Medullary Thick Ascending Limb Cells Research Articles

A comparative modeling study of the mitochondrial function of the proximal tubule and thick ascending limb cells in the rat kidney.

To reabsorb >99% of the glomerular filtrate, the metabolic demand of the kidney is high. Interestingly, renal blood flow distribution exhibits marked inhomogeneity, with typical tissue oxygen tension (Po2) of 50-60 mmHg in the well-perfused cortex and 10-20 mmHg in the inner medulla. Cellular fluid composition and acidity also varies substantially. To understand how different renal epithelial cells adapt to their local environment, we have developed and applied computational models of mitochondrial function of proximal convoluted tubule cell (baseline Po2 = 50 mmHg, cytoplasmic pH = 7.20) and medullary thick ascending limb (mTAL) cell (baseline Po2 = 10 mmHg, cytoplasmic pH = 6.85). The models predict key cellular quantities, including ATP generation, P/O (phosphate/oxygen) ratio, proton motive force, electrical potential gradient, oxygen consumption, the redox state of key electron carriers, and ATP consumption. Model simulations predict that close to their respective baseline conditions, the proximal tubule and mTAL mitochondria exhibit qualitatively similar behaviors. Nonetheless, because the mTAL mitochondrion has adapted to a much lower Po2, it can sustain a sufficiently high ATP production at Po2 as low as 4-5 mmHg, whereas the proximal tubule mitochondria would not. Also, because the mTAL cytosol is already acidic under baseline conditions, the proton motive force (pmf) exhibits higher sensitivity to further acidification. Among the different pathways that lead to oxidative phosphorylation impairment, the models predict that both the proximal tubule and mTAL mitochondria are most sensitive to reductions in Complex III activity.NEW & NOTEWORTHY Tissue fluid composition varies substantially within the mammalian kidney. The renal cortex is well perfused and pH neutral, whereas some medullary regions are hypoxic and acidic. How do these environments affect the mitochondrial function of proximal convoluted tubule and medullary thick ascending limb cells, which reside in the cortex and medulla, respectively? This computational modeling study demonstrates that these mitochondria can adapt to their contrasting environments and exhibit different sensitivities to perturbations to local environments.

MicroRNA-195a-5p Regulates Blood Pressure by Inhibiting NKCC2A.

Previous studies showed that miR-195a-5p was among the most abundant microRNAs (miRNAs) expressed in the kidney. Lentivirus silencing of tumor necrosis factor-α (TNF) was performed in vivo and in vitro. Luciferase reporter assays confirmed that bumetanide-sensitive Na+-K+-2Cl- cotransporter isoform A (NKCC2A) mRNA is targeted and repressed by miR-195a-5p. Radiotelemetry was used to measure mean arterial pressure. TNF upregulates mmu-miR-195a-5p, and -203 and downregulates mmu-miR-30c and -100 in the medullary thick ascending limb of male mice. miR-195a-5p was >3-fold higher in the renal outer medulla of mice given an intrarenal injection of murine recombinant TNF, whereas silencing TNF inhibited miR-195a-5p expression by ≈51%. Transient transfection of a miR-195a-5p mimic into medullary thick ascending limb cells suppressed NKCC2A mRNA by ≈83%, whereas transfection with Anti-miR-195a-5p increased NKCC2A mRNA. Silencing TNF in medullary thick ascending limb cells prevented increases in miR-195 induced by 400 mosmol/kg H2O medium, an effect reversed by transfection with a miR-195a-5p mimic. Expression of phosphorylated NKCC2 increased 1.5-fold in medullary thick ascending limb cells transfected with Anti-miR-195a-5p and a miR-195a-5p mimic prevented the increase, which was induced by silencing TNF in cells exposed to 400 mosmol/kg H2O medium after osmolality was increased by adding NaCl. Intrarenal injection of TNF suppressed NKCC2A mRNA, whereas injection of miR-195a-5p prevented the increase of NKCC2A mRNA abundance and phosphorylated NKCC2 expression when TNF was silenced. Intrarenal injection with miR-195a-5p markedly attenuated MAP after renal silencing of TNF in mice given 1% NaCl. The study identifies miR-195a-5p as a salt-sensitive and TNF-inducible miRNA that attenuates NaCl-mediated increases in blood pressure by inhibiting NKCC2A.

