Terminal Inner Medullary Collecting Duct Research Articles

Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts.

Hypertonicity increases water permeability, independently of vasopressin, in the inner medullary collecting duct (IMCD) by increasing aquaporin-2 (AQP2) membrane accumulation. We investigated whether protein kinase C (PKC) and adenosine monophosphate kinase (AMPK) are involved in hypertonicity-regulated water permeability. Increasing perfusate osmolality from 150 to 290 mosmol/kgH2O and bath osmolality from 290 to 430 mosmol/kgH2O significantly stimulated osmotic water permeability. The PKC inhibitors chelerythrine (10 µM) and rottlerin (50 µM) significantly reversed the increase in osmotic water permeability stimulated by hypertonicity in perfused rat terminal IMCDs. Chelerythrine significantly increased phosphorylation of AQP2 at S261 but not at S256. Previous studies show that AMPK is stimulated by osmotic stress. We tested AMPK phosphorylation under hypertonic conditions. Hypertonicity significantly increased AMPK phosphorylation in inner medullary tissues. Blockade of AMPK with Compound C decreased hypertonicity-stimulated water permeability but did not alter phosphorylation of AQP2 at S256 and S261. AICAR, an AMPK stimulator, caused a transient increase in osmotic water permeability and increased phosphorylation of AQP2 at S256. When inner medullary tissue was treated with the PKC activator phorbol dibutyrate (PDBu), the AMPK activator metformin, or both, AQP2 phosphorylation at S261 was decreased with PDBu or metformin alone, but there was no additive effect on phosphorylation with PDBu and metformin together. In conclusion, hypertonicity regulates water reabsorption by activating PKC. Hypertonicity-stimulated water reabsorption by PKC may be related to the decrease in endocytosis of AQP2. AMPK activation promotes water reabsorption, but the mechanism remains to be determined. PKC and AMPK do not appear to act synergistically to regulate water reabsorption.

Metformin, an AMPK activator, stimulates the phosphorylation of aquaporin 2 and urea transporter A1 in inner medullary collecting ducts.

Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations.

Thienoquinolins exert diuresis by strongly inhibiting UT-A urea transporters.

Urea transporters (UT) play an important role in the urine concentration mechanism by mediating intrarenal urea recycling, suggesting that UT inhibitors could have therapeutic use as a novel class of diuretic. Recently, we found a thienoquinolin UT inhibitor, PU-14, that exhibited diuretic activity. The purpose of this study was to identify more potent UT inhibitors that strongly inhibit UT-A isoforms in the inner medullary collecting duct (IMCD). Efficient thienoquinolin UT inhibitors were identified by structure-activity relationship analysis. Urea transport inhibition activity was assayed in perfused rat terminal IMCDs. Diuretic activity of the compound was determined in rats and mice using metabolic cages. The results show that the compound PU-48 exhibited potent UT-A inhibition activity. The inhibition was 69.5% with an IC50 of 0.32 μM. PU-48 significantly inhibited urea transport in perfused rat terminal IMCDs. PU-48 caused significant diuresis in UT-B null mice, which indicates that UT-A is the target of PU-48. The diuresis caused by PU-48 did not change blood Na(+), K(+), or Cl(-) levels or nonurea solute excretion in rats and mice. No toxicity was detected in cells or animals treated with PU-48. The results indicate that thienoquinolin UT inhibitors induce a diuresis by inhibiting UT-A in the IMCD. This suggests that they may have the potential to be developed as a novel class of diuretics with fewer side effects than classical diuretics.

