Monophosphoryl lipid A pretreatment suppresses sepsis- and LPS-induced proinflammatory cytokine production in the medullary thick ascending limb.

Sepsis is the leading cause of acute kidney injury in critically ill patients. Tumor necrosis factor-α (TNF-α) has been implicated in the pathogenesis of septic kidney injury; however, the sites and mechanisms of renal TNF-α production during sepsis remain to be defined. In the present study, we showed that TNF-α expression is increased in medullary thick ascending limbs (MTALs) of mice with sepsis induced by cecal ligation and puncture. Treatment with lipopolysaccharide (LPS) for 3 h in vitro also increased MTAL TNF-α production. Sepsis and LPS increased MTAL TNF-α expression through activation of the myeloid differentiation factor 88 (MyD88)-IL-1 receptor-associated kinase 1-ERK signaling pathway. Pretreatment with monophosphoryl lipid A (MPLA), a nontoxic immunomodulator that protects against bacterial infection, eliminated the sepsis- and LPS-induced increases in MTAL TNF-α production. The suppressive effect of MPLA on TNF-α was mediated through activation of a phosphatidylinositol 3-kinase-dependent pathway that inhibits MyD88-dependent ERK activation. This likely involves MPLA-phosphatidylinositol 3-kinase-mediated induction of Tollip, which negatively regulates the MyD88-ERK pathway by inhibiting activation of IL-1 receptor-associated kinase 1. These regulatory mechanisms are similar to those previously shown to mediate the effect of MPLA to prevent sepsis-induced inhibition of MTAL [Formula: see text] absorption. These results identify the MTAL as a site of local TNF-α production in the kidney during sepsis and identify molecular mechanisms that can be targeted to attenuate renal TNF-α expression. The ability of MPLA pretreatment to suppress MyD88-dependent ERK signaling in the MTAL during sepsis has the dual beneficial effects of protecting tubule transport functions and attenuating harmful proinflammatory responses.

Superoxide increases surface NKCC2 in the rat thick ascending limbs via PKC.

The apical Na+-K+-2Cl- cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The free radical superoxide ( O2- ) stimulates TAL NaCl absorption by enhancing NKCC2 activity. In contrast, nitric oxide (NO) scavenges O2- and inhibits NKCC2. NKCC2 activity depends on the number of NKCC2 transporters in the TAL apical membrane and its phosphorylation. We hypothesized that O2- stimulates NKCC2 activity by enhancing apical surface NKCC2 expression. We measured surface NKCC2 expression in rat TALs by surface biotinylation and Western blot analysis. Treatment of TALs with O2- produced by exogenous xanthine oxidase (1 mU/ml) and hypoxanthine (500 µM) stimulated surface NKCC2 expression by ~18 ± 5% (P < 0.05). O2- -stimulated surface NKCC2 expression was blocked by the O2- scavenger tempol (50 µM). Scavenging H2O2 with 100 U/ml catalase did not block the stimulatory effect of xanthine oxidase-hypoxanthine (22 ± 8% increase from control, P < 0.05). Inhibition of endogenous NO production with Nω-nitro-l-arginine methyl ester enhanced surface NKCC2 expression by 21 ± 6% and, when added together with xanthine oxidase-hypoxanthine, increased surface NKCC2 by 41 ± 10% (P < 0.05). Scavenging O2- with superoxide dismutase (300 U/ml) decreased this stimulatory effect by 60% (39 ± 4% to 15 ± 10%, P < 0.05). Protein kinase C inhibition with Gö-6976 (100 nM) blocked O2- -stimulated surface NKCC2 expression (P < 0.05). O2- did not affect NKCC2 phosphorylation at Thr96/101 or its upstream kinases STE20/SPS1-related proline/alanine-rich kinase-oxidative stress-responsive kinase 1. We conclude that O2- increases surface NKCC2 expression by stimulating protein kinase C and that this effect is blunted by endogenous NO. O2- -stimulated apical trafficking of NKCC2 may be involved in the enhanced surface NKCC2 expression observed in Dahl salt-sensitive rats.

Fructose acutely stimulates NKCC2 activity in rat thick ascending limbs by increasing surface NKCC2 expression.

