contrast to the kidney where responses to succinate alone and succinate + rotenone were similar indicating that the reverse electron transfer via complex I (blocked by rotenone) may play a less prominent regulatory role in the kidney than in the heart. Alternately, oxaloacetate, a TCA cycle intermediate, may accumulate faster in the heart compared to the kidney, thereby inhibiting succinate oxidation. To better understand these substrate and tissue specific effects of mitochondrial respiration, thermodynamically‐constrained mechanistic computational models were developed integrating the kinetics of TCA cycle, ETC/OxPhos, and substrate transport/oxidation, incorporating published mitochondrial bioenergetic data along with our respirometry data. Intrinsic model parameters such as Michaelis‐Menten constants were assumed identical and fixed for the kidney and heart, while extrinsic model parameters such as maximal enzymatic rates were separately estimated based on available experimental data for the heart and kidney mitochondrial bioenergetics. Simulations generated using these computational models have provided us with a deeper quantitative understanding of the key determinants of the kinetic and molecular mechanisms involved in the differential regulation of substrate‐dependent mitochondrial respiration in the heart and kidney (OM & cortex). These models will be essential for understanding the regulation of substrate utilization and ATP synthesis in different tissues under normal and pathological conditions.Support or Funding InformationNIH P01‐GM066730, P01‐HL116264, and U01‐HL122199

Prolactin Signaling Pathways Determining Its Direct Effects on Kidneys in the Cholestasis of Pregnancy Model.

Cholestasis of pregnancy is a pathology associated with disruptions in the bile flow and dysregulation of salt and water homeostasis. Prolactin is one of the most important regulators of salt and water balance. Changes in the expression of long and short isoforms of the prolactin receptor (PrlR) and mediators of prolactin signaling were studied by immunoblotting and RT-qPCR in the rat kidney cortex and outer medulla in the model of cholestasis of pregnancy. Both PrlR isoforms were shown to participate in the effects of prolactin in cholestasis of pregnancy. Direct impact of prolactin on the kidney has been demonstrated: (i) mRNA expression of both PrlR isoforms in the kidney depended on the physiological conditions and prolactin levels; (ii) expression of pSTAT5, a key mediator of the long PrlR isoform signaling, was increased in animals with cholestasis of pregnancy; (iii) in the case of long PrlR isoform predomination, expression of mRNAs for the prolactin signaling inhibitors SOCS3 and PIAS3 was upregulated (the genes of these regulators contain STAT-responsive elements in their promoters); (iv) expression of the mRNA for galactose-1-phosphate uridyltransferase (GALT), a molecular target of the PrlR short isoform, was decreased in the kidney outer medulla.

Calcium Stimulation of Mitochondrial Respiration is Substrate Dependent and Tissue Specific

Mitochondrial enzymes, such as pyruvate dehydrogenase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase, have been shown to be stimulated by calcium ion in isolated enzyme experimental systems. However, the extent of Ca2+ stimulation of these enzymes and the resulting integrated stimulatory effect on mitochondrial energy metabolism in in-situ and in-vivo states in the heart and kidney remains to be clarified. In this study, we hypothesized that Ca2+ stimulation of mitochondrial respiration and ATP synthesis is substrate dependent and tissue-specific. To test this hypothesis, we isolated mitochondria from heart and kidney outer medulla from Sprague-Dawley rats. Oxygen consumption rates were determined under different respiratory states: leak state (energized mitochondria in absence of ADP) and the ADP-stimulated state (energized mitochondria in presence of saturating concentration of ADP) with/without bolus injection of different concentrations of CaCl2 using an Oroboros Oxygraph-2k Instrument with various respiratory substrates, including pyruvate+malate (PM), glutamate+malate (GM), alpha-ketoglutarate+malate (AM), palmitoyl-L-carnitine+malate (PCM), and succinate (SUC). The results demonstrated that the various respiratory substrates yielded differences in ADP consumption rates and hence, led to dramatically different respiratory rates in isolated mitochondria from the heart and kidney. Importantly, it was found that Ca2+ significantly increased mitochondrial state III respiration with GM and AM substrates compared to the other substrates in both organs. Moreover, Ca2+ effects on mitochondrial respiration were found to be biphasic, i.e. a small increases in [Ca2+] had a stimulatory effect while large increases had inhibitory effect. These results suggest that energized isolated mitochondria in presence of Ca2+ do not respond the same extent when different substrates are used. We conclude that isolated mitochondria require a suitable substrate combination, optimal [Ca2+], and specific tissue source of mitochondria to achieve a level of energy metabolism and ATP production comparable to in-vivo conditions.