Protein kinase C-α comes to the rescue of aquaporin-2

the article by Thai et al. ([12][1]) in an issue of the American Journal of Physiology-Renal Physiology brings to the forefront a previously unrecognized role of PKC-α in preventing downregulation of aquaporin (AQP)2 by ANG II. Regulated transport of water, sodium, and urea in the renal medulla is

Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts

Urea plays a critical role in the concentration of urine, thereby regulating water balance. Vasopressin, acting through cAMP, stimulates urea transport across rat terminal inner medullary collecting ducts (IMCD) by increasing the phosphorylation and accumulation at the apical plasma membrane of UT-A1. In addition to acting through protein kinase A (PKA), cAMP also activates Epac (exchange protein activated by cAMP). In this study, we tested whether the regulation of urea transport and UT-A1 transporter activity involve Epac in rat IMCD. Functional analysis showed that an Epac activator significantly increased urea permeability in isolated, perfused rat terminal IMCD. Similarly, stimulating Epac by adding forskolin and an inhibitor of PKA significantly increased urea permeability. Incubation of rat IMCD suspensions with the Epac activator significantly increased UT-A1 phosphorylation and its accumulation in the plasma membrane. Furthermore, forskolin-stimulated cAMP significantly increased ERK 1/2 phosphorylation, which was not prevented by inhibiting PKA, indicating that Epac mediated this phosphorylation of ERK 1/2. Inhibition of MEK 1/2 phosphorylation decreased the forskolin-stimulated UT-A1 phosphorylation. Taken together, activation of Epac increases urea transport, accumulation of UT-A1 at the plasma membrane, and UT-A1 phosphorylation, the latter of which is mediated by the MEK-ERK pathway.

Epac Regulation of Urea Transport and the UT‐A1 Urea Transporter in Rat Inner Medullary Collecting Duct.

Urea is critical to the urinary concentrating mechanism and to the regulation of water balance. Vasopressin, through cAMP, stimulates urea transport across perfused rat terminal inner medullary collecting ducts (IMCD) by increasing UT‐A1 phosphorylation and membrane accumulation. Cyclic AMP is known to act through protein kinase A (PKA); however, it can also activate Epac (exchange protein activated by cAMP). We tested whether Epac is involved in regulation of urea transport and UT‐A1 transporter activity in rat IMCD. Functional analysis showed that the Epac activator, Sp‐8‐pCPT‐2 O‐Me‐cAMPS, significantly increased urea permeability in isolated, perfused rat terminal IMCDs by 29%. Incubation of rat IMCD suspensions with the Epac activator significantly increased UT‐A1 in the plasma membrane (biotinylated UT‐A1) and UT‐A1 phosphorylation. To further explore the signaling pathway, we tested whether forskolin could stimulate ERK 1/2 phosphorylation. Forskolin increased ERK 1/2 phosphorylation 25%; this increase was not inhibited by a PKA inhibitor, H‐89, indicating that ERK 1/2 phosphorylation was through Epac. The MEK 1/2 inhibitor, U0126, inhibited forskolin‐stimulated UT‐A1 phosphorylation. We conclude that activation of Epac increases urea transport, UT‐A1 phosphorylation, and UT‐A1 plasma membrane accumulation. The increase in UT‐A1 phosphorylation is mediated by the MEK‐ERK pathway.

Urea transporters UT-A1 and UT-A3 accumulate in the plasma membrane in response to increased hypertonicity

The UT-A1 and UT-A3 urea transporters are expressed in the terminal inner medullary collecting duct (IMCD) and play an important role in the production of concentrated urine. We showed that both hyperosmolarity and vasopressin increase urea permeability in perfused rat terminal IMCDs and that UT-A1 and UT-A3 accumulate in the plasma membrane in response to vasopressin. In this study, we investigated whether hyperosmolarity causes UT-A1 and/or UT-A3 to accumulate in the plasma membrane or represents a complimentary stimulatory pathway. Rat IMCD suspensions were incubated in 450 vs. 900 mosM solutions. We biotinylated the IMCD surface proteins, collected, and analyzed them. Membrane accumulation was assessed by Western blotting of the biotinylated protein pool probed with anti-UT-A1 or anti-UT-A3. We studied the effect of NaCl, urea, and sucrose as osmotic agents. Membrane-associated UT-A1 and UT-A3 increased relative to control levels when either NaCl (UT-A1 increased 37 +/- 6%; UT-A3 increased 46 +/- 13%) or sucrose (UT-A1 increased 81 +/- 13%; UT-A3 increased 60 +/- 8%) was used to increase osmolarity. There was no increase in membrane UT-A1 or UT-A3 when urea was added. Analogously, UT-A1 phosphorylation was increased in NaCl- and sucrose- but not in urea-based hyperosmolar solutions. Hypertonicity also increased UT-A3 phosphorylation. We conclude that the increase in the urea permeability in response to hyperosmolarity reflects both UT-A1 and UT-A3 movement to the plasma membrane and may be a direct response to tonicity. Furthermore, this movement is accompanied by, and may require, increased phosphorylation in response to hypertonicity.

