Renomedullary Interstitial Cells Research Articles

Deletion of AT1a Receptors Selectively in Renomedullary Interstitial Cells Attenuates Basal, Angiotensin II-, or Vasopressin-induced Vasoactive Factors' mRNA Expression in The Mouse Kidney Medulla

We recently shown that deletion of the vasoactive peptide angiotensin II (Ang II) AT1a receptors selectively in renomedullary interstitial cells (RMICs) of the kidney medulla significantly lowered basal blood pressure and impaired urine concentration in mice. However, the underlying signaling mechanisms involved remains poorly understood. In the present study, AT1a receptors were selectively deleted in the RMICs of the mouse kidney medulla using tamoxifen-inducible Tenascin-C-CreER2/Agtr1af/f approach. Three groups of adult male wild-type (WT) and RMIC- Agtr1a−/− mice (n=6-10) were pretreated with or without tamoxifen (150 mg/kg, i.p., for 5 days) before Ang II was infused to induce Ang II-dependent hypertension (~0.75 mg/kg/day, i.p., for 2 weeks). Systolic, diastolic, and mean arterial blood pressure and urine osmolality were determined before and after Ang II treatment. At the end of the experiment, the kidneys were harvested, and the inner and outer medulla were dissected out for the measurement of the mRNA expression of key vasoactive factors that are associated with the regulation of blood pressure and urine concentration using digital droplet PCR. Deletion of AT1a receptors in RMICs significantly decreased AT1a receptor mRNA expression by 41% (WT: 554 ± 49 vs. RMIC- Agtr1a−/−: 327 ± 10 copies/ng RNA; P<0.01) and bradykinin B2 receptor mRNA expression by 52% (WT: 10.10 ± 0.75 vs. RMIC- Agtr1a−/−: 4.90 ± 0.56 copies/ng RNA; P<0.01), without altering the expression of endothelin ETB (WT: 466 ± 21 vs. RMIC- Agtr1a−/−: 393 ± 24 copies/ng RNA; n.s.) and vasopressin V2 receptor mRNAs in the kidney medulla (WT: 580 ± 60 vs. RMIC- Agtr1a−/−: 586 ± 61 copies/ng RNA; n.s.). Deletion of AT1a receptors in RMICs also significantly decreased the expression of eNOS (WT: 87.0 ± 3.0 vs. RMIC- Agtr1a−/−: 61.2 ± 2.8 copies/ng RNA; P<0.01) and aquaporin 2 (AQP2) mRNAs (WT: 12463 ± 2015 vs. RMIC- Agtr1a−/−: 6613 ± 692 copies/ng RNA; P<0.01). Interestingly, deletion of AT1a receptors in RMICs did not alter the mRNA expression of cyclooxygenase-2 (COX-2, n.s.), prostaglandin E2 (PGE2, n.s.), epithelial sodium channel (ENaC, n.s.) or prorenin receptors (PRR, n.s.) in the kidney medulla. In response to Ang II infusion, deletion of AT1a receptors in RMICs significantly attenuated Ang II-induced expression of bradykinin B2 receptors, COX-2, eNOS, and AQP2 mRNAs ( P<0.01), respectively. Furthermore, deletion of AT1a receptors in RMICs also significantly attenuated vasopressin-induced bradykinin B2 receptor, eNOS, and AQP2 mRNA expression in the kidney medulla ( P<0.01). We concluded that AT1a receptors in RMICs in the kidney medulla play an important role in maintaining physiological blood pressure homeostasis and urine concentration process likely via AT1a receptor/bradykinin B2 receptor/eNOS/AQP2-mediated signaling pathways. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract P433: Evidence For An Important Role Of AT1a Receptors In The Renomedullary Interstitial Cells In Basal Blood Pressure Homeostasis And The Development Of Angiotensin Ii-induced Hypertension