The kidney protects against sepsis by producing systemic uromodulin.

Sepsis is a significant cause of mortality in hospitalized patients. Concomitant development of acute kidney injury (AKI) increases sepsis mortality through unclear mechanisms. Although electrolyte disturbances and toxic metabolite buildup during AKI could be important, it is possible that the kidney produces a protective molecule lost during sepsis with AKI. We have previously demonstrated that systemic Tamm-Horsfall protein (THP; uromodulin), a kidney-derived protein with immunomodulatory properties, falls in AKI. Using a mouse sepsis model without severe kidney injury, we showed that the kidney increases circulating THP by enhancing the basolateral release of THP from medullary thick ascending limb cells. In patients with sepsis, changes in circulating THP were positively associated with a critical illness. THP was also found de novo in injured lungs. Genetic ablation of THP in mice led to increased mortality and bacterial burden during sepsis. Consistent with the increased bacterial burden, the presence of THP in vitro and in vivo led macrophages and monocytes to upregulate a transcriptional program promoting cell migration, phagocytosis, and chemotaxis, and treatment of macrophages with purified THP increases phagocytosis. Rescue of septic THP-/- mice with exogenous systemic THP improved survival. Together, these findings suggest that through releasing THP, the kidney modulates the immune response in sepsis by enhancing mononuclear phagocyte function, and systemic THP has therapeutic potential in sepsis.NEW & NOTEWORTHY Specific therapies to improve outcomes in sepsis with kidney injury have been limited by an unclear understanding of how kidney injury increases sepsis mortality. Here, we identified Tamm-Horsfall protein, known to protect in ischemic acute kidney injury, as protective in preclinical sepsis models. Tamm-Horsfall protein also increased in clinical sepsis without severe kidney injury and concentrated in injured organs. Further study could lead to novel sepsis therapeutics.

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through induction of Tollip and negative regulation of IRAK-1.

LPS inhibits absorption in the medullary thick ascending limb (MTAL) through a Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-extracellular signal-regulated kinase (ERK) pathway that is upregulated by sepsis. Pretreatment with the nontoxic immunomodulator monophosphoryl lipid A (MPLA) prevents inhibition by LPS through activation of a TLR4-TIR-domain-containing adaptor-inducing interferon-β (TRIF)-phosphatidylinositol 3-kinase (PI3K) pathway that prevents LPS-induced ERK activation. Here, we identified the molecular mechanisms that underlie the protective inhibitory interaction between the MPLA-PI3K and LPS-ERK pathways. Treatment of mouse MTALs with LPS in vitro increased phosphorylation of IL-1 receptor-associated kinase (IRAK)-1, a critical mediator of LPS signaling downstream of TLR4-MyD88. Activation of ERK by LPS was eliminated by a selective IRAK-1 inhibitor, establishing IRAK-1 as the upstream mediator of ERK activation. Pretreatment of MTALs with MPLA in vitro prevented LPS-induced IRAK-1 activation; this effect was dependent on PI3K. Treatment of MTALs with MPLA increased expression of Toll-interacting protein (Tollip), an inducible protein that negatively regulates LPS signaling by inhibiting IRAK-1. The MPLA-induced increase in Tollip protein level was prevented by PI3K inhibitors. In coimmunoprecipitation experiments, MPLA increased the amount of Tollip stably bound to IRAK-1, an interaction that inhibits IRAK-1 activation. These results support a mechanism whereby MPLA increases Tollip expression in the MTAL through a PI3K-dependent pathway. Tollip, in turn, inhibits LPS-induced TLR4 signaling by suppressing activation of IRAK-1, thereby preventing activation of ERK that inhibits absorption. These studies show that MPLA induces reprogramming of MTAL cells that protects against LPS stimulation and identify IRAK-1 and Tollip as new therapeutic targets to prevent renal tubule dysfunction in response to infectious and inflammatory stimuli.