Acute regulation of aquaporin-2 phosphorylation at Ser-264 by vasopressin

By phosphoproteome analysis, we identified a phosphorylation site, serine 264 (pS264), in the COOH terminus of the vasopressin-regulated water channel, aquaporin-2 (AQP2). In this study, we examined the regulation of AQP2 phosphorylated at serine 264 (pS264-AQP2) by vasopressin, using a phospho-specific antibody (anti-pS264). Immunohistochemical analysis showed pS264-AQP2 labeling of inner medullary collecting duct (IMCD) from control mice, whereas AQP2 knockout mice showed a complete absence of labeling. In rat and mouse, pS264-AQP2 was present throughout the collecting duct system, from the connecting tubule to the terminal IMCD. Immunogold electron microscopy, combined with double-labeling confocal immunofluorescence microscopy with organelle-specific markers, determined that the majority of pS264 resides in compartments associated with the plasma membrane and early endocytic pathways. In Brattleboro rats treated with [deamino-Cys-1, d-Arg-8]vasopressin (dDAVP), the abundance of pS264-AQP2 increased 4-fold over controls. Additionally, dDAVP treatment resulted in a time-dependent change in the distribution of pS264 from predominantly intracellular vesicles, to both the basolateral and apical plasma membranes. Sixty minutes after dDAVP exposure, a proportion of pS264-AQP2 was observed in clathrin-coated vesicles, early endosomal compartments, and recycling compartments, but not lysosomes. Overall, our results are consistent with a dynamic effect of AVP on the phosphorylation and subcellular distribution of AQP2.

Dynamics of aquaporin-2 serine-261 phosphorylation in response to short-term vasopressin treatment in collecting duct

We recently identified a novel phosphorylation site, serine-261 (pS261), in the COOH-terminus of the vasopressin-regulated water channel, aquaporin-2 (AQP2). To address whether phosphorylation at this site is regulated by vasopressin, a rabbit polyclonal phospho-specific antibody was generated. Dot blot and immunoblot analysis demonstrated that this antibody specifically recognizes AQP2 phosphorylated at pS261, and that phosphorylation of S256 (pS256), a site already known to be regulated by vasopressin, does not interfere with antibody recognition. Immunohistochemical analysis revealed intense pS261 labeling of inner medullary collecting duct (IMCD) from wild-type mice, while sections from AQP2 knockout animals showed a general absence of labeling. AQP2 pS261 was present in principal cells of all mouse and rat distal tubule segments from the connecting tubule to the terminal IMCD. Co-immunolabeling of collecting duct with phospho-specific and total AQP2 antibodies revealed that pS261 and pS256 have distinct subcellular distributions. Levels of pS256 increased, while the amount of pS261 significantly decreased in freshly isolated rat IMCD samples incubated with 1 nM [deamino-Cys(1),D-Arg(8)]vasopressin for 30 min. Similarly, based on immunohistochemical labeling, the amount of pS261 was reduced in all collecting duct segments of Brattleboro rats treated with [deamino-Cys(1),D-Arg(8)]vasopressin for 2 h. This study reveals a reciprocal change in S256 and S261 phosphorylation in response to short-term vasopressin exposure, suggesting that these residues may serve distinct roles in regulation of AQP2 subcellular distribution and collecting duct water permeability.