The renomedullary interstitial cells (RMICs) are highly specialized lipid-laden interstitial fibroblasts, which form characteristic "ladder-like" structural associations with adjacent microvessels and renal tubules in the kidney medulla. We have previously reported that RMICs express abundant AT 1 (AT 1a ) receptors in vitro and in vivo , but the physiological role of AT 1 (AT 1a ) receptors in RMICs has not been determined. In the present study, we generated and phenotyped for the 1 st time a new mutant mouse model with RMIC-selective deletion of AT 1a receptors in the kidney medulla, RMIC- Agtr1a -/- , using an innovative inducible Tenascin-C-CreER2/Agtr1a f/f approach. Deletion of AT 1a receptors selectively in RMICs did not alter the development and cellular structures in the kidney medulla of RMIC- Agtr1a -/- mice. The heart wt. to body wt. ratio was lower ( P <0.01), whereas the kidney wt. to body wt. ratio was significantly higher ( P <0.01) in RMIC- Agtr1a -/- than wild-type (WT) mice without altering heart rate. Compared with WT mice, basal systolic, diastolic, and mean arterial blood pressure, as measured by the telemetry technique, were significantly lower in adult male RMIC- Agtr1a -/- mice (WT: 122 ± 3 mmHg vs. RMIC- Agtr1a -/- : 103 ± 4 mmHg, P <0.01). The decreases in basal blood pressure in RMIC- Agtr1a -/- mice was associated with significantly lower urine excretion (WT: 1.56 ± 0.15 mL/24 h vs. RMIC- Agtr1a -/- : 0.98 ± 0.13 mL/24 h, P <0.01), urinary sodium (WT: 207.9 ± 7.3 μmol/24 h vs. RMIC- Agtr1a -/- : 145.2 ± 5.4 μmol/24 h, P <0.01), potassium excretion (WT: 221.0 ± 6.7 μmol/24 h vs. RMIC- Agtr1a -/- : 160.8 ± 10.6 μmol/24 h, P <0.01), and markedly decreased urine osmolality in RMIC- Agtr1a -/- mice (WT: 1724 ± 335 mOsm/Kg H 2 O vs. RMIC- Agtr1a -/- : 1128 ± 102 mOsm/Kg H 2 O, P <0.01). Ang II infusion (0.5 mg/kg/day, i.p., 2 weeks) increased blood pressure to 148 ± 5 mmHg in WT mice, as expected, but it increased only to 129 ± 8 mmHg without further altering urine osmolality from controls in RMIC- Agtr1a -/- mice ( P <0.01). In conclusion, the present study provides new evidence for the 1st time for an important role of AT 1a receptors in RMICs in maintaining basal blood pressure homeostasis and the development of Ang II-induced hypertension.

AT1a receptor knockout selectively in renomedullary interstitial cells of the kidney lowers basal blood pressure, impairs urine concentration, and attenuates angiotensin II-induced hypertension in RMIC-Agtr1a-/- mice

We previously reported that global deletion of angiotensin II (Ang II) AT1a receptors significantly lowered basal blood pressure and impaired urine concentration in mice. As we also demonstrated that renomedullary interstitial cells (RMICs) of the kidney expressed a high level of AT1 (AT1a) receptors, we hypothesized that AT1a receptors in RMICs may play an important physiological role in maintaining basal blood pressure and urine concentration homeostasis. To test the hypothesis, we generated a novel mouse model with deletion of AT1a receptors selectively in the RMICs of the kidney medulla using inducible Tenascin-C-CreER2/Agtr1af/f recombination approach (RMIC- Agtr1a-/- ) in the present study. Adult male wildtype (WT) and RMIC- Agtr1a-/- mice (n=10-18 per group) were phenotyped under basal conditions and treated with or without a slow pressor dose of Ang II infusion via an osmotic minipump (~500 μg/kg/day, i.p.) for 2 weeks to determine the roles of AT1a receptors in RMICs in maintaining basal blood pressure and urine concentration homeostasis and in the development of Ang II-induced hypertension. Under basal conditions, telemetry systolic blood pressure was significantly lower in adult male RMIC- Agtr1a-/- than WT mice (WT: 122 ± 4 mmHg vs. RMIC-Agtr1a-/-: 102 ± 3 mmHg, P<0.01). This hypotensive phenotype in RMIC- Agtr1a-/- mice was associated with significant decreases in 24 h urine output (WT: 1.56 ± 0.15 mL/24 h vs. RMIC- Agtr1a-/- : 0.98 ± 0.13 mL/24 h, P<0.01), urinary sodium (WT: 207.9 ± 7.3 μmol/24 h vs. RMIC- Agtr1a-/- : 145.2 ± 5.4 μmol/24 h, P<0.01), and potassium excretion (WT: 221.0 ± 6.7 μmol/24 h vs. RMIC-Agtr1a-/-: 160.8 ± 10.6 μmol/24 h, P<0.01), respectively. Moreover, urine osmolality was markedly decreased in RMIC- Agtr1a-/- mice even though 24 h urine output was lowered (WT: 1724 ± 335 mOsm/Kg H2O vs. RMIC- Agtr1a-/- : 1128 ± 102 mOsm/Kg H2O, P<0.01). In response to Ang II infusion, telemetry systolic blood pressure increased to 148 ± 5 mmHg in WT mice, but it increased only to 129 ± 8 mmHg in RMIC- Agtr1a-/- mice ( P<0.01). Furthermore, Ang II infusion for 2 weeks failed to alter urine osmolality in RMIC- Agtr1a-/- mice (1226 ± 110 mOsm/Kg H2O, n.s.). In conclusion, we demonstrated for the 1st time that AT1a receptors in RMICs of the kidney medulla play a key role in physiologically maintaining basal blood pressure homeostasis and urine concentration, as well as in the development of Ang II-induced hypertension. R01DK123144, R01DK067299, R01DK102429 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Renomedullary Interstitial Cell Endothelin A Receptors Regulate BP and Renal Function.