The thick ascending limb (TAL) reabsorbs 25% of the filtered NaCl through the Na+-K+-2Cl- cotransporter (NKCC2). NKCC2 activity is directly related to surface NKCC2 expression and phosphorylation. Higher NaCl reabsorption by TALs is linked to salt-sensitive hypertension, which is linked to consumption of fructose in the diet. However, little is known about the effects of fructose on renal NaCl reabsorption. We hypothesized that fructose, but not glucose, acutely enhances TAL-dependent NaCl reabsorption by increasing NKCC2 activity via stimulation of surface NKCC2 levels and phosphorylation at Thr96/101. We found that fructose (5 mM) increased transport-related O2 consumption in TALs by 11.1 ± 3.2% ( P < 0.05). The effect of fructose on O2 consumption was blocked by furosemide. To study the effect of fructose on NKCC2 activity, we measured the initial rate of NKCC2-dependent thallium influx. We found that 20 min of treatment with fructose (5 mM) increased NKCC2 activity by 58.5 ± 16.9% ( P < 0.05). We then used surface biotinylation to measure surface NKCC2 levels in rat TALs. Fructose increased surface NKCC2 expression in a concentration-dependent manner (22 ± 5, 49 ± 10, and 101 ± 59% of baseline with 1, 5, and 10 mM fructose, respectively, P < 0.05), whereas glucose or a glucose metabolite did not. Fructose did not change NKCC2 phosphorylation at Thre96/101 or total NKCC2 expression. We concluded that acute fructose treatment increases NKCC2 activity by enhancing surface NKCC2 expression, rather than NKCC2 phosphorylation. Our data suggest that fructose consumption could contribute to salt-sensitive hypertension by stimulating NKCC2-dependent NaCl reabsorption in TALs.

cGMP induces degradation of NKCC2 in the thick ascending limb via the ubiquitin-proteasomal system.

The thick ascending limb of Henle's loop (TAL) reabsorbs NaCl via the apical Na+-K+-2Cl- cotransporter (NKCC2). NKCC2 activity is regulated by surface NKCC2 levels. The second messenger cGMP decreases NKCC2 activity by decreasing surface NKCC2 levels. We found that surface NKCC2 undergoes constitutive degradation. Therefore, we hypothesized that cGMP decreases NKCC2 levels by increasing NKCC2 ubiquitination and proteasomal degradation. We measured surface NKCC2 levels by biotinylation of surface proteins, immunoprecipitation of NKCC2, and ubiquitin in TALs. First, we found that inhibition of proteasomal degradation blunts the cGMP-dependent decrease in surface NKCC2 levels [vehicle: 100%, db-cGMP (500 µM): 70.3 ± 9.8%, MG132 (20 µM): 97.7 ± 5.0%, and db-cGMP + MG132: 103.3 ± 3.4%, n = 5, P < 0.05]. We then found that cGMP decreased the internalized NKCC2 pool and that this effect was prevented by inhibition of the proteasome but not the lysosome. Finally, we found that NKCC2 is constitutively ubiquitinated in TALs and that cGMP enhances the rate of NKCC2 ubiquitination [vehicle: 59 ± 14% and db-cGMP (500 µM): 111 ± 25%, n = 5, P < 0.05]. We conclude that NKCC2 is constitutively ubiquitinated and that cGMP stimulates NKCC2 ubiquitination and proteasomal degradation. Our data suggest that the cGMP-induced NKCC2 ubiquitination and degradation may contribute to the cGMP-induced decrease of the NKCC2-dependent NaCl reabsorption in TALs.

Claudin-19 mediates the effects of NO on the paracellular pathway in thick ascending limbs.

Claudins are a family of tight junction proteins that provide size and charge selectivity to solutes traversing the paracellular space. Thick ascending limbs (TALs) express numerous claudins, including claudin-19. Nitric oxide (NO), via cGMP, reduces dilution potentials in perfused TALs, a measure of paracellular permeability, but the role of claudin-19 is unknown. We hypothesized that claudin-19 mediates the effects of NO/cGMP on the paracellular pathway in TALs via increases in plasma membrane expression of this protein. We measured the effect of the NO donor spermine NONOate (SPM) on dilution potentials with and without blocking antibodies and plasma membrane expression of claudin-19. During the control period, the dilution potential was -18.2 ± 1.8 mV. After treatment with 200 µmol/l SPM, it was -14.7 ± 2.0 mV (P < 0.04). In the presence of claudin-19 antibody, the dilution potential was -12.7 ± 2.1 mV. After SPM, it was -12.9 ± 2.4 mV, not significantly different. Claudin-19 antibody alone had no effect on dilution potentials. In the presence of Tamm-Horsfall protein antibody, SPM reduced the dilution potential from -9.7 ± 1.0 to -6.3 ± 1.1 mV (P < 0.006). Dibutyryl-cGMP (500 µmol/l) reduced the dilution potential from -19.6 ± 2.6 to -17.2 ± 2.3 mV (P < 0.002). Dibutyryl-cGMP increased expression of claudin-19 in the plasma membrane from 29.9 ± 3.8% to 65.9 ± 10.1% of total (P < 0.011) but did not change total expression. We conclude that claudin-19 mediates the effects of the NO/cGMP signaling cascade on the paracellular pathway.