A two-hit mechanism for sepsis-induced impairment of renal tubule function

Renal insufficiency is a common and severe complication of sepsis, and the development of kidney dysfunction increases morbidity and mortality in septic patients. Sepsis is associated with a variety of defects in renal tubule function, but the underlying mechanisms are incompletely understood. We used a cecal ligation and puncture (CLP) model to examine mechanisms by which sepsis influences the transport function of the medullary thick ascending limb (MTAL). MTALs from sham and CLP mice were studied in vitro 18 h after surgery. The results show that sepsis impairs the ability of the MTAL to absorb HCO(3)(-) through two distinct mechanisms. First, sepsis induces an adaptive decrease in the intrinsic capacity of the tubules to absorb HCO(3)(-). This effect is associated with an increase in ERK phosphorylation in MTAL cells and is prevented by pretreatment of CLP mice with a MEK/ERK inhibitor. The CLP-induced reduction in intrinsic HCO(3)(-) absorption rate appears to involve loss of function of basolateral Na(+)/H(+) exchange. Second, sepsis enhances the ability of LPS to inhibit HCO(3)(-) absorption, mediated through upregulation of Toll-like receptor 4 (TLR4)-ERK signaling in the basolateral membrane. The two inhibitory mechanisms are additive and thus can function in a two-hit capacity to impair renal tubule function in sepsis. Both effects depend on ERK and are eliminated by interventions that prevent ERK activation. Thus the TLR4 and ERK signaling pathways represent potential therapeutic targets to treat or prevent sepsis-induced renal tubule dysfunction.

Increased Proliferative Cells in the Medullary Thick Ascending Limb of the Loop of Henle in the Dahl Salt-Sensitive Rat

Studies of transcriptome profiles have provided new insights into mechanisms underlying the development of hypertension. Cell type heterogeneity in tissue samples, however, has been a significant hindrance in these studies. We performed a transcriptome analysis in medullary thick ascending limbs of the loop of Henle isolated from Dahl salt-sensitive rats. Genes differentially expressed between Dahl salt-sensitive rats and salt-insensitive consomic SS.13(BN) rats on either 0.4% or 7 days of 8.0% NaCl diet (n=4) were highly enriched for genes located on chromosome 13, the chromosome substituted in the SS.13(BN) rat. A pathway involving cell proliferation and cell cycle regulation was identified as one of the most highly ranked pathways based on differentially expressed genes and by a Bayesian model analysis. Immunofluorescent analysis indicated that just 1 week of a high-salt diet resulted in a severalfold increase in proliferative medullary thick ascending limb cells in both rat strains, and that Dahl salt-sensitive rats exhibited a significantly greater proportion of medullary thick ascending limb cells in a proliferative state than in SS.13(BN) rats (15.0±1.4% versus 10.1±0.6%; n=7-9; P<0.05). The total number of cells per medullary thick ascending limb section analyzed was not different between the 2 strains. The study revealed alterations in regulatory pathways in Dahl salt-sensitive rats in tissues highly enriched for a single cell type, leading to the unexpected finding of a greater increase in the number of proliferative medullary thick ascending limb cells in Dahl salt-sensitive rats on a high-salt diet.

Sodium–bicarbonate cotransporter NBCn1 in the kidney medullary thick ascending limb cell line is upregulated under acidic conditions and enhances ammonium transport

In this study, we examined the effect of bicarbonate transporters on ammonium/ammonia uptake in the medullary thick ascending limb cell line ST-1. Cells were treated with 1 mm ouabain and 0.2 mM bumetanide to minimize carrier-mediated NH(4)(+) transport, and the intracellular accumulation of (14)C-methylammonium/methylammonia ((14)C-MA) was determined. In CO(2)/HCO(3)(-)-free solution, cells at normal pH briefly accumulated (14)C-MA over 7 min and reached a plateau. In CO(2)/HCO(3)(-) solution, however, cells markedly accumulated (14)C-MA over the experimental period of 30 min. This CO(2)/HCO(3)(-)-dependent accumulation was reduced by the bicarbonate transporter blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS; 0.5 mM). Replacing Cl(-) with gluconate reduced the accumulation, but the reduction was more substantial in the presence of DIDS. Incubation of cells at pH 6.8 (adjusted with NaHCO(3) in 5% CO(2)) for 24 h lowered the mean steady-state intracellular pH to 6.96, significantly lower than 7.28 for control cells. The presence of DIDS reduced (14)C-MA accumulation in control conditions but had no effect after acidic incubation. Immunoblotting showed that NBCn1 was upregulated after acidic incubation and in NH(4)Cl-containing media. The Cl(-)-HCO(3)(-) exchanger AE2 was present, but its expression remained unaffected by acidic incubation. Expressed in Xenopus oocytes, NBCn1 increased carrier-mediated (14)C-MA transport, which was abolished by replacing Na(+). Two-electrode voltage clamp of oocytes exhibited negligible current after NH(4)Cl application. These results suggest that DIDS-sensitive HCO(3)(-) extrusion normally governs NH(4)(+)/NH(3) uptake in the medullary thick ascending limb cells. We propose that, in acidic conditions, DIDS-sensitive HCO(3)(-) extrusion is inactivated, while NBCn1 is upregulated to stimulate NH(4)(+) transport.