The physiologic role of renomedullary interstitial cells, which are uniquely and abundantly found in the renal inner medulla, is largely unknown. Endothelin A receptors regulate multiple aspects of renomedullary interstitial cell function in vitro. To assess the effect of targeting renomedullary interstitial cell endothelin A receptors in vivo, we generated a mouse knockout model with inducible disruption of renomedullary interstitial cell endothelin A receptors at 3 months of age. BP and renal function were similar between endothelin A receptor knockout and control mice during normal and reduced sodium or water intake. In contrast, on a high-salt diet, compared with control mice, the knockout mice had reduced BP; increased urinary sodium, potassium, water, and endothelin-1 excretion; increased urinary nitrite/nitrate excretion associated with increased noncollecting duct nitric oxide synthase-1 expression; increased PGE2 excretion associated with increased collecting duct cyclooxygenase-1 expression; and reduced inner medullary epithelial sodium channel expression. Water-loaded endothelin A receptor knockout mice, compared with control mice, had markedly enhanced urine volume and reduced urine osmolality associated with increased urinary endothelin-1 and PGE2 excretion, increased cyclooxygenase-2 protein expression, and decreased inner medullary aquaporin-2 protein content. No evidence of endothelin-1-induced renomedullary interstitial cell contraction was observed. Disruption of renomedullary interstitial cell endothelin A receptors reduces BP and increases salt and water excretion associated with enhanced production of intrinsic renal natriuretic and diuretic factors. These studies indicate that renomedullary interstitial cells can modulate BP and renal function under physiologic conditions.

Renomedullary Interstitial Cell Endothelin A Receptors Regulated Blood Pressure and Renal Function

Renomedullary interstitial cells (RMIC) may modulate blood pressure (BP) and renal function through release of natriuretic factors and through direct effects on medullary blood flow (RMIC encircle vasa recta). Endothelin‐1 (ET‐1) modulates RMIC vasoactive factor release and contraction via ETA receptors (ETAR) in vitro. Consequently, we tested the hypothesis that knockout of ETAR in RMIC regulates renal function and BP in vivo. We bred ETARf/f mice containing loxP‐flanked exons 6–8 of the EDNRA gene with tamoxifen‐inducible tenascin‐C‐CreER2 knockin mice. Tenascin‐C‐CreER2:ETARf/f mice were injected with tamoxifen (160 mg/kg IP) for 5 consecutive days at 3 months of age to obtain RMIC ETAR KO. Littermates of the same genotype without injection served as controls. EDNRA gene recombination occurred in inner medulla, but not cortex. Inner medulla ETAR mRNA content fell by 80%, while ETBR mRNA levels were unaffected. ETRA KO did not affect BP in mice on normal (0.3% Na+) or low (0.03% Na+) salt diets. In contrast, ETAR KO reduced salt‐sensitive systolic BP elevation in mice on a high (3.2% Na+) salt diet (ETAR KO 127±3 vs. control 134±2 mmHg, n=13, 15, p<0.05). Urine volume and urinary Na+ and K+ excretion were higher in high‐salt diet‐treated ETAR KO mice compared with controls during the first 3 days of high salt intake. Urinary ET‐1, nitrite/nitrate, and prostaglandin E2 excretion were increased in high salt diet‐treated ETAR KO mice vs. controls. No significant differences were found in GFR, vasopressin, aldosterone, or renin levels between high salt fed ETAR KO and control mice. Inner medullary protein and mRNA levels of total NKCC2 and ENaC‐α and ‐β were decreased, and inner medullary cyclooxygenase‐2 (COX‐2), but not COX‐1, protein levels were increased, in high salt fed ETAR KO vs. control mice (n=8). Exogenous ET‐1 did not detectably alter RMIC intracellular Ca2+ concentration or cause RMIC contraction as assessed by ex vivo inner medulla confocal imaging. We conclude that disruption of RMIC ETAR reduces salt‐sensitive hypertension and this is associated with reductions in NKCC2 and ENaC together with elevations in several autocrine and paracrine natriuretic factors. These effects of RMIC ETAR, at least based on ex vivo studies, may not involve RMIC contraction. While further study is required, our data suggest a novel regulatory pathway for maintaining water/electrolyte homeostasis.Support or Funding InformationNHLBI P01 HL136267