Ca2+-Dependent Mitochondrial Permeability Transition Pore Opening in Kidney is Substrate Dependent

Oxidative stress within the kidney is associated with disease states including hypertension, renal diseases. The metabolic functions of the mammalian kidneys result in consumption of nearly 20% of total oxygen utilization. There is limited information regarding the effects of oxygen free radicals upon kidney mitochondrial functions. Not only mitochondria oxidize numerous metabolic substrates but also play a key role in maintaining cellular Ca2+ homeostasis. Failure of cellular Ca2+ homeostasis and excessive mitochondrial Ca2+ (mCa2+) lead to opening of a mitochondrial permeability transition pore (mPTP), which is associated with loss of IMM potential and uncoupling of oxidative phosphorylation. We investigated whether mitochondrial metabolic substrates have a differential impact on the Ca2+-induced mPTP opening in the kidney, which plays a critical role in hypertension. For this purpose, mitochondria isolated from Sprague Dawley (SD) rat kidney outer medulla (OM) were energized using mechanistically distinct substrate combinations, Pyruvate+Malate, Glutamate+Malate(GM), Succinate+Rotenone, Succinate, α-KetoGlutarate+Malate, or PalmitoylCarnitine+Malate. Fluorescence-based analysis using Ca2+ indicator Fura-4F assessed the functional consequences of Ca2+-induced mPTP opening. Results show that in the presence of GM, mitochondria can tolerate significantly greater mCa2+ loads as compared to that in the presence of other substrates. Succinate+Rotenone addition significantly delayed mPTP opening over controls (succinate alone). A pore inhibitor CyclosporinA(CsA) and Na+/Ca2+exchanger inhibitor(CGP) were used with all substrates for comparisons. The present study reports variations in mitochondrial substrates sensitivity to Ca2+-induced mPTP opening and that the metabolic fluxes determine the mitochondrial functional responses to mCa2+ overload. The results underscore the effects of various mitochondrial substrates and their electron flux in regulating mPTP opening, which is a determinant of cell death in many diseases. These data are important in order to understand the different pathways/proteins involved in mPTP opening to identify effective interventions to prevent/limit it.

Apelin/APJ system: A novel potential therapy target for kidney disease

Apelin is an endogenous ligand of seven-transmembrane G protein-coupled receptor APJ. Apelin and APJ are distributed in various tissues, including the heart, lung, kidney, and even in tumor tissues. Studies show that apelin mRNA is highly expressed in the inner stripe of kidney outer medulla, which plays an important role in process of water and sodium balance. Additionally, more studies also indicate that apelin/APJ system exerts a broad range of activities in kidney. Therefore, we review the role of apelin/APJ system in kidney diseases such as renal fibrosis, renal ischemia/reperfusion injury, diabetic nephropathy, polycystic kidney disease, and hemodialysis (HD). Apelin/APJ system can improve renal interstitial fibrosis by reducing the deposition of extracellular matrix. Apelin/APJ system significantly reduces renal ischemia/reperfusion injury by inhibiting renal cell death. Apelin/APJ system involves the progression of diabetic nephropathy (DN). Apelin/APJ system also predicts the process of polycystic kidney disease. Besides, apelin/APJ system prevents some dialysis complications in HD patients. And apelin/APJ system alleviates chronic kidney disease (CKD) by inhibiting vascular calcification (VC). Overall, apelin/APJ system plays diversified roles in kidney disease and may be a potential target for the treatment of kidney disease.