Fluoxetine inhibits arginine vasopressin (AVP)‐stimulated water permeability in rat inner medullary collecting duct (IMCD) via alpha‐2 mechanism

Fluoxetine is widely used as an anti-depressant drug that inhibits reuptake of serotonin. The drug also induces hyponatremia by an unknown mechanism. Evidence shows that fluoxetine does not promote the release of vasopressin that would lead to hyponatremia (Marar IE and Amico JA, Endocrine, 8: 13–18, 1998). Effects of fluoxetine on renal physiology are not known. The purpose of this study was to determine if fluoxetine affects water permeability in the IMCD. Terminal IMCDs from male Wistar rats were isolated and perfused via standard methods. To measure osmotic water permeability (Pf, μm/sec), bath fluid was hypertonic to luminal fluid (545 vs 300 mOsm/Kg H2O). Agents were added to the bath – AVP at 220 pM and fluoxetine at 2.5 μM. In one set of experiments (n=4), fluoxetine reduced AVP-stimulated Pf from 1235±135 to 587±220 (p<.01). This inhibitory effect was reversible. In another protocol (n=5) fluoxetine failed to affect basal Pf (29±9). Subsequent addition of AVP increased Pf to 93±18 and the removal of fluoxetine raised Pf to 591±200 (p<.01). Fluoxetine failed to decrease Pf stimulated by the nonhydrolyzable cAMP analog 8-chlorophenylthio cAMP (n=4). To investigate the inhibitory mechanism, we used the selective alpha-2 antagonist atipamezole. In this protocol (n=4) atipamezole reversed the fluoxetine-induced inhibition of AVP-stimulated Pf from 235±92 to 657±153 (p<05). We conclude that fluoxetine inhibits AVP-stimulated Pf in rat IMCD via a pre-cAMP-dependent mechanism mediated by alpha-2 adrenoceptors. The mechanism for fluoxetine-induced hyponatremia remains unknown.

Angiotensin II increases vasopressin-stimulated facilitated urea permeability in rat terminal IMCDs.

Angiotensin II receptors are present along the rat inner medullary collecting duct (IMCD), although their physiological role is unknown. Because urea is one of the major solutes transported across the terminal IMCD, we measured angiotensin II's effect on urea permeability. In the perfused rat terminal IMCD, angiotensin II had no effect on basal urea permeability but significantly increased vasopressin-stimulated urea permeability by 55%. Angiotensin II, both without and with vasopressin, also increased the amount of (32)P incorporated into urea transporter (UT)-A1 in inner medullary tissue exposed to these hormones ex vivo. Because angiotensin II activates protein kinase C, we tested the effect of staurosporine (SSP). In the absence of angiotensin II, SSP had no effect on vasopressin-stimulated urea permeability in the perfused terminal IMCD. However, SSP completely and reversibly blocked the angiotensin II-mediated increase in vasopressin-stimulated urea permeability. SSP and chelerythrine reduced the angiotensin II-stimulated (32)P incorporation into UT-A1 in inner medullary tissue exposed ex vivo. We conclude that angiotensin II increases vasopressin-stimulated facilitated urea permeability and (32)P incorporation into the 97- and 117-kDa UT-A1 proteins via a protein kinase C-mediated signaling pathway. These data suggest that angiotensin II augments vasopressin-stimulated facilitated urea transport in the rat terminal IMCD and may play a physiological role in the urinary concentrating mechanism by augmenting the maximal response to vasopressin.

Role of PGE(2) in alpha(2)-induced inhibition of AVP- and cAMP-stimulated H(2)O, Na(+), and urea transport in rat IMCD.