A PF‐3845‐induced novel lipid fraction in mouse medullary interstitial cells causes diuresis and natriuresis via a CB1‐mediated pathway

The relationship between the endocannabinoid system in the renal medulla and the long‐term regulation of blood pressure is not understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase (FAAH), PF‐3845. Treatment of MMICs with PF‐3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. HPLC analyses of lipid extracts of MMIC culture medium revealed a 205nm‐absorbing peak that showed responsiveness to PF‐3845 treatment. The biologic activities of the PF‐3845‐induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically‐instrumented mice. Intravenous and intramedullary infusions of PIP at low doses resulted in a vasodepressor, diuretic and natriuretic effects. Pharmacological and genetic ablation of cannabinoid 1 receptors resulted in the loss of the vasodepressor, diuretic and natriuretic effect of PIP. Radioactive binding assays showed that PIP competes for binding to CB1 receptors in the presence of a full agonist (CP 55,940). PIP did not elevate urine and sodium excretion in a genetic knockout mouse model of FAAH. A PIP‐induced decrease in blood pressure at high doses of PIP was similar between FAAH WT and FAAH KO mice. These data support a model whereby PF‐3845 treatment of MMICs results in increased secretion of a neutral lipid which acts directly to promote diuresis and natriuresis via a CB1‐mediated mechanism. The diuretic and natriuretic responses to PIP are eliminated by inhibition of FAAH, suggesting an undergoing sensitization to PIP in FAAH KO mice. Efforts to identify the structure of the PF‐3845‐induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.Support or Funding InformationThis work was supported by the National Institute of Diabetes and Digestive and Kidney Disorders grant [grant R01DK102539] and the National Institute on Drug Abuse [grant P30DA033934]. S.D. was supported by the National Institute on Drug Abuse [training grant T32DA007027].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diuretic, Natriuretic, and Vasodepressor Activity of a Lipid Fraction Enhanced in Medium of Cultured Mouse Medullary Interstitial Cells by a Selective Fatty Acid Amide Hydrolase Inhibitor.

The relationship between the endocannabinoid system in the renal medulla and the long-term regulation of blood pressure is not yet understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured, and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase, PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide). Treatment of MMICs with PF-3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. High-performance liquid chromatography (HPLC) analyses of lipid extracts of MMIC culture medium revealed a 205-nm absorbing peak that showed responsiveness to PF-3845 treatment. The biologic activities of the PF-3845-induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically instrumented mice. Intramedullary and intravenous infusion of PIP at low dose rates (0.5-1 area units under the peak/10 min) stimulated diuresis and natriuresis, whereas these parameters returned toward baseline at higher doses but mean arterial pressure (MAP) was lowered. Whereas intravenous bolus doses of PIP stimulated diuresis, the glomerular filtration rate, and medullary blood flow (MBF) and reduced or had no effect on MAP, an intraperitoneal bolus injection of PIP reduced MAP, increased MBF, and had no effect on urine parameters. These data support a model whereby PF-3845 treatment of MMICs results in increased secretion of a neutral lipid that acts directly to promote diuresis and natriuresis and indirectly through metabolites to produce vasodepression. Efforts to identify the structure of the PF-3845-induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.

Hyaluronan Production by Renomedullary Interstitial Cells: Influence of Endothelin, Angiotensin II and Vasopressin.