Na/Bicarbonate Cotransporter NBCn1 is Upregulated under Acidic Conditions and Enhances Ammonium Transport in the Kidney Medullary Thick Ascending Limb Cell Line

The sodium/bicarbonate cotransporter NBCn1 regulates intracellular pH (pHi) and transepithelial HCO–3 movement. We examined NBCn1 expression and activity under acidic conditions and its contribution to ammonium movement in the mouse medullary thick ascending limb (MTAL) cell line ST‐1. Incubating cells at pH 6.8 for 24 h lowered the mean steady‐state pHi from 7.10 to 6.56. Immunoblot showed NBCn1 upregulation after acidic incubation. The Cl/HCO3 exchanger AE2 was present but its expression remained unaffected by acidic incubation. Acidic incubation decreased 14C‐methylammonium/ammonia (MA) accumulation by 22%. The 14C‐MA accumulation after acidic incubation was unaffected by 0.5 mM DIDS, whereas the accumulation in controls was reduced by DIDS. In CO2/HCO–3‐free solution, cells at normal pH accumulated 14C‐MA over 7 min and reached a plateau. In CO2/HCO–3 solution, however, cells markedly accumulated 14C‐MA over 30 min. This CO2/HCO–3‐dependent accumulation was reduced by DIDS. Cl– removal reduced the accumulation and this reduction was more substantial with DIDS. Expressed in Xenopus oocytes, NBCn1 increased CO2/HCO–3‐dependent 14C‐MA accumulation, which was abolished by replacing Na+. Two‐electrode voltage clamp exhibited negligible current upon NH4Cl application. We conclude that NBCn1 in the MTAL is upregulated under acidic conditions and the upregulation enhances ammonium uptake.

Nox2 is required for amiloride‐sensitive H+ efflux in a rat medullary thick ascending limb cell line

We have recently demonstrated that an amiloride (NMA)‐sensitive outward H+ current is present in mTAL of salt‐sensitive (SS) but not salt‐resistant (SS.13BN) rats and that when activated, this stimulates the production of excess superoxide (O2−). The identity of the H+ transporter remains unclear, however it displays many of the characteristics of a nox2 associated H+ current characterized in macrophages. The aim of this study was to determine which subunits of NAD(P)H oxidase are expressed in a rat mTAL cell line and whether a similar H+ transport pathway exists in these cells. mTAL cells were cultured, transfected with nox2, loaded with BCECF dye, imaged on a inverted microscope and intracellular pH (pHi) recovery determined following an NH4Cl prepulse in 0 Na+ solution with BaCl2. pHi recovery following an NH4Cl prepulse was absent in control transfected mTAL (δpHi/sec ‐1.9±2*10−5) however, in mTAL transfected with nox2, amiloride‐sensitive pHi recovery could be detected (δpHi/sec 7.9±3.2*10−5 vs ‐1.6±2*10−5 with NMA; P&lt;0.03). Cellular mRNA expression of endogenous NAD(P)H oxidase subunits are shown in the table below. We conclude that although nox4 is the primary gp91 homolog expressed in mTAL cells, nox2 over‐expression is required to produce the outward NMA‐sensitive H+ current observed in SS mTAL.mTAL copy # per 106 18S RNA (n=4)

Calcium-sensing receptor signaling pathways in medullary thick ascending limb cells mediate COX-2-derived PGE2production: functional significance