Urea Transporter B and MicroRNA-200c Differ in Kidney Outer Versus Inner Medulla Following Dehydration

BackgroundUrea transporters (UTs) are important in urine concentration and in urea recycling, and UT-B has been implicated in both. In kidney, UT-B was originally localized to outer medullary descending vasa recta, and more recently detected in inner medullary descending vasa recta. Endogenously produced microRNAs (miRs) bind to the 3′UTR of genes and generally inhibit their translation, thus playing a pivotal role gene regulation. MethodsMice were dehydrated for 24 hours then sacrificed. Inner and outer medullas were analyzed by polymerase chain reaction (PCR) and quantitative PCR for miRNA expression and analyzed by western blotting for protein abundance. ResultsMiRNA sequencing analysis of mouse inner medullas showed a 40% increase in miRNA-200c in dehydrated mice compared with controls. An in silico analysis of the targets for miR-200c revealed that miRNA-200c could directly target the gene for UT-B. PCR confirmed that miR-200c is up-regulated in the inner medullas of dehydrated mice while western blot showed that UT-B protein abundance was down-regulated in the same portion of the kidney. However, in the outer medulla, miR-200c was reduced and UT-B protein was increased in dehydrated mice. ConclusionsThis is the first indication that UT-B protein and miR-200c may each be differentially regulated by dehydration within the kidney outer and inner medulla. The inverse correlation between the direction of change in miR-200c and UT-B protein abundance in both the inner and outer medulla suggests that miR-200c may be associated with the change in UT-B protein in these 2 portions of the kidney medulla.

Toxicologic assessment of Paecilomyces tenuipes in rats: Renal toxicity and mutagenic potential

Paecilomyces tenuipes is entomogenous fungus that is called snow-flake Dongchunghacho in Korea. Although it is widely used in traditional medicines, its safety has not yet been comprehensively investigated. Therefore, the aim of this study was to evaluate the genotoxicity, acute and subchronic toxicity of P. tenuipes. The acute oral LD50 of P. tenuipes extract in rats was estimated to be greater than 2000mg/kg of body weight. In the subchronic study, the oral treatment of rats with 500, 1000 or 2000mg/kg P. tenuipes extract daily for 13weeks did not induce any dose-related changes (body weight, food consumption, clinical observation, urinalysis, hematology, clinical chemistry and organ weight). In contrast, histopathological observation revealed that P. tenuipes extract induced karyomegaly in outer medulla of kidney in all treated rats. Importantly, P. tenuipes extract exerted the mutagenic potential in Ames assay. Since karyomegalic alterations have been known to be associated with carcinogenicity, our finding on the mutagenicity of P. tenuipes extract supports the possibility on the potential involvement of P. tenuipes in carcinogenicity at least partially. In conclusion, the subchronic oral exposure of P. tenuipes may induce kidney abnormality at the concentration higher than 500mg/kg body weight, although further studies using other animal models are needed to identify the toxicity of P. tenuipes.