The content of hyaluronan (HA) in the interstitium of the renal medulla changes in relation to body hydration status. We investigated if hormones of central importance for body fluid homeostasis affect HA production by renomedullary interstitial cells in culture (RMICs). Simultaneous treatment with vasopressin and angiotensin II (Ang II) reduced HA by 69%. No change occurred in the mRNA expressions of hyaluronan synthase 2 (HAS2) or hyaluronidases (Hyals), while Hyal activity in the supernatant increased by 67% and CD44 expression reduced by 42%. The autocoid endothelin (ET-1) at low concentrations (10−10 and 10−8 M) increased HA 3-fold. On the contrary, at a high concentration (10−6 M) ET-1 reduced HA by 47%. The ET-A receptor antagonist BQ123 not only reversed the reducing effect of high ET-1 on HA, but elevated it to the same level as low concentration ET-1, suggesting separate regulating roles for ET-A and ET-B receptors. This was corroborated by the addition of ET-B receptor antagonist BQ788 to low concentration ET-1, which abolished the HA increase. HAS2 and Hyal2 mRNA did not alter, while Hyal1 mRNA was increased at all ET-1 concentrations tested. Hyal activity was elevated the most by high ET-1 concentration, and blockade of ET-A receptors by BQ123 prevented about 30% of this response. The present study demonstrates an important regulatory influence of hormones involved in body fluid balance on HA handling by RMICs, thereby supporting the concept of a dynamic involvement of interstitial HA in renal fluid handling.

The effects of different inhibitory pathways of prostaglandin E2 biosynthesis on renomedullary interstitial cells in rats: a multidisciplinary study

Abstract Renomedullary interstitial cells (RMICs) are the most dominant cell type in inner renal medulla. Their most distinct characteristic is the presence of multiple lipid droplets in their cytoplasm. These lipid droplets are

Renomedullary Interstitial Cell Tumors: Pathologic Features and Clinical Correlations.

Renomedullary interstitial cell tumors are common incidental findings in kidney specimens. Despite their frequency, little is known about their morphology and pathogenesis. Kidneys from 402 unselected autopsies were sectioned at 1 to 2 mm intervals, and all lesions were examined histologically. A total of 421 renomedullary interstitial cell tumors were present in 150 patients (37%), ranging from 1 to 23 tumors per patient (mean=3). There was no statistically significant difference in age, sex, hypertension, heart weight, tobacco smoking, diabetes mellitus, and renal function between patients with renomedullary interstitial cell tumors and those without. Almost half the patients with renomedullary interstitial cell tumors (41%) had bilateral tumors, and they were older than patients with unilateral tumors (P=0.0007). The tumors ranged in size from 0.5 to 6 mm (mean 1.7 mm). The lesions varied in cellularity: fibrous stroma was found in older patients, whereas cellular and hypocellular stroma predominated in younger patients (P=0.001 and P<0.0001, respectively). Entrapped renal tubules were found throughout the tumor in younger patients and smaller tumors, whereas the absence of entrapped tubules or their location only at the periphery of the lesion were common in older patients and larger tumors (P=0.02 and P<0.0001, respectively). Ropey brightly eosinophilic material, found in 26% of tumors, was not amyloid but collagen type III. This material was observed in older patients (P<0.0001) with larger tumors (P<0.0001) and was also correlated to higher heart weight (P=0.003) but not to hypertension (P=0.11). On the basis of our findings, renomedullary interstitial cell tumors appear to originate as a proliferation of renomedullary interstitial cells between medullary tubules. As their size increases, cellularity decreases, ropey eosinophilic material is deposited, and tubules disappear.

Endothelin, Ang II, vasopressin and osmolality regulate hyaluronan turnover by renomedullary interstitial cells suggesting an involvement of this matrix component in fluid handling (860.9)