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

LPS inhibits HCO3- 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-ß (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 HCO3- 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.

RNA-Seq and protein mass spectrometry in microdissected kidney tubules reveal signaling processes initiating lithium-induced nephrogenic diabetes insipidus.

Lithium salts, used for treating bipolar disorder, frequently induce nephrogenic diabetes insipidus (NDI) thereby limiting therapeutic success. NDI is associated with loss of expression of the gene coding for the molecular water channel, aquaporin-2, in the renal collecting duct (CD). Here, we use systems biology methods in a well-established rat model of lithium-induced NDI to identify signaling pathways activated at the onset of polyuria. Using single-tubule RNA-Seq, full transcriptomes were determined in microdissected cortical collecting ducts (CCDs) of rats after 72 hours without or with initiation of lithium chloride administration. Transcriptome-wide changes in mRNA abundances were mapped to gene sets associated with curated canonical signaling pathways, showing evidence for activation of NF-κB signaling with induction of genes coding for multiple chemokines and most components of the Major Histocompatibility Complex Class I antigen-presenting complex. Administration of anti-inflammatory doses of dexamethasone to lithium chloride-treated rats countered the loss of aquaporin-2. RNA-Seq also confirmed prior evidence of a shift from quiescence into the cell cycle with arrest. Time course studies demonstrated an early (12 hour) increase in multiple immediate early response genes including several transcription factors. Protein mass spectrometry in microdissected CCDs provided corroborative evidence and identified decreased abundance of several anti-oxidant proteins. Thus, in the context of prior observations, our study can be best explained by a model in which lithium increases ERK activation leading to induction of NF-κB signaling and an inflammatory-like response that represses Aqp2 transcription.

Diuretic state affects ascending thin limb tight junctions.

The nephron segments in the inner medulla are part of the urine concentrating mechanism. Depending on the diuretic state, they are facing a large range of extracellular osmolality. We investigated whether water homeostasis affects tubular transport and permeability properties in inner medullary descending thin limb (IMdTL) and ascending thin limb (IMaTL). Three experimental groups of rats under different diuretic states were investigated on metabolic cages: waterload, furosemide-induced diuresis, and control (antidiuresis). Urine production and osmolalities reflected the 3-day treatment. To functionally investigate tubular epithelial properties, we performed experiments in freshly isolated inner medullary thin limbs from these animals. Tubular segments were acutely dissected and investigated for trans- and paracellular properties by in vitro perfusion and electrophysiological analysis. IMdTL and IMaTL were distinguished by morphological criteria. We confirmed absence of transepithelial electrogenic transport in thin limbs. Although diffusion potential measurements showed no differences between treatments in IMdTLs, we observed increased paracellular cation selectivity under waterload in IMaTLs. NaCl diffusion potential was -5.64 ± 1.93 mV under waterload, -1.99 ± 1.72 mV under furosemide-induced diuresis, and 0.27 ± 0.40 mV under control. The corresponding permeability ratio PNa/Cl was 1.53 ± 0.21 (waterload), 1.22 ± 0.18 (furosemide-induced diuresis), and 0.99 ± 0.02 (control), respectively. Claudins are main constituents of the tight junction responsible for paracellular selectivity; however, immunofluorescence did not show qualitative differences in claudin 4, 10, and 16 localization. Our results show that IMaTLs change tight junction properties in response to diuretic state to allow adaptation of NaCl reabsorption.

Monophosphoryl lipid A prevents impairment of medullary thick ascending limb [Formula: see text] absorption and improves plasma [Formula: see text] concentration in septic mice.