We determined the functional implications of calcium-sensing receptor (CaR)-dependent, Gq- and Gi-coupled signaling cascades, which work in a coordinated manner to regulate activity of nuclear factor of activated T cells and tumor necrosis factor (TNF)-alpha gene transcription that cause expression of cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) synthesis by rat medullary thick ascending limb cells (mTAL). Interruption of Gq, Gi, protein kinase C (PKC), or calcineurin (CaN) activities abolished CaR-mediated COX-2 expression and PGE2 synthesis. We tested the hypothesis that these pathways contribute to the effects of CaR activation on ion transport in mTAL cells. Ouabain-sensitive O2 consumption, an in vitro correlate of ion transport in the mTAL, was inhibited by approximately 70% in cells treated for 6 h with extracellular Ca2+ (1.2 mM), an effect prevented in mTAL cells transiently transfected with a dominant negative CaR overexpression construct (R796W), indicating that the effect was initiated by stimulation of the CaR. Pretreatment with the COX-2-selective inhibitor, NS-398 (1 microM), reversed CaR-activated decreases in ouabain-sensitive O2 consumption by approximately 60%, but did not alter basal levels of ouabain-sensitive O2 consumption. Similarly, inhibition of either Gq, Gi, PKC, or CaN, which are components of the mechanism associated with CaR-stimulated COX-2-derived PGE2 synthesis, reversed the inhibitory effects of CaR on O2 consumption without affecting basal O2 consumption. Our findings identified signaling elements required for CaR-mediated TNF production that are integral components regulating mTAL function via a mechanism involving COX-2 expression and PGE2 production.

PGE2 inhibits basolateral 50 pS potassium channels in the thick ascending limb of the rat kidney

To study the inhibition of the inwardly rectifying basolateral 50 pS potassium channels by PGE(2) we performed patch-clamp studies on the basolateral membrane of the rat kidney thick ascending limb. PGE(2)'s effect was mimicked by the selective EP1- and EP3-receptor agonist, sulprostone, but was prevented by inhibiting protein kinase-C with calphostin-C. The mitogen-activated protein kinase inhibitor PD98059 (ERK) or SB203580 (p38) increased basal channel activity; however, while neither alone prevented the inhibitory effect of PGE(2), but using both of them together completely abolished PGE(2)'s effect on channel activity. Treatment with PGE(2) stimulated phosphorylation of both p38 and ERK in primary cultures of medullary thick ascending limb cells. The PGE(2)-mediated mitogen-activated protein kinase activation was not affected by indomethacin, but was completely blocked by calphostin-C. These studies show that inhibition of basolateral 50 pS potassium channels by PGE(2) is mediated by protein kinase-C, which in turn stimulates mitogen-activated protein kinases in the thick ascending limb of the rat kidney.

Na–Picotransporter type I activity causes a transient intracellular alkalinization during ATP depletion in rabbit medullary thick ascending limb cells

The cellular pathophysiology of renal ischemia-reperfusion injury was investigated in primary cell cultures from rabbit medullary thick ascending limb (MTAL). Metabolic inhibition (MI) was achieved with cyanide and 2-deoxyglucose. Sixty minutes of MI caused a profound but reversible decrease in intracellular concentration of ATP ([ATP]i). Intracellular pH (pHi) first decreased after initiation of MI, followed by a transient alkalinization. When [ATP]i reached its lowest value (<1% of control), the cells slowly acidified to reach a stable pHi of 6.92 after 50 min of MI. In the presence of EIPA (10 micromol/L), the pattern of changes in pHi was unchanged and acidification was not increased, indicating that the Na+/H+ exchangers were inactive during ATP depletion. When inorganic phosphate (P(i)) or Na+ was omitted from the apical solutions during MI, the transient alkalinization was no longer observed and the cytosol slowly acidified. Experiments on Na+-dependent alkalinizations revealed the presence of a Na-P(i) cotransporter in the apical cell membrane. With indirect immunofluorescence, the Na-P(i) cotransporter expressed in these primary cell cultures could be identified as Na-P(i) type I. Although the exact physiological role of Na-P(i) type I still is unresolved, these experiments demonstrate that apical Na-P(i) type I activity is increased at the onset of ATP depletion in MTAL cells.