The content of the matrix component hyaluronan (HA) change in the medullary interstitium in relation to hydration status in vivo: elevated during hydration, reduced during dehydration suggesting a role in fluid handling by affecting interstitial fluid flux. We examined the effect of osmolality and fluid regulating hormones on HA‐turnover by RMIC in culture. Reducing media osmolality to simulate hydration, increased supernatant HA 4‐fold. Cell HA synthase (HAS) 2 and Hyaluronidase (Hyal) 2 mRNA expressions were unaltered while Hyal 1 mRNA was reduced by 35%. Supernatant Hyal activity was reduced by 44%. An inhibitor of Hyal activity (Asc‐P) elevated HA to similar high levels as low osmolality. AngII reduced HA by 58% and combined treatment with vasopressin reduced HA even further (69%). HAS and Hyal mRNA were stable while Hyal activity increased by 38% by AngII and 67% by the combination. Low dose endothelin (ET, 10‐10M) increased HA 3‐fold which was inhibited by ET‐B receptor blocker BQ788. On the contrary, high dose ET (10‐6M) reduced HA by 47%. The ET‐A receptor antagonist BQ123 not only reversed the reducing effect of high dose ET on HA but elevated it to the same level as low dose ET suggesting importance of ET‐B receptors. The study demonstrates a regulatory influence of osmolality and fluid regulating hormones on HA turnover, supporting the concept of an involvement of medullary interstitial HA in renal fluid handling by affecting interstitial permeability.Grant Funding Source: Supported by the Swedish Research Council‐Medicine

Structural Characteristics of Renomedullary Interstitial Cells of Hypertensive ISIAH Rats

The structural characteristics of the renal medulla and its interstitial cells were studied in hypertensive ISIAH rats (in comparison with normotensive WAG rats) in order to clear out the role of the renomedullary interstitial cells in the mechanisms of AP regulation. Morphometric electron microscopic analysis and immunohistochemical studies in ISIAH rats detected the initial signs of the renomedullary sclerosis. The renomedullary interstitial cells of ISIAH rats were characterized by higher numerical density and were larger in size, with a higher volumic share of their secretory granules. These structural features of the studied cells were regarded as signs of their more intense functional activity aimed at hypertension suppression.

Mechanically‐induced intercellular calcium waves in renal medullary interstitial cells

In addition to providing structural support, renomedullary interstitial cells (RMIC) are thought to play an active physiological role in regulating renal medullary function. RMICs have been shown to have contractile properties, however, the cellular and molecular mechanisms of this action remain unknown. We aimed to identify the existence of molecular elements of an intercellular calcium wave in cultured rat RMICs using Fluo‐4/Fura red and confocal fluorescence microscopy. RMICs expressed connexins (Cx) Cx30 and Cx45 and P2X4 and P2Y2 purinergic receptors measured by RT‐PCR and immunohistochemistry. Mechanical stimulation of a single RMIC caused a 4±0.2‐fold increase in [Ca2+]i (n=45) which was propagated throughout the monolayer (16 ± 1.2 um/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances &gt;800 um. The gap junction uncoupler 18a‐glycyrrhetinic acid (18a‐GA, 25 uM), breakdown of extracellular ATP (apyrase and hexokinase, 50 U/mL each), or P2 receptor inhibition (suramin, 50 uM) all decreased the speed and D[Ca2+]i of the calcium wave. Physical contact between cells was not required for calcium wave propagation. These studies suggest that mechanosensitive, ATP‐releasing Cx hemichannels and cell‐to‐cell purinergic signaling are key molecular elements of the RMIC calcium wave, which may serve an important role in RMIC‐mediated renal medullary function.

Hormonal regulation of renomedullary hyaluronan

Hyaluronan (HA) is involved in renomedullary water handling through its water-binding capacity. This study addressed the effect of hormones involved in regulating fluid-electrolyte homeostasis on renomedullary HA content in vivo and in vitro. The kidneys from rats treated with L-NAME, indomethacin, vasopressin (AVP) or methylprednisolone (MP) during euvolaemia or water loading were analysed for HA by RIA, ELISA and histochemical staining. HA was measured in renomedullary interstitial cells treated with AVP, angiotensin II (Ang II) or a combination of AVP and Ang II. Baseline renal cortical and medullary HA content was unaffected by 2 h of intravenous treatment with L-NAME (NOS inhibitor) or indomethacin (cyclo-oxygenase inhibitor), whereas AVP reduced medullary HA by 33%. During 2 h of acute water loading, diuresis was accompanied by an increase in renomedullary HA (+45%), but cortical HA was unaffected. In both L-NAME- and indomethacin-treated animals, the water loading-induced increase in renomedullary HA was absent, indicating involvement of NO and prostaglandins. After 7 days of MP treatment, medullary HA was reduced by 40%, but the water loading-induced elevation in HA remained. In cultured renomedullary interstitial cells, AVP reduced the HA content in the supernatant by 63%, and simultaneous treatment with Ang II reduced the HA content even further (95%). AVP reduces HA content, and NO and prostaglandins are needed for the increase in HA during water loading.