Metabolic acidosis is the most common acid-base disorder in septic patients and is associated with increased mortality. Previously, we demonstrated that sepsis induced by cecal ligation and puncture (CLP) impairs [Formula: see text] absorption in the medullary thick ascending limb (MTAL) by 1) decreasing the intrinsic [Formula: see text] absorptive capacity and 2) enhancing inhibition of [Formula: see text] absorption by LPS through upregulation of Toll-like receptor (TLR) 4 signaling. Both effects depend on ERK activation. Monophosphoryl lipid A (MPLA) is a detoxified TLR4 agonist that enhances innate antimicrobial immunity and improves survival following sepsis. Pretreatment of MTALs with MPLA in vitro prevents LPS inhibition of [Formula: see text] absorption. Here we examined whether pretreatment with MPLA would protect the MTAL against sepsis. Vehicle or MPLA was administered to mice 48 h before sham or CLP surgery, and MTALs were studied in vitro 18 h postsurgery. Pretreatment with MPLA prevented the effects of sepsis to decrease the basal [Formula: see text] absorption rate and enhance inhibition by LPS. These protective effects were mediated through MPLA stimulation of a Toll/IL-1 receptor domain-containing adaptor-inducing IFN-ß-(TRIF)-dependent phosphatidylinositol 3-kinase-Akt pathway that prevents sepsis- and LPS-induced ERK activation. The effects of MPLA to improve MTAL [Formula: see text] absorption were associated with marked improvement in plasma [Formula: see text] concentration, supporting a role for the kidneys in the pathogenesis of sepsis-induced metabolic acidosis. These studies support detoxified TLR4-based immunomodulators, such as MPLA, that enhance antimicrobial responses as a safe and effective approach to prevent or treat sepsis-induced renal tubule dysfunction and identify cell signaling pathways that can be targeted to preserve MTAL [Formula: see text] absorption and attenuate metabolic acidosis during sepsis.

Nitric oxide reduces paracellular resistance in rat thick ascending limbs by increasing Na+ and Cl- permeabilities.

About 50% of the Na+ reabsorbed in thick ascending limbs traverses the paracellular pathway. Nitric oxide (NO) reduces the permselectivity of this pathway via cGMP, but its effects on absolute Na+ ([Formula: see text]) and Cl- ([Formula: see text]) permeabilities are unknown. To address this, we measured the effect of l-arginine (0.5 mmol/l; NO synthase substrate) and cGMP (0.5 mmol/l) on [Formula: see text] and [Formula: see text] calculated from the transepithelial resistance (Rt) and [Formula: see text]/[Formula: see text] in medullary thick ascending limbs. Rt was 7,722 ± 1,554 ohm·cm in the control period and 6,318 ± 1,757 ohm·cm after l-arginine treatment (P < 0.05). [Formula: see text]/[Formula: see text] was 2.0 ± 0.2 in the control period and 1.7 ± 0.1 after l-arginine (P < 0.04). Calculated [Formula: see text] and [Formula: see text] were 3.52 ± 0.2 and 1.81 ± 0.10 × 10-5 cm/s, respectively, in the control period. After l-arginine they were 6.65 ± 0.69 (P < 0.0001 vs. control) and 3.97 ± 0.44 (P < 0.0001) × 10-5 cm/s, respectively. NOS inhibition with Nω-nitro-l-arginine methyl ester (5 mmol/l) prevented l-arginine's effect on Rt Next we tested the effect of cGMP. Rt in the control period was 7,592 ± 1,470 and 4,796 ± 847 ohm·cm after dibutyryl-cGMP (0.5 mmol/l; db-cGMP) treatment (P < 0.04). [Formula: see text]/[Formula: see text] was 1.8 ± 0.1 in the control period and 1.6 ± 0.1 after db-cGMP (P < 0.03). [Formula: see text] and [Formula: see text] were 4.58 ± 0.80 and 2.66 ± 0.57 × 10-5 cm/s, respectively, for the control period and 9.48 ± 1.63 (P < 0.007) and 6.01 ± 1.05 (P < 0.005) × 10-5 cm/s, respectively, after db-cGMP. We modeled NO's effect on luminal Na+ concentration along the thick ascending limb. We found that NO's effect on the paracellular pathway reduces net Na+ reabsorption and that the magnitude of this effect is similar to that due to NO's inhibition of transcellular transport.

Calcineurin inhibitor cyclosporine A activates renal Na-K-Cl cotransporters via local and systemic mechanisms.