Structural Analysis of N-glycans attached to Pig Kidney Na+/K+-ATPase

Na+/K+-ATPase is a membrane glycoprotein composed of α, β, and γ subunits, generating ion gradients across plasma membranes. Ion pumping is mainly accomplished by the α subunit, while the glycosylated β subunit binds tightly to the α subunit to assemble the pump and plays an essential role in the stabilization and maturation of Na+/K+-ATPase. Accumulating evidence suggests that the N-glycans of the β subunit contribute to cell-cell interaction and the tightness of cell contacts is modulated by N-glycan branching. However, N-glycan function is not fully understood due to a lack of detailed information on the oligosaccharide structure. We, here, perform glycosylation profi ling of the N-glycans attached to pig kidney Na+/K+-ATPase in order to better understand the mechanism of Na+/K+-ATPase-mediated cell adhesion. We purifi ed Na+/K+-ATPase from pig kidney outer medulla to homogeneity and solubilized it with the detergent C12E8. The enzyme thus obtained was identifi ed as α1β1 subtype by LC-MS/MS analysis of tryptic digests. During the course of MS analysis, we found that Lys456 of the α subunit was partially modifi ed with 4-hydroxynonenal, an aldehydric lipid peroxidation product. Three N-glycosylation sites on the β1 subunit were confi rmed to be fully occupied by time course analysis of enzymatic deglycosylation monitored by SDS-PAGE. HPLC profi ling of pyridylaminated oligosaccharides derived from Na+/K+-ATPase showed that high-mannose type oligosaccharides predominate while most of the less abundant complex-type oligosaccharides are capped with galactose residues. No glycans could be detected on the four consensus N-glycosylation sites on the α1 subunit. We briefl y discuss the possibility that oligomerization of Na+/K+- ATPase via β-β interactions assembles the N-glycans and promotes cell-cell adhesion.

Angiotensin II stimulates superoxide production in the thick ascending limb by activating NOX4

Angiotensin II (ANG II) stimulates production of superoxide (O(2)(-)) by NADPH oxidase (NOX) in medullary thick ascending limbs (TALs). There are three isoforms of the catalytic subunit (NOX1, 2, and 4) known to be expressed in the kidney. We hypothesized that NOX2 mediates ANG II-induced O(2)(-) production by TALs. To test this, we measured NOX1, 2, and 4 mRNA and protein by RT-PCR and Western blot in TAL suspensions from rats and found three catalytic subunits expressed in the TAL. We measured O(2)(-) production using a lucigenin-based assay. To assess the contribution of NOX2, we measured ANG II-induced O(2)(-) production in wild-type and NOX2 knockout mice (KO). ANG II increased O(2)(-) production by 346 relative light units (RLU)/mg protein in the wild-type mice (n = 9; P < 0.0007 vs. control). In the knockout mice, ANG II increased O(2)(-) production by 290 RLU/mg protein (n = 9; P < 0.007 vs. control). This suggests that NOX2 does not contribute to ANG II-induced O(2)(-) production (P < 0.6 WT vs. KO). To test whether NOX4 mediates the effect of ANG II, we selectively decreased NOX4 expression in rats using an adenovirus that expresses NOX4 short hairpin (sh)RNA. Six to seven days after in vivo transduction of the kidney outer medulla, NOX4 mRNA was reduced by 77%, while NOX1 and NOX2 mRNA was unaffected. In control TALs, ANG II stimulated O(2)(-) production by 96%. In TALs transduced with NOX4 shRNA, ANG II-stimulated O(2)(-) production was not significantly different from the baseline. We concluded that NOX4 is the main catalytic isoform of NADPH oxidase that contributes to ANG II-stimulated O(2)(-) production by TALs.

Fasting induces the expression of PGC-1α and ERR isoforms in the outer stripe of the outer medulla (OSOM) of the mouse kidney.

BackgroundPeroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) is a member of the transcriptional coactivator family that plays a central role in the regulation of cellular energy metabolism under various physiological stimuli. During fasting, PGC-1α is induced in the liver and together with estrogen-related receptor a and γ (ERRαand ERRγ, orphan nuclear receptors with no known endogenous ligand, regulate sets of genes that participate in the energy balance program. We found that PGC-1α, ERRα and ERRγ was highly expressed in human kidney HK2 cells and that PGC-1α induced dynamic protein interactions on the ERRα chromatin. However, the effect of fasting on the expression of endogenous PGC-1α, ERRα and ERRγ in the kidney is not known.Methodology/Principal FindingsIn this study, we demonstrated by qPCR that the expression of PGC-1α, ERRα and ERRγ was increased in the mouse kidney after fasting. By using immunohistochemistry (IHC), we showed these three proteins are co-localized in the outer stripe of the outer medulla (OSOM) of the mouse kidney. We were able to collect this region from the kidney using the Laser Capture Microdissection (LCM) technique. The qPCR data showed significant increase of PGC-1α, ERRα and ERRγ mRNA in the LCM samples after fasting for 24 hours. Furthermore, the known ERRα target genes, mitochondrial oxidative phosphorylation gene COX8H and the tricarboxylic acid (TCA) cycle gene IDH3A also showed an increase. Taken together, our data suggest that fasting activates the energy balance program in the OSOM of the kidney.