Mechanism of Vasopressin-Induced Contraction of Renal Medullary Interstitial Cells

Background/Aims: Previous studies have identified a contractile function for renomedullary interstitial cells (RMIC). Such studies focused on the mechanism of endothelin-1-induced RMIC contraction; however, vasopressin (AVP) was also noted to contract RMIC. Since AVP-induced RMIC contraction may be relevant to the medullary effects of AVP on urinary concentration, these initial observations have been extended to examination of the mechanism of AVP-induced RMIC contraction. Methods: Cultured rat RMIC were exposed to AVP and other agents, and examined using video microscopy. Results: AVP caused a slowly developing and dose-dependent reduction in RMIC surface area. AVP-induced RMIC contraction was abolished by blockade of V₁, but not V₂, receptors. Nifedipine and nickel reduced AVP-stimulated RMIC contraction, indicating that this effect is dependent upon dihydropyridine-sensitive calcium channels. H7, a protein kinase C inhibitor, completely abrogated AVP action, while the nitric oxide synthase inhibitor, NMMA, had no effect. Indomethacin enhanced AVP-induced RMIC contraction, and addition of PGE₂ together with indomethacin reduced AVP action. Conclusion: These data indicate that AVP potently contracts RMIC via V₁ receptor stimulation of PKC and intracellular calcium accumulation, and that AVP-stimulated prostaglandin production downregulates the contractile effect of AVP on RMIC. AVP modulation of RMIC contraction may be involved in the regulation of urinary concentration.

Posttranscriptional mechanisms contribute to osmotic regulation of ANG type 1 receptors in cultured rat renomedullary interstitial cells

Previously, we showed that ANG II receptors in cultured rat renomedullary interstitial cells (RMICs) are osmotically regulated (19). The current study examined the mechanisms underlying this osmotic regulation in RMICs cultured in isoosmotic (300 mosmol/kgH2O) and hyperosmotic (600 mosmol/kgH2O) conditions. Radioligand competition analysis coupled with RNase protection assays (RPA) and ligand-mediated receptor internalization studies revealed that RMICs primarily express the type 1a angiotensin receptor (AT(1a)R). When cultured under hyperosmotic conditions, the density (B(max)) of AT1R in RMIC membranes decreased by 31% [B(max) (pmol/mg protein): 300 mosmol/kgH2O, 6.44 +/- 0.46 vs. 600 mosmol/kgH2O, 4.42 +/- 0.37, n = 8, P < 0.01], under conditions in which no detectable changes in AT(1a)R mRNA expression or in the kinetics of ligand-mediated AT1R internalization were observed. RNA electromobility shift assays showed that RNA protein complex (RPC) formation between RMIC cytosolic RNA binding proteins and the 5' leader sequence (5'LS) of the AT(1a)R was increased 1.5-fold under hyperosmotic conditions [5'LS RPC (arbitrary units): 300 mosmol/kgH2O, 0.79 +/- 0.08 vs. 600 mosmol/kgH2O, 1.17 +/- 0.07, n = 4, P < 0.01]. These results suggest that the downregulation of AT(1a)R expression in RMICs cultured under hyperosmotic conditions is regulated at the posttranscriptional level by RNA binding proteins that interact within the 5'LS of the AT(1a)R mRNA. The downregulation of AT(1a)R expression under hyperosmotic conditions may be an important mechanism by which the activity of ANG II is regulated in the hyperosmotic renal medulla.

Regulation of cyclooxygenase expression by vasopressin in rat renal medulla.