Calcineurin dephosphorylates nuclear factor of activated T cells transcription factors, thereby facilitating T cell-mediated immune responses. Calcineurin inhibitors are instrumental for immunosuppression after organ transplantation but may cause side effects, including hypertension and electrolyte disorders. Kidneys were recently shown to display activation of the furosemide-sensitive Na-K-2Cl cotransporter (NKCC2) of the thick ascending limb and the thiazide-sensitive Na-Cl cotransporter (NCC) of the distal convoluted tubule upon calcineurin inhibition using cyclosporin A (CsA). An involvement of major hormones like angiotensin II or arginine vasopressin (AVP) has been proposed. To resolve this issue, the effects of CsA treatment in normal Wistar rats, AVP-deficient Brattleboro rats, and cultured renal epithelial cells endogenously expressing either NKCC2 or NCC were studied. Acute administration of CsA to Wistar rats rapidly augmented phosphorylation levels of NKCC2, NCC, and their activating kinases suggesting intraepithelial activating effects. Chronic CsA administration caused salt retention and hypertension, along with stimulation of renin and suppression of renal cyclooxygenase 2, pointing to a contribution of endocrine and paracrine mechanisms at long term. In Brattleboro rats, CsA induced activation of NCC, but not NKCC2, and parallel effects were obtained in cultured cells in the absence of AVP. Stimulation of cultured thick ascending limb cells with AVP agonist restored their responsiveness to CsA. Our results suggest that the direct epithelial action of calcineurin inhibition is sufficient for the activation of NCC, whereas its effect on NKCC2 is more complex and requires concomitant stimulation by AVP.

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through a TLR4-TRIF-PI3K signaling pathway.

Monophosphoryl lipid A (MPLA) is a detoxified derivative of LPS that induces tolerance to LPS and augments host resistance to bacterial infections. Previously, we demonstrated that LPS inhibits [Formula: see text] absorption in the medullary thick ascending limb (MTAL) through a basolateral Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-ERK pathway. Here we examined whether pretreatment with MPLA would attenuate LPS inhibition. MTALs from rats were perfused in vitro with MPLA (1 µg/ml) in bath and lumen or bath alone for 2 h, and then LPS was added to (and MPLA removed from) the bath solution. Pretreatment with MPLA eliminated LPS-induced inhibition of [Formula: see text] absorption. In MTALs pretreated with MPLA plus a phosphatidylinositol 3-kinase (PI3K) or Akt inhibitor, LPS decreased [Formula: see text] absorption. MPLA increased Akt phosphorylation in dissected MTALs. The Akt activation was eliminated by a PI3K inhibitor and in MTALs from TLR4-/- or Toll/IL-1 receptor domain-containing adaptor-inducing IFN-ß (TRIF)-/- mice. The effect of MPLA to prevent LPS inhibition of [Formula: see text] absorption also was TRIF dependent. Pretreatment with MPLA prevented LPS-induced ERK activation; this effect was dependent on PI3K. MPLA alone had no effect on [Formula: see text] absorption, and MPLA pretreatment did not prevent ERK-mediated inhibition of [Formula: see text] absorption by aldosterone, consistent with MPLA's low toxicity profile. These results demonstrate that pretreatment with MPLA prevents the effect of LPS to inhibit [Formula: see text] absorption in the MTAL. This protective effect is mediated directly through MPLA stimulation of a TLR4-TRIF-PI3K-Akt pathway that prevents LPS-induced ERK activation. These studies identify detoxified TLR4-based immunomodulators as novel potential therapeutic agents to prevent or treat renal tubule dysfunction in response to bacterial infections.

Corticomedullary difference in the effects of dietary Ca²âº on tight junction properties in thick ascending limbs of Henle's loop.