Post-transcriptional Control of Na,K-ATPase Activity and Cell Growth by a Splice Variant of FXYD2 Protein with Modified mRNA

In kidney, FXYD proteins regulate Na,K-ATPase in a nephron segment-specific way. FXYD2 is the most abundant renal FXYD but is not expressed in most renal cell lines unless induced by hypertonicity. Expression by transfection of FXYD2a or FXYD2b splice variants in NRK-52E cells reduces the apparent Na(+) affinity of the Na,K-ATPase and slows the cell proliferation rate. Based on RT-PCR, mRNAs for both splice variants were expressed in wild type NRK-52E cells as low abundance species. DNA sequencing of the PCR products revealed a base alteration from C to T in FXYD2b but not FXYD2a from both untreated and hypertonicity-treated NRK-52E cells. The 172C→T sequence change exposed a cryptic KKXX endoplasmic reticulum retrieval signal via a premature stop codon. The truncation affected trafficking of FXYD2b and its association with Na,K-ATPase and blocked its effect on enzyme kinetics and cell growth. The data may be explained by altered splicing or selective RNA editing of FXYD2b, a supplementary process that would ensure that it was inactive even if transcribed and translated, in these cells that normally express only FXYD2a. 172C→T mutation was also identified after mutagenesis of FXYD2b by error-prone PCR coupled with a selection for cell proliferation. Furthermore, the error-prone PCR alone introduced the mutation with high frequency, implying a structural peculiarity. The data confirm truncation of FXYD2b as a potential mechanism to regulate the amount of FXYD2 at the cell surface to control activity of Na,K-ATPase and cell growth.

Transcriptional inhibition of progressive renal disease by gene silencing pyrrole–imidazole polyamide targeting of the transforming growth factor-β1 promoter

Pyrrole-imidazole (PI) polyamides are small synthetic molecules that recognize and attach to the minor groove of DNA, thereby inhibiting gene transcription by blocking transcription factor binding. These derivatives can act as gene silencers inhibiting target gene expression under stimulatory conditions such as disease. To evaluate PI polyamides as treatments for the progression of renal diseases, we examined morphological effects, pharmacological properties, and the specificity of PI polyamides targeted to the transforming growth factor (TGF)-β1 promoter during salt-induced hypertensive nephrosclerosis in Dahl salt-sensitive rats. The targeted PI polyamide markedly reduced glomerulosclerosis and interstitial fibrosis without side effects. PI polyamide significantly decreased expression of TGF-β1 and extracellular matrix in the renal cortex. Microarray analysis found that only 3% of the transcripts were affected by PI polyamide, but this included decreased expression of extracellular matrix, TGF-β1-related cytokines, angiogenic, and cell stabilizing factors, proteinases, and renal injury-related factors. Thus, targeted PI polyamides are potential gene silencers for diseases not treatable by current remedies.