Investigation of the Ba2+-Sensitive NH4+ Transport Pathways in the Apical Cell Membrane of Primary Cultured Rabbit MTAL Cells

Background: Several apical ammonium (NH₄⁺/NH₃) transport pathways have been described in medullary thick ascending limb (MTAL) cells. The exact nature and importance of some of these pathways remain controversial. Methods: Ammonium transport in primary cultured rabbit MTAL cells was investigated by measuring intracellular pH (pH_i). Results: To create physiological conditions, experiments were performed in the symmetrical presence of NH₄Cl, which acidified the cells to pH_i 6.89. When blockers of apical NH₄⁺ transport were used, the cells alkalinized due to a decreased NH₄⁺ loading. The following values (pH units) were observed: bumetanide, +0.05; verapamil, +0.04; Ba²⁺ and Cs⁺, +0.19; tertiapin, +0.09. Tetraethylammonium had no effect. Depolarizing the cells by increasing the K⁺ concentration alkalinized the cells by 0.16 pH units. Because NH₄⁺ might enter through nonspecific channels, ammonium pulse experiments were performed: an NH₄Cl pulse acidified controls as well as depolarized cells. In contrast, when Ba²⁺, Cs⁺ or tertiapin were present, an NH₄Cl pulse alkalinized the cells. The pharmacological profile of this apical NH₄⁺ transport pathway correlates with the renal outer medullary K⁺ (ROMK) channel. Indirect immunofluorescence showed the presence of the ROMK protein. Conclusion: In these MTAL cells the Ba²⁺-sensitive component of NH₄⁺ transport is predominant and consists of permeation of NH₄⁺ through an apical ROMK-related channel.

Hypertonicity increases CLC-5 expression in mouse medullary thick ascending limb cells.

Genetic studies indicated that mutations of the chloride channel CLC-5 in the kidney are responsible for a group of clinical disorders, collectively called Dent's disease. In the kidney, CLC-5 was found to be expressed in the proximal tubule, medullary thick ascending limb (mTAL) of loop of Henle, and intercalated cells of the collecting tubule. In proximal tubular cells, CLC-5 was found to play an important role in receptor-mediated endocytosis. However, the functional roles of CLC-5 in mTAL and collecting tubules remain unclear. Because mTAL is normally exposed to a hypertonic environment, we aimed to examine the effect of hypertonicity on CLC-5 expression in this nephron segment. Our studies revealed that exposure to hypertonicity (up to 550 mosM) increased CLC-5 mRNA and protein levels in a murine mTAL cell line (MTAL) but not in an opossum kidney (OK) proximal tubular cell line. A similar effect was also found in mouse kidneys, where CLC-5 expression was enhanced in renal medulla, but not cortex, after 48 h of water deprivation. We also tested the effect of hypertonicity on endocytotic activity and found that exposure to hypertonicity caused a significant decrease in cellular uptake of FITC-labeled albumin in OK but not in MTAL cells. Our results suggest that CLC-5 expression is upregulated by hypertonicity in mTAL cells but not in proximal tubular cells. We speculate that the increased CLC-5 levels in mTAL may serve to maintain the endocytotic activity in a hypertonic environment.

Ischemia-reperfusion induces G-CSF gene expression by renal medullary thick ascending limb cells in vivo and in vitro.

Ischemic acute renal failure involves not only the kidney but also extrarenal organs such as the bone marrow that produces inflammatory cells. By ELISA and RNase protection assays, we now show that renal ischemia-reperfusion increases serum concentrations of granulocyte macrophage colony-stimulating factor (G-CSF) protein and increases both G-CSF mRNA and protein in the ischemic kidney. In situ hybridization localized the increased G-CSF mRNA to tubule cells, including medullary thick ascending limb cells (mTAL), in the outer medulla. We also show that mTAL produce G-CSF protein and increase G-CSF mRNA after stimulation by reactive oxygen species in vitro. The production of G-CSF by the kidney after ischemia-reperfusion provides a means of communication from the injured kidney to the bone marrow. This supports the known inflammatory response to ischemia.

Cyclosporin increases the density of angiotensin II subtype 1 (AT1) receptors in mouse medullary thick ascending limb cells.