PGE(2) inhibits osmotic water permeability (P(f)) in the rat inner medullary collecting duct (IMCD) via cellular events occurring after the stimulation of cAMP, i.e., post-cAMP-dependent events. The alpha(2)-agonists also inhibit P(f) in the rat IMCD via post-cAMP-dependent events. The purpose of this study was to determine whether PGE(2) plays a role in alpha(2)-mediated inhibition of P(f), Na(+), and urea transport in the rat IMCD. Isolated terminal IMCDs from Wistar rats were perfused to measure, in separate experiments, P(f), lumen-to-bath (22)Na(+) transport (J(lb)), and urea permeability (P(u)). Transport was stimulated with 220 pM arginine vasopressin (AVP) or 0.1 mM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP). Indomethacin was used to inhibit endogenous prostaglandin synthesis, and the alpha(2)-agonists clonidine, oxymetazoline, and dexmedetomidine were used to test the role of PGE(2) in the alpha(2)-mediated mechanism that inhibits transport. All agents were added to the bath. Indomethacin at 5 microM significantly elevated CPT-cAMP-stimulated P(f), J(lb), and P(u), and subsequent addition of 100 nM PGE(2) reduced these transport parameters. Indomethacin reversed alpha(2) inhibition of CPT-cAMP-stimulated P(f), J(lb), and P(u), and subsequent addition of PGE(2) reduced transport in each case. Indomethacin partially reversed alpha(2) inhibition of AVP-stimulated P(f), J(lb), and P(u), and PGE(2) reduced transport back to the alpha(2)-inhibited level. These results indicate that PGE(2) is a second messenger involved in the mechanism of transport inhibition mediated by alpha(2)-adrenoceptors via post-cAMP-dependent events in the rat IMCD.

Regulation of renal urea transporters.

Urea is important for the conservation of body water due to its role in the production of concentrated urine in the renal inner medulla. Physiologic data demonstrate that urea is transported by facilitated and by active urea transporter proteins. The facilitated urea transporter (UT-A) in the terminal inner medullary collecting duct (IMCD) permits very high rates of transepithelial urea transport and results in the delivery of large amounts of urea into the deepest portions of the inner medulla where it is needed to maintain a high interstitial osmolality for concentrating the urine maximally. Four isoforms of the UT-A urea transporter family have been cloned to date. The facilitated urea transporter (UT-B) in erythrocytes permits these cells to lose urea rapidly as they traverse the ascending vasa recta, thereby preventing loss of urea from the medulla and decreasing urine-concentrating ability by decreasing the efficiency of countercurrent exchange, as occurs in Jk null individuals (who lack Kidd antigen). In addition to these facilitated urea transporters, three sodium-dependent, secondary active urea transport mechanisms have been characterized functionally in IMCD subsegments: (1) active urea reabsorption in the apical membrane of initial IMCD from low-protein fed or hypercalcemic rats; (2) active urea reabsorption in the basolateral membrane of initial IMCD from furosemide-treated rats; and (3) active urea secretion in the apical membrane of terminal IMCD from untreated rats. This review focuses on the physiologic, biophysical, and molecular evidence for facilitated and active urea transporters, and integrative studies of their acute and long-term regulation in rats with reduced urine-concentrating ability.

Colonic H+-K+-ATPase is induced and mediates increased [formula omitted] reabsorption in inner medullary collecting duct in potassium depletion

Potassium depletion increases HCO3- reabsorption in outer medullary collecting duct (OMCD) by activation of colonic (c) H-K-ATPase (HKA). The purpose of the current experiments was to examine the role of the isoforms of HKA in HCO3- reabsorption by terminal inner medullary collecting duct (IMCD) cells in potassium depletion. Sprague-Dawley rats were fed a potassium-free diet and studied after 8 to 10 days. mRNA expression of HKA isoforms in terminal portion of inner medulla was examined and correlated with HCO3- reabsorption in the terminal IMCD. Gastric (g) HKA mRNA decreased whereas colonic (c) HKA mRNA expression was heavily induced in terminal portion of inner medulla in potassium depleted rats. Net HCO3- flux (JtCO2) in terminal IMCD increased in potassium depletion (4.56 to 7.06 pmol/min/mm tubule length, P < 0.001). In normal rats, 1 mM ouabain in perfusate had no effect on JtCO2, whereas 10 microM Schering 28080 (SCH) decreased JtCO2 to 2.4 (P < 0.002). In KD rats, 1 mM ouabain decreased JtCO2 to 4.9 (P < 0.005) and 10 microM SCH decreased JtCO2 to 3.3 (P < 0.001). However, the inhibitory effects of SCH and ouabain on JtCO2 in potassium depleted animals were not additive. The data indicate that gHKA is suppressed whereas cHKA is induced in potassium depletion and mediates increased HCO3- reabsorption in terminal IMCD. The results further indicate that cHKA in vivo is sensitive to both SCH and ouabain.