Time Course of Urinary Concentrating Defect in Vitamin D‐Induced Hypercalcemic Rats

We studied the onset of hypercalcemia‐induced nephrogenic diabetes insipidus and its relationship to aquaporin‐2 (AQP2) and bumetanide‐sensitive Na‐K‐2Cl cotransporter (NKCC2) expression. Rats were treated with or without food supplement of 1mg dihydrotachysterol (DHT)/kg BW/day for 1, 2, 7 days. After treatment for 1 day, serum calcium was significantly increased (control: 9.7±0.3 vs. DHT: 10.8±0.2 mg/dl). Urine volume was also significantly increased (0.27±0.18 vs. 0.83±0.14 ml/gram BW/day). Urine osmolality was decreased after 2 days of DHT ingestion (1.53±0.5 vs. 0.91±0.00 osmol/kgH2O, P &lt; 0.05), suggesting DHT rats lose their ability to concentrate urine. Immunoblotting showed that NKCC2 abundance in kidney outer medulla of DHT‐treated rats was decreased by 59% after 1 day treatment and remained decreased until day 7. The AQP2 abundance in inner medulla was decreased by 44% after 2 days of DHT diet and remained decreased until day 7. In conclusion, the onset of polyuria and hypercalcemia were found to occur within 1 day after DHT treatment. The early development of polyuria in association with hypercalcemia was most likely due to a downregulation of NKCC2 in medullary thick ascending limb, whereas the urinary concentrating defect after 2 days DHT treatment involved downregulation of both NKCC2 and AQP2.This study was supported by a grant from Thammasat University to SK and PU.

Cell Volume and Sodium Content in Rat Kidney Collecting Duct Principal Cells During Hypotonic Shock

The purpose of this study was to investigate the time course of the volume-regulatory response and intracellular sodium concentration ([Na+]i) in the principal cells of rat kidney outer medulla collecting duct (OMCD) epithelia during acute swelling in hypotonic medium. Hypotonic shock was created by PBS diluted with 50% of water. Changes in cell volume were measured with calcein quenching method. Intracellular sodium concentration was studied with fluorescence dye Sodium Green. Principal cells of microdissected OMCD fragments swelled very fast. The characteristic time of swelling (τ 1) was 0.65 ± 0.05 seconds, and the volume increased more than 60% (92.9 ± 5.6 and 151.3 ± 9.8 μm3 control and peak volumes correspondently, P < .01). After cell volume reached the peak of swelling, the RVD began without lag period. The characteristic time of volume decreasing to new steady-state level (τ 2) was 8.9 ± 1.1 seconds. In hypoosmotic medium, cell volume stabilized on higher level in comparison with control (110.3 ± 8.3 μm3, P < .01). After restoration of the medium osmolality to normotonic, cell volume stabilized on significantly low level in comparison with control level (71.4 ± 6.1 μm3, P < .01). During the hypoosmotic shock, [Na+]i decreased from control level in isotonic PBS to the low level in hypoosmotic solution (27.7 ± 1.4 and 5.8 ± 0.23 mM, P < .01). Calculation of sodium content per cell has shown the significant sodium entry into the cells, which caused a temporary increase correlated with the peak of cell volume caused by swelling. The conclusion is made that in our model of hypoosmotic shock, swelling activates transporters with high permeability for Na+ that provides sodium flux into the cells.

Fluorescence Spectroscopic Studies of Pressure Effects on Na+,K+-ATPase Reconstituted into Phospholipid Bilayers and Model Raft Mixtures