The antagonism between prostaglandin and vasopressin represents a classic negative feedback loop. It is not clear whether cyclooxygenase (COX)-2 and/or COX-1 expression is involved in elevated prostaglandin production stimulated by vasopressin in vivo. In the present study, we explored vasopressin regulation of medullary COX-2 and COX-1 expression acutely and chronically in rats. Medullary COX-1 expression was moderately lower and COX-2 expression was significantly lower in adult male Brattleboro rats than age-matched Long-Evans controls. Chronic treatment of Brattleboro rats with vasopressin for 1 wk led to a decrease in urine volume and a moderate increase in medullary COX-1; in contrast, medullary COX-2 expression was almost undetectable in untreated rats but was dramatically up-regulated with vasopressin treatment and was accompanied by increased urinary prostaglandin E(2) excretion. Further investigation revealed that both V1 and V2 receptors were involved in chronic medullary COX-1 and COX-2 up-regulation. Acute treatment with specific V1 or V2 receptor agonists resulted in specific increases in medullary COX-2, which was prevented by furosemide. Vasopressin did not affect COX-2 expression in cultured renomedullary interstitial cells. These data demonstrate that vasopressin stimulates medullary COX-2 expression through activation of both V1 and V2 receptors, and this stimulation is indirect and probably involves increased medullary electrolyte tonicity.

Low endogenous glucocorticoid allows induction of kidney cortical cyclooxygenase-2 during postnatal rat development.

In postnatal weeks 2-4, cyclooxygenase-2 (COX-2) is induced in the rat kidney cortex where it is critically involved in final stages of kidney development. We examined whether changes in circulating gluco- or mineralocorticosteroids or in their renal receptors regulate postnatal COX-2 induction. Plasma corticosterone concentration peaked at birth, decreased to low levels at days 3-13, and increased to adult levels from day 22. Aldosterone peaked at birth and then stabilized at adult levels. Gluco- and mineralocorticoid receptor (GR and MR) mRNAs were expressed stably in kidney before, during, and after COX-2 induction. 11 beta-hydroxysteroid dehydrogenase 2 was induced shortly after birth and was widely distributed in the whole collecting duct system in the suckling period and then returned to an adult pattern. Supplementation with corticosterone (20 mg.kg-1.day-1) or GR-specific dexamethasone (1 mg.kg-1.day-1) during low endogenous corticosterone suppressed renal COX-2 mRNA and protein and led to a restricted distribution of COX-2 immunolabeling. The ability of glucocorticoids to affect COX-2 was reflected in colocalization of GR-alpha and COX-2 immunoreactivity and mRNAs in thick ascending limb of Henle's loop. The MR antagonist potassium canrenoate (20 mg.kg-1.day-1) enhanced COX-2 expression from days 5 to 10, but low MR-specific concentrations of DOCA (1 mg.kg-1.day-1) had no effect on COX-2. Renomedullary interstitial cells expressed GR-alpha and COX-2. Dexamethasone suppressed COX-2 in these cells. Thus low plasma concentrations of corticosterone allowed for cortical and medullary COX-2 induction during postnatal kidney development. Increased circulating glucocorticoid in the postnatal period may damage late renal development through inhibition of COX-2.

Changes in mean arterial pressure predict degranulation of renomedullary interstitial cells.

1. Renomedullary interstitial cells (RMIC) are characterized by numerous intracellular granules thought to contain renal medullary antihypertensive substances. However, the nature of the trigger for RMIC degranulation remains to be elucidated. The present study examines the effects of acute alterations in mean arterial pressure (MAP) and medullary blood flow (MBF) on RMIC granulation. 2. Basal MAP and MBF in anaesthetized Sprague-Dawley rats (n = 4/group) were altered by intravenous infusions of vasoactive agents, including angiotensin II alone or with a nitric oxide (NO) synthase inhibitor (N-omega-nitro-l-arginine) or NO donor (sodium nitroprusside), noradrenaline and by carotid artery clamping. Following these treatments, kidneys were examined by electron microscopy and the absolute volume of granules in the renal medulla was calculated using unbiased stereological methods. 3. Acute increases in MAP, regardless of the treatment causing the increase, were associated with a reduction in the absolute volume of granules in the range of 42-67%. Regression analysis revealed that only increases in MAP, but not MBF, strongly predict RMIC degranulation. 4. Despite previous reports that changes in MBF activate renomedullary antihypertensive activity, we conclude that the change in MAP is an important determinant of the activity of the blood pressure-lowering mechanism of the renal medulla, with the assumption that the medullary lipids mediate the antihypertensive property of the renal medulla.

ER and PR in Renomedullary Interstitial Cells During Syrian Hamster Estrogen-Induced Tumorigenesis: Evidence for Receptor-Mediated Oncogenesis

ER and PR in Renomedullary Interstitial Cells During Syrian Hamster Estrogen-Induced Tumorigenesis: Evidence for Receptor-Mediated Oncogenesis