The thick ascending limb of Henle's loop (TAL) drives an important part of the reabsorption of divalent cations. This reabsorption occurs via the paracellular pathway formed by the tight junction (TJ), which in the TAL shows cation selectivity. Claudins, a family of TJ proteins, determine the permeability and selectivity of this pathway. Mice were fed with normal or high-Ca(2+) diet, and effects on the reabsorptive properties of cortical and medullary TAL segments were analysed by tubule microdissection and microperfusion. Claudin expression was investigated by immunostaining and quantitative PCR. We show that the TAL adapted to high Ca(2+) load in a sub-segment-specific manner. In medullary TAL, transcellular NaCl transport was attenuated. The transepithelial voltage decreased from 10.9 ± 0.6 mV at control diet to 8.3 ± 0.5 mV at high Ca(2+) load, thereby reducing the driving force for Ca(2+) and Mg(2+) uptake. Cortical TAL showed a reduction in paracellular Ca(2+) and Mg(2+) permeabilities from 8.2 ± 0.7 to 6.2 ± 0.5 ∙ 10(-4) cm/s and from 4.8 ± 0.5 to 3.0 ± 0.2 · 10(-4) cm/s at control and high-Ca(2+) diet, respectively. Expression, localisation and regulation of claudins 10, 14, 16 and 19 differed along the corticomedullary axis: Towards the cortex, the main site of divalent cation reabsorption in TAL, high-Ca(2+) intake led to a strong upregulation of claudin-14 within TAL TJs while claudin-16 and -19 were unaltered. Towards the inner medulla, only claudin-10 was present in TAL TJ strands. In summary, high-Ca(2+) diet induced a reduction of divalent cation reabsorption via a diminution of NaCl transport and driving force in mTAL and via decreased paracellular permeabilities in cTAL. We reveal an important regulatory pattern along the corticomedullary axis and improve the understanding how the kidney disposes of detrimental excess Ca(2+).

Nitric oxide inhibits basolateral 10-pS Cl- channels through the cGMP/PKG signaling pathway in the thick ascending limb of C57BL/6 mice.

We used patch-clamp techniques to examine whether nitric oxide (NO) decreases NaCl reabsorption by suppressing basolateral 10-pS Cl- channels in the thick ascending limb (TAL). Both the NO synthase substrate l-arginine (l-Arg) and the NO donor S-nitroso-N-acetylpenicillamine significantly inhibited 10-pS Cl- channel activity in the TAL. The inhibitory effect of l-Arg on Cl- channels was completely abolished in the presence of the NO synthase inhibitor or NO scavenger. Moreover, inhibition of soluble guanylyl cyclase abrogated the effect of l-Arg on Cl- channels, whereas the cGMP analog 8-bromo-cGMP (8-BrcGMP) mimicked the effect of l-Arg and significantly decreased 10-pS Cl- channel activity, indicating that NO inhibits basolateral Cl- channels by increasing cGMP production. Furthermore, treatment of the TAL with a PKG inhibitor blocked the effect of l-Arg and 8-BrcGMP on Cl- channels, respectively. In contrast, a phosphodiesterase 2 inhibitor had no significant effect on l-Arg or 8-BrcGMP-induced inhibition of Cl- channels. Therefore, we conclude that NO decreases basolateral 10-pS Cl- channel activity through a cGMP-dependent PKG pathway, which may contribute to the natriuretic and diuretic effects of NO in vivo.

Angiotensin II-mediated hypertension impairs nitric oxide-induced NKCC2 inhibition in thick ascending limbs.

In thick ascending limbs (THALs), nitric oxide (NO) decreases NaCl reabsorption via cGMP-mediated inhibition of Na-K-2Cl cotransporter (NKCC2). In angiotensin (ANG II)-induced hypertension, endothelin-1 (ET-1)-induced NO production by THALs is impaired. However, whether this alters NO's natriuretic effects and the mechanisms involved are unknown. In other cell types, ANG II augments phosphodiesterase 5 (PDE5)-mediated cGMP degradation. We hypothesized that NO-mediated inhibition of NKCC2 activity and stimulation of cGMP synthesis are blunted via PDE5 in ANG II-induced hypertension. Sprague-Dawley rats were infused with vehicle or ANG II (200 ng·kg-1·min-1) for 5 days. ET-1 reduced NKCC2 activity by 38 ± 13% (P < 0.05) in THALs from vehicle-treated rats but not from ANG II-hypertensive rats (Δ: -9 ± 13%). A NO donor yielded similar results as ET-1. In contrast, dibutyryl-cGMP significantly decreased NKCC2 activity in both vehicle-treated and ANG II-hypertensive rats (control: Δ-44 ± 15% vs. ANG II: Δ-41 ± 10%). NO increased cGMP by 2.08 ± 0.36 fmol/µg protein in THALs from vehicle-treated rats but only 1.06 ± 0.25 fmol/µg protein in ANG II-hypertensive rats (P < 0.04). Vardenafil (25 nM), a PDE5 inhibitor, restored NO's ability to inhibit NKCC2 activity in THALs from ANG II-hypertensive rats (Δ: -60 ± 9%, P < 0.003). Similarly, NO's stimulation of cGMP was also restored by vardenafil (vehicle-treated: 1.89 ± 0.71 vs. ANG II-hypertensive: 2.02 ± 0.32 fmol/µg protein). PDE5 expression did not differ between vehicle-treated and ANG II-hypertensive rats. We conclude that NO-induced inhibition of NKCC2 and increases in cGMP are blunted in ANG II-hypertensive rats due to PDE5 activation. Defects in the response of THALs to NO may enhance NaCl retention in ANG II-induced hypertension.