To contribute to the understanding of membrane protein function upon application of pressure as relevant for understanding, for example, the physiology of deep sea organisms or for baroenzymological biotechnical processes, we investigated the influence of hydrostatic pressure on the activity of Na+,K+-ATPase enriched in the plasma membrane from rabbit kidney outer medulla using a kinetic assay that couples ATP hydrolysis to NADH oxidation. The data show that the activity of Na+,K+-ATPase is reversibly inhibited by pressures below 2 kbar. At higher pressures, the enzyme is irreversibly inactivated. To be able to explore the effect of the lipid matrix on enzyme activity, the enzyme was also reconstituted into various lipid bilayer systems of different chain length, conformation, phase state, and heterogeneity including model raft mixtures. To yield additional information on the conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined as well. Incorporation of the enzyme leads to a significant increase of the lipid chain order. Generally, similar to the enzyme activity in the natural plasma membrane, high hydrostatic pressures lead to a decline of the activity of the enzyme reconstituted into the various lipid bilayer systems, and in most cases, a multi-phasic behavior is observed. Interestingly, in the low-pressure region, around 100 bar, a significant increase of activity is observed for the enzyme reconstituted into DMPC and DOPC bilayers. Above 100-200 bar, this activity enhancement is followed by a steep decrease of activity up to about 800 bar, where a more or less broad plateau value is reached. The enzyme activity decreases to zero around 2 kbar for all reconstituted systems measured. A different scenario is observed for the effect of pressure on the enzyme activity in the model raft mixture. The coexistence of liquid-ordered and liquid-disordered domains with the possibility of lipid sorting in this lipid mixture leads to a reduced pressure sensitivity in the medium-pressure range. The decrease of ATPase activity may be induced by an increasing hydrophobic mismatch, leading to a decrease of the conformational dynamics of the protein and eventually subunit rearrangement. High pressures, above about 2.2 kbar, irreversibly change protein conformation, probably because of the dissociation and partial unfolding of the subunits.

Redundant roles ofSox17andSox18in postnatal angiogenesis in mice

Sox7, Sox17 and Sox18 constitute group F of the Sox family of HMG box transcription factor genes. Dominant-negative mutations in Sox18 underlie the cardiovascular defects observed in ragged mutant mice. By contrast, Sox18(-/-) mice are viable and fertile, and display no appreciable anomaly in their vasculature, suggesting functional compensation by the two other SoxF genes. Here, we provide direct evidence for redundant function of Sox17 and Sox18 in postnatal neovascularization by generating Sox17(+/-) -Sox18(-/-) double mutant mice. Whereas Sox18(-/-) and Sox17(+/-) -Sox18(+/-) mice showed no vascular defects, approximately half of the Sox17(+/-) -Sox18(-/-) pups died before postnatal day 21 (P21). They showed reduced neovascularization in the liver sinusoids and kidney outer medulla vasa recta at P7, which most likely caused the ischemic necrosis observed by P14 in hepatocytes and renal tubular epithelia. Those that survived to adulthood showed similar, but milder, vascular anomalies in both liver and kidney, and females were infertile with varying degrees of vascular abnormalities in the reproductive organs. These anomalies corresponded with sites of expression of Sox7 and Sox17 in the developing postnatal vasculature. In vitro angiogenesis assays, using primary endothelial cells isolated from the P7 livers, showed that the Sox17(+/-) -Sox18(-/-) endothelial cells were defective in endothelial sprouting and remodeling of the vasculature in a phenotype-dependent manner. Therefore, our findings indicate that Sox17 and Sox18, and possibly all three SoxF genes, are cooperatively involved in mammalian vascular development.

Binding of a Single Rb+ Increases Na+/K+-ATPase, Activating Dephosphorylation without Stoichiometric Occlusion

We used partially purified Na+/K+-ATPase from pig kidney to study dephosphorylation, occlusion, and ATPase activity in the same enzyme preparation and in media of identical composition containing 10 microM ATP and different concentrations of Rb+, used as a K+ congener. The experiments were performed using a rapid-mixing apparatus with a time resolution of 3.5 ms. The main findings were as follows. (i) At sufficiently low Rb+ concentration the initial rate of dephosphorylation was higher than that of occlusion, (ii) as [Rb+] tended to zero the slope of the time course of occlusion but not that of the time course of dephosphorylation approached zero and, (iii) as Rb+ concentration increased, ATPase activity first increased and, after passing through a maximum, tended to a value that was lower than that observed in media without Rb+. None of these results is compatible with the currently held idea that binding of a single Rb+ to the E2P conformer of the ATPase does not modify the rate of dephosphorylation and strongly suggest that a single Rb+ does promote dephosphorylation through a mechanism that is not stoichiometrically coupled to Rb+ occlusion. If this mechanism is included in the currently accepted scheme for ATP hydrolysis by the Na+/K+-ATPase, a reasonable prediction of the experimental results is obtained.