Renal tubular and vascular urea transporters: influence of antidiuretic hormone on messenger RNA expression in Brattleboro rats.

In the kidney, facilitated urea transport in precise vascular and tubular structures is mainly involved in water conservation. Three urea transporters have been cloned: UT2-long expressed in terminal inner medullary collecting duct (IMCD), UT2-short expressed in thin descending limb, and UT11 in descending vasa recta. The effect of arginine vasopressin (AVP) administration on mRNA expression of these three transporters was examined in Brattleboro rats with diabetes insipidus. V2 effects were discriminated from combined V1 + V2 effects by comparing treatments with 1-deamino-8-D-AVP (dDAVP) (selective V2 agonism) and AVP (V1 and V2 agonism). Acute and chronic treatments were studied. Abundance of specific mRNA was assessed by quantitative Northern blot analysis of RNA extracted from two regions of inner stripe of outer medulla and from two regions of inner medulla (IM). The results show that mRNA of these urea transporters are differently regulated by AVP. (1) Long-term treatment with either AVP or dDAVP does not alter UT2-long mRNA in tip IM (terminal IMCD) except for a transient initial decrease. (2) Unlike AVP, dDAVP induces the appearance of significant expression of UT2-long mRNA in base IM (initial IMCD), indicating a major V2 effect. (3) UT2-short mRNA in deep inner stripe of outer medulla and base IM (thin descending limb of short and long loops, respectively) is progressively upregulated with duration of AVP or dDAVP treatment. (4) The much higher changes in UT2-long and UT2-short induced by dDAVP compared with AVP suggest that they are dependent mainly on V2 agonism, and likely attenuated by V1 agonism. (5) UT11 mRNA expression in tip IM is equally depressed by AVP and dDAVP, indicating that this vascular transporter is also influenced by AVP and/or urine-concentrating activity, via an indirect mechanism that remains to be determined.

Long-term regulation of inner medullary collecting duct urea transport in rat.

Facilitated urea transport is regulated acutely by arginine vasopressin (AVP) and hyperosmolality in rat terminal inner medullary collecting duct (IMCD). This study tested whether chronic diuresis or antidiuresis regulates facilitated urea transport. Basal and AVP-stimulated urea permeabilities (Purea) were measured in perfused IMCD subsegments. Rats were made: (1) diuretic by giving them sugar water (with or without food) or furosemide; or (2) antidiuretic by water deprivation. They were then compared with untreated rats given food and water ad libitum. Terminal IMCD from untreated rats had a high basal Purea that was significantly increased by AVP. Diuresis significantly increased basal Purea in terminal IMCD in all five diuresis protocols. Water deprivation for 1 or 3 d had no effect on basal or AVP-stimulated Purea in the IMCD2 subsegment of the terminal IMCD. In contrast, 3 d of water deprivation significantly increased both basal and AVP-stimulated Purea in the IMCD3 subsegment; 1 d of water deprivation had no effect on basal or AVP-stimulated Purea. Next, initial IMCD (IMCD1) were studied. Initial IMCD from untreated rats had a low basal Purea that was not increased by AVP (10 nM). Water diuresis (with or without food) for 3 to 5 d had no effect on basal Purea but significantly increased AVP-stimulated Purea. Furosemide diuresis and water diuresis for 1 or 7 d had no effect on either basal or AVP-stimulated Purea in initial IMCD. Water deprivation for 2 to 3 d, but not for 1 d, significantly increased basal Purea in initial IMCD, whereas water deprivation for 1 d increased AVP-stimulated Purea. It is concluded that chronic changes in hydration cause heterogeneous changes in facilitated urea transport in rat IMCD subsegments.