Furosemide-induced urinary acidification is caused by pronounced H+ secretion in the thick ascending limb.

The loop diuretic furosemide inhibits NaCl reabsorption in the thick ascending limb (TAL). In addition, furosemide acidifies the urine, which is traditionally explained by increased Na+ loading to the distal tubule causing an activation of H+ secretion via H+-ATPase in α-intercalated cells. The inability to acidify urine in response to furosemide serves to diagnose distal renal tubular acidosis (dysfunction of α-intercalated cells). Since the TAL is important for acid/base regulation, we speculated that it is involved in furosemide-induced urinary acidification. Luminal furosemide (100 µM) caused major, stable, and reversible intracellular alkalization (7.27 ± 0.06 to 7.6 ± 0.04) in isolated perfused murine medullary TAL and pronounced H+ secretion. This H+ secretion was fully inhibited with luminal amiloride (1 mM) and the Na+/H+ exchanger (NHE)3-specific antagonist #4167 (1 µM). Moreover, furosemide triggered a substantial drop of intracellular Na+ concentration in the medullary TAL. These results suggest that the furosemide-induced H+ secretion is a consequence of a drop in intracellular Na+ concentration, increasing the driving force for NHE3. Intriguingly, in whole animal experiments, furosemide-induced urinary acidification and net acid excretion were markedly reduced by specific NHE3 inhibition. Furthermore, the furosemide-induced urinary acidification was partially preserved during epithelial Na+ channel inhibition with benzamil. These results provide new insights in the mechanism of furosemide-induced urinary acidification and emphasize the role of the TAL in renal acid/base handling.

Alternative channels for urea in the inner medulla of the rat kidney.

The ascending thin limbs (ATLs) and lower descending thin limbs (DTLs) of Henle's loop in the inner medulla of the rat are highly permeable to urea, and yet no urea transporters have been identified in these sections. We hypothesized that novel, yet-unidentified transporters in these tubule segments could explain the high urea permeability. cDNAs encoding for Na(+)-glucose transporter 1a (SGLT1a), Na(+)-glucose transporter 1 (NaGLT1), urea transporter (UT)-A2c, and UT-A2d were isolated and cloned from the Munich-Wistar rat inner medulla. SGLT1a is a novel NH2-terminal truncated variant of SGLT1. NaGLT1 is a Na(+)-dependent glucose transporter primarily located in the proximal tubules and not previously described in the thin limbs. UT-A2c and UT-A2d are novel variants of UT-A2. UT-A2c is truncated at the COOH terminus, and UT-A2d has one exon skipped. When rats underwent water restriction for 72 h, mRNA levels of SGLT1a increased in ATLs, NaGLT1 levels increased in both ATLs and DTLs, and UT-A2c increased in ATLs. [(14)C]urea uptake assays performed on Xenopus oocytes heterologously expressing these proteins revealed that despite having structural differences from their full-length versions, SGLT1a, UT-A2c, and UT-A2d enhanced urea uptake. NaGLT1 also facilitated urea uptake. Uptakes were Na(+) independent and inhibitable by phloretin and/or phloridzin. Our data indicate that there are several alternative channels for urea in the rat inner medulla that could potentially contribute to the high urea permeabilities in thin limb segments.

Iodinated contrast media cause direct tubular cell damage, leading to oxidative stress, low nitric oxide, and impairment of tubuloglomerular feedback.