Cyclosporin A (CsA), a potent immunosuppressive agent, can be nephrotoxic. Because clinical studies have suggested that the intrarenal renin-angiotensin system may be involved in the mechanism responsible for CsA nephrotoxicity, we have analysed the effects of CsA on angiotensin II (Ang II) receptors in medullary thick ascending limb (mTAL) cells known to be sensitive to the action of CsA. Experiments were carried out on subcultured mouse mTAL cells. The expression of mRNA of Ang II subtype 1 and 2 (AT(1) and AT(2)) receptors was investigated using reverse transcription-polymerase chain reaction (RT-PCR). [(3)H]Ang II was used for radioligand and binding studies. Fluorimetric recordings using the fluorescent dye fura-2/AM were performed to determine the effect of CsA on the intracellular calcium ([Ca(2+)]i) content of untreated and Ang II-treated mTAL cells. Subcultured mTAL cells expressed AT(1) and AT(2) Ang II receptor mRNAs, and binding studies revealed that the AT(1) receptors were the predominant Ang II receptor subtype ( approximately 90%) in mTAL cells. CsA (100 ng/ml, 24 h) increased (1.7-fold) the number of Ang II receptors (untreated, 315.8; +CsA, 543.6 fmol/mg protein) without altering the K(D) (untreated, 7.16; +CsA, 7.06 nM). CsA also significantly increased the level of [Ca(2+)]i measured in cultured mTAL cells both in the basal state (-CSA, 72.2; +CsA, 93.4 nM/10(6) cells) and in the presence of Ang II (-CSA, 97.8; +CsA, 206.3 nM/10(6) cells). These findings suggest that the increase in Ang II AT(1) receptors and [Ca(2+)]i caused by CsA may be involved in the mechanism(s) responsible for CsA nephrotoxicity.

PH dependence of Na+/myo-inositol cotransporters in rat thick limb cells

To balance medullary interstitium hypertonicity generated by transepithelial NaCl absorption, medullary thick ascending limb (MTAL) cells accumulate myo-inositol (MI). Expression of Na+-MI cotransporter (SMIT) mRNA in TAL is correlated with the NaCl absorption rate. Our present study aimed to determine the plasma membrane location and functional properties of the Na+-MI cotransporter in MTAL cells. Preparation of basolateral (BLMV) and luminal (LMV) membrane vesicles were simultaneously isolated from purified rat MTAL suspension, and uptake of [3H]myo-inositol ([3H]MI) was used to assess Na+-MI cotransport activity. In the presence of an inside-negative membrane potential, imposing an inwardly-directed Na+-gradient versus tetramethylammonium (TMA) stimulated the initial [3H]MI uptake in BLMV and LMV. Phlorizin inhibited Na+ gradient-dependent initial [3H]MI uptake in both preparations, with IC50 values of 565 and 29 micromol/L in BLMV and LMV, respectively. 2-0,C-methylene myo-inositol (MMI), a competitive inhibitor of MI transport, only inhibited the BLMV Na+-MI cotransporter. Phlorizin-sensitive Na+ gradient-dependent initial [3H]MI uptake showed Michaelis-Menten kinetics in both preparations, with similar Vmax but different Km values of 51 and 107 micromol/L in BLMV and LMV, respectively. Finally, BLMV but not LMV Na+-MI cotransporter exhibited a marked pH dependence with sigmoidal patterns of activation, as intravesicular pH (pHi) was decreased from 8.0 to 6.0 at extravesicular pH (pHe) 8.0, and as pHe was increased from 6.0 to 8.0 at pHi 6.0. Maximal activation was observed at pHi 6.5 and pHe 7.5. In rat MTAL cells, Na+-MI cotransporter activity is present in both BLM and LM, and has markedly different functional properties, indicating the presence of distinct transporters. Basolateral Na+-MI cotransporter activity is maximal at physiological pH values of MTAL cells and interstitium, and a powerful modulation of the transporter activity may be exerted by pHe and pHi.