Dysregulation of cytokine expression in tubulointerstitial nephritis associated with murine malaria

Dysregulation of cytokine expression in tubulointerstitial nephritis associated with murine malaria

Indomethacin and staurosporine reverse α2 inhibition of water transport in rat IMCD

These studies were conducted to determine if the prostaglandin-synthesis inhibitor indomethacin or the protein kinase C (PKC) inhibitor staurosporine affect the inhibition of osmotic water permeability (Pf) by the alpha-2 (alpha 2) agonist dexmedetomidine in the rat inner medullary collecting duct (IMCD). Terminal IMCDs from Wistar rats were perfused and Pf was increased with either 220 pM arginine vasopressin (AVP) or 0.1 mM 8-chlorophenylthio cyclic adenosine monophosphate (8CPTcAMP). All agents were added to the bathing solution. Dexmedetomidine at 100 nM inhibited both AVP- and 8CPTcAMP-stimulated Pf. When Pf was increased by AVP, indomethacin at 0.1 mM or 5 microM reversed the dexmedetomidine-induced inhibition by 68% and 43%, respectively. When Pf was increased by 8CPTcAMP, indomethacin at 0.1 mM or 5 microM reversed inhibition by 83% and 70%, respectively. Indomethacin increased AVP and 8CPTcAMP-stimulated Pf by 20 to 30% and dexmedetomidine inhibited the AVP+ indomethacin-stimulated Pf. Staurosporine at 10 nM yielded similar results. Results suggest that PKC and prostaglandins are involved in the alpha 2 mediated mechanism, and staurosporine and indomethacin-sensitive cellular mediators modulate basal Pf.

Microlocalization and effects of adrenomedullin in nephron segments and in mesangial cells of the rat.

We examined microlocalization of mRNA coding for adrenomedullin (AM), using reverse transcription-polymerase chain reaction (RT-PCR), and the effects of AM on adenosine 3',5'-cyclic monophosphate (cAMP) generation and water transport in microdissected rat nephron segments. We also examined intraglomerular site of the expression of AM and AM-stimulated cAMP generation in cultured rat mesangial cells (MC). RT-PCR demonstrated the signals for AM mRNA in glomerulus (Glm), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD) but not in proximal convoluted tubule (PCT) or medullary thick ascending limb (MTAL). AM (10(-7) M) stimulated cAMP generation in Glm >> CCD = IMCD > OMCD but not in PCT or MTAL, which corresponded to the results of the expression of AM mRNA. AM (10(-8) M) slightly increased osmotic water permeability by 24% in perfused terminal IMCD. Northern blot analysis revealed high expression of AM mRNA in MC. AM (10(-7) M) stimulated cAMP generation in MC both in the presence and absence of fetal calf serum, suggesting that AM-dependent cAMP generation was evident both in cycling MC and in quiescent MC. AM may work as a diuretic peptide mainly by increasing glomerular filtration rate via cAMP in MC.

Glucocorticoids downregulate the vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts.

This study tested whether glucocorticoids regulate tubular urea transport. Urea permeability was measured in perfused inner medullary collecting duct (IMCD) subsegments from rats that underwent adrenalectomy, adrenalectomy plus replacement with a physiologic dose of glucocorticoid (dexamethasone), or sham operation. Compared with sham rats, basal urea permeability in terminal IMCD was significantly increased in adrenalectomized rats and reduced in dexamethasone-treated rats. Vasopressin significantly increased urea permeability in all three groups. In contrast, there was no difference in basal or vasopressin-stimulated urea permeability in initial IMCD between the three groups. Next, membrane and vesicle fraction proteins were isolated from inner medullary tip or base and Western analysis was performed by use of an antibody to the rat vasopressin-regulated urea transporter. Vasopressin-regulated urea transporter protein was significantly increased in both membrane and vesicle fractions from the inner medullary tip of adrenalectomized rats. There was no change in vasopressin-regulated urea transporter protein in the inner medullary base, and Northern analysis showed no change in urea transporter mRNA abundance in either inner medullary region. It was concluded that glucocorticoids can downregulate function and expression of the vasopressin-regulated urea transporter in rat terminal IMCD.