Iodinated contrast media (CM) have adverse effects that may result in contrast-induced acute kidney injury. Oxidative stress is believed to play a role in CM-induced kidney injury. We test the hypothesis that oxidative stress and reduced nitric oxide in tubules are consequences of CM-induced direct cell damage and that increased local oxidative stress may increase tubuloglomerular feedback. Rat thick ascending limbs (TAL) were isolated and perfused. Superoxide and nitric oxide were quantified using fluorescence techniques. Cell death rate was estimated using propidium iodide and trypan blue. The function of macula densa and tubuloglomerular feedback responsiveness were measured in isolated, perfused juxtaglomerular apparatuses (JGA) of rabbits. The expression of genes related to oxidative stress and the activity of superoxide dismutase (SOD) were investigated in the renal medulla of rats that received CM. CM increased superoxide concentration and reduced nitric oxide bioavailability in TAL. Propidium iodide fluorescence and trypan blue uptake increased more in CM-perfused TAL than in controls, indicating increased rate of cell death. There were no marked acute changes in the expression of genes related to oxidative stress in medullary segments of Henle's loop. SOD activity did not differ between CM and control groups. The tubuloglomerular feedback in isolated JGA was increased by CM. Tubular cell damage and accompanying oxidative stress in our model are consequences of CM-induced direct cell damage, which also modifies the tubulovascular interaction at the macula densa, and may therefore contribute to disturbances of renal perfusion and filtration.

Dynamin2, clathrin, and lipid rafts mediate endocytosis of the apical Na/K/2Cl cotransporter NKCC2 in thick ascending limbs.

Steady-state surface levels of the apical Na/K/2Cl cotransporter NKCC2 regulate NaCl reabsorption by epithelial cells of the renal thick ascending limb (THAL). We reported that constitutive endocytosis of NKCC2 controls NaCl absorption in native THALs; however, the pathways involved in NKCC2 endocytosis are unknown. We hypothesized that NKCC2 endocytosis at the apical surface depends on dynamin-2 and clathrin. Measurements of steady-state surface NKCC2 and the rate of NKCC2 endocytosis in freshly isolated rat THALs showed that inhibition of endogenous dynamin-2 with dynasore blunted NKCC2 endocytosis by 56 ± 11% and increased steady-state surface NKCC2 by 67 ± 27% (p < 0.05). Expression of the dominant negative Dyn2K44A in THALs slowed the rate of NKCC2 endocytosis by 38 ± 8% and increased steady-state surface NKCC2 by 37 ± 8%, without changing total NKCC2 expression. Inhibition of clathrin-mediated endocytosis with chlorpromazine blunted NKCC2 endocytosis by 54 ± 6%, while preventing clathrin from interacting with synaptojanin also blunted NKCC2 endocytosis by 52 ± 5%. Disruption of lipid rafts blunted NKCC2 endocytosis by 39 ± 4% and silencing caveolin-1 by 29 ± 4%. Simultaneous inhibition of clathrin- and lipid raft-mediated endocytosis completely blocked NKCC2 internalization. We concluded that dynamin-2, clathrin, and lipid rafts mediate NKCC2 endocytosis and maintain steady-state apical surface NKCC2 in native THALs. These are the first data identifying the endocytic pathway for apical NKCC2 endocytosis.

Insulin-like growth factor-1 (IGF-1) inhibits the basolateral Cl channels in the thick ascending limb of the rat kidney.

The aim of the present study is to test the hypothesis that insulin-like-growth factor-1 (IGF-1) plays a role in the regulation of basolateral Cl channels in the thick ascending limb (TAL). The patch-clamp experiments demonstrated that application of IGF-I or insulin inhibited the basolateral 10-pS Cl channels. However, the concentration of insulin required for the inhibition of the Cl channels by 50% (K(1/2)) was ten times higher than those of IGF-1. The inhibitory effect of IGF-I on the 10-pS Cl channels was blocked by suppressing protein tyrosine kinase or by blocking phosphoinositide 3-kinase (PI3K). In contrast, inhibition of phospholipase C (PLC) failed to abolish the inhibitory effect of IGF-1 on the Cl channels in the TAL. Western blot analysis demonstrated that IGF-1 significantly increased the phosphorylation of phospholipid-dependent kinase (PDK) at serine residue 241 (Ser(241)) and AKT at Ser(473) in the isolated medullary TAL. Moreover, inhibition of PI3K with LY294002 abolished the effect of IGF-1 on the phosphorylation of PDK and AKT. The notion that the effect of IGF-1 on the 10-pS Cl channels was induced by stimulation of PDK-AKT-mTOR pathway was further suggested by the finding that rapamycin completely abolished the effect of IGF-1 on the 10-pS Cl channels in the TAL. We conclude that IGF-1 inhibits the basolateral Cl channels by activating PI3K-AKT-mTOR pathways. The inhibitory effect of IGF-1 on the Cl channels may play a role in ameliorating the ischemia-induced renal injury through IGF-1 administration.