Mycophenolic acid reduces renin-angiotensin-system activity in cultured mouse medullary thick ascending limb cells

Abstract Mycophenolate mofetil (MMF) is a promising immunosuppressive agent. The intra-renal reninangiotensin system (RAS) plays an important role in the regulation of intra-renal hemodynamics. Mycophenolic acid (MPA) is the bioactive metabolite of MMF. The interaction between MPA and intra-renal RAS is still unclear. We hypothesized that MPA might affect intra-renal RAS activity. We chose models of cultured mouse medullary thick ascending limb (mTAL) cells for the experiments, as the mTAL is one of the major sites of intra-renal RAS. We investigated the angiotensin-converting enzyme (ACE) activity by means of enzymatic assay, and the angiotensin-receptor activity by means of a binding study with radiolabeled angiotensin II, and measured the intracellular calcium concentration in cultured cells treated with and without MPA. ACE activity changed neither in cells incubated with MPA nor in those treated without MPA. The binding study also indicated decreased angiotensin-II binding in MPA-treated (MPA 10−7 M) cells, up to 43.7%. The decreased intracellular calcium concentration in MPA-treated cells further confirmed the MPA-inhibitory effect on the angiotensin receptor. We conclude that MPA reduces intra-renal RAS activity mainly through the decrease of AT1 receptor activity without affecting ACE activity. The results suggest that the inhibitory effect of MPA in the intra-renal RAS might play a role in the extra-immunosuppressive effect of MMF.

Mycophenolic acid reduces renin-angiotensin-system activity in cultured mouse medullary thick ascending limb cells.

Mycophenolate mofetil (MMF) is a promising immunosuppressive agent. The intra-renal renin-angiotensin system (RAS) plays an important role in the regulation of intra-renal hemodynamics. Mycophenolic acid (MPA) is the bioactive metabolite of MMF. The interaction between MPA and intra-renal RAS is still unclear. We hypothesized that MPA might affect intra-renal RAS activity. We chose models of cultured mouse medullary thick ascending limb (mTAL) cells for the experiments, as the mTAL is one of the major sites of intra-renal RAS. We investigated the angiotensin-converting enzyme (ACE) activity by means of enzymatic assay, and the angiotensin-receptor activity by means of a binding study with radiolabeled angiotensin II, and measured the intracellular calcium concentration in cultured cells treated with and without MPA. ACE activity changed neither in cells incubated with MPA nor in those treated without MPA. The binding study also indicated decreased angiotensin-II binding in MPA-treated (MPA 10(-7) M) cells, up to 43.7%. The decreased intracellular calcium concentration in MPA-treated cells further confirmed the MPA-inhibitory effect on the angiotensin receptor. We conclude that MPA reduces intra-renal RAS activity mainly through the decrease of AT1 receptor activity without affecting ACE activity. The results suggest that the inhibitory effect of MPA in the intra-renal RAS might play a role in the extra-immunosuppressive effect of MMF.

Calcium-sensing receptor-mediated TNF production in medullary thick ascending limb cells

Medullary thick ascending limb (mTAL) cells in primary culture express the Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor that senses changes in extracellular Ca(2+) (Ca(o)(2+)) concentration, resulting in increases of intracellular Ca(2+) concentration and PKC activity. Exposure of mTAL cells to either Ca(o)(2+) or the CaR-selective agonist poly-L-arginine increased TNF-alpha synthesis. Moreover, the response to Ca(o)(2+) was enhanced in mTAL cells transfected with a CaR overexpression vector. Transfection of mTAL cells with a TNF promoter construct revealed an increase in reporter gene activity after exposure of the cells to Ca(o)(2+), suggesting that intracellular signaling pathways initiated by means of activation of a CaR contribute to TNF synthesis by a mechanism that involves transcription of the TNF gene. Neutralization of TNF activity with an anti-TNF antibody attenuated Ca(2+)-mediated increases in cyclooxygenase-2 (COX-2) protein expression and PGE(2) synthesis, suggesting that TNF exerts an autocrine effect in the mTAL, which contributes to COX-2-mediated PGE(2) production. Preincubation with the PKC inhibitor bisindolylmaleimide I inhibited Ca(2+)-mediated TNF production. Significant inhibition of COX-2 protein expression and PGE(2) synthesis also was observed when cells were challenged with Ca(o)(2+) in the presence of bisindolylmaleimide I. The data suggest that increases in TNF production subsequent to activation of the CaR may be the basis of an important renal mechanism that regulates salt and water excretion.