Isolation and perfusion of rat inner medullary vasa recta.

Outer medullary isolated descending vasa recta have proven to be experimentally tractable, and consequently much has been learned about outer medullary vasa recta endothelial transport, pericyte contractile mechanisms, and tubulovascular interactions. In contrast, inner medullary vasa recta have never been isolated from any species, and therefore isolated vasa recta function has never been subjected to in vitro quantitative evaluation. As we teased out inner medullary thin limbs of Henle's loops from the Munich-Wistar rat, we found that vasa recta could be isolated using similar protocols. We isolated ∼30 inner medullary vasa recta from 23 adult male Munich-Wistar rats and prepared them for brightfield or electron microscopy, gene expression analysis by RT-PCR, or isolated tubule microperfusion. Morphological characteristics include branching and nonbranching segments exhibiting a thin endothelium, axial surface filaments radiating outward giving vessels a hairy appearance, and attached interstitial cells. Electron microscopy shows multiple cells, tight junctions, and either continuous or fenestrated endothelia. Isolated vasa recta express genes encoding the urea transporter UT-B and/or the fenestral protein PV-1, genes expressed in descending or ascending vasa recta, respectively. The transepithelial NaCl permeability (383.3 ± 60.0 × 10(-5) cm/s, mean ± SE, n = 4) was determined in isolated perfused vasa recta. Future quantitative analyses of isolated inner medullary vasa recta should provide structural and functional details important for more fully understanding fluid and solute flows through the inner medulla and their associated regulatory pathways.

Dicer1 activity in the stromal compartment regulates nephron differentiation and vascular patterning during mammalian kidney organogenesis.

MicroRNAs, activated by the enzyme Dicer1, control post-transcriptional gene expression. Dicer1 has important roles in the epithelium during nephrogenesis, but its function in stromal cells during kidney development is unknown. To study this, we inactivated Dicer1 in renal stromal cells. This resulted in hypoplastic kidneys, abnormal differentiation of the nephron tubule and vasculature, and perinatal mortality. In mutant kidneys, genes involved in stromal cell migration and activation were suppressed as were those involved in epithelial and endothelial differentiation and maturation. Consistently, polarity of the proximal tubule was incorrect, distal tubule differentiation was diminished, and elongation of Henle's loop attenuated resulting in lack of inner medulla and papilla in stroma-specific Dicer1 mutants. Glomerular maturation and capillary loop formation were abnormal, whereas peritubular capillaries, with enhanced branching and increased diameter, formed later. In Dicer1-null renal stromal cells, expression of factors associated with migration, proliferation, and morphogenic functions including α-smooth muscle actin, integrin-α8, -ß1, and the WNT pathway transcriptional regulator LEF1 were reduced. Dicer1 mutation in stroma led to loss of expression of distinct microRNAs. Of these, miR-214, -199a-5p, and -199a-3p regulate stromal cell functions ex vivo, including WNT pathway activation, migration, and proliferation. Thus, Dicer1 activity in the renal stromal compartment regulates critical stromal cell functions that, in turn, regulate differentiation of the nephron and vasculature during nephrogenesis.

Spatial organization of the vascular bundle and the interbundle region: three-dimensional reconstruction at the inner stripe of the outer medulla in the mouse kidney.

The vascular bundle (VB) is a complex structure that resides in the inner stripe of the outer medulla. At present, the tubulovascular spatial organization of the VB, which is crucial for the formation of the osmolarity gradient and for solute transport, is still under debate. In this study, we used computer-assisted digital tracing combined with aquaporin-1 immunohistochemistry to reconstruct all tubules and vessels in the VB of the mouse kidney. We found, first, that the descending and ascending vasa recta travelled exclusively through the VB. The ascending vasa recta received no tributaries (no branches) along their entire path in the medulla and were not connected with the capillary plexus in the interbundle region. Second, a specific group of the descending vasa recta were closely accompanied by the longest ascending vasa recta, which connected only to the capillary plexus at the tip of the papilla. Third, the descending thin limbs of all short-looped nephrons travelled exclusively through the outer part of the VB. The loops of these nephrons (both descending and ascending parts) were distributed in a regular pattern based on their length. Finally, the thick ascending limbs of all long-looped nephrons were located at the margin of the VB (except a few within the VB), which formed a layer separating the VB from the interbundle region. In conclusion, our three-dimensional analysis of the VB strongly suggest a lateral osmolarity heterogeneity across the inner stripe of the outer medulla, which might work as a driving force for water and solute transport.

Isolated interstitial nodal spaces may facilitate preferential solute and fluid mixing in the rat renal inner medulla.

Recent anatomic findings indicate that in the upper inner medulla of the rodent kidney, tubules, and vessels are organized around clusters of collecting ducts (CDs). Within CD clusters, CDs and some of the ascending vasa recta (AVR) and ascending thin limbs (ATLs), when viewed in transverse sections, form interstitial nodal spaces, which are arrayed at structured intervals throughout the inner medulla. These spaces, or microdomains, are bordered on one side by a single CD, on the opposite side by one or more ATLs, and on the other two sides by AVR. To study the interactions among these CDs, ATLs, and AVR, we have developed a mathematical compartment model, which simulates steady-state solute exchange through the microdomain at a given inner medullary level. Fluid in all compartments contains Na(+), Cl(-), urea and, in the microdomain, negative fixed charges that represent macromolecules (e.g., hyaluronan) balanced by Na(+). Fluid entry into AVR is assumed to be driven by hydraulic and oncotic pressures. Model results suggest that the isolated microdomains facilitate solute and fluid mixing among the CDs, ATLs, and AVR, promote water withdrawal from CDs, and consequently may play an important role in generating the inner medullary osmotic gradient.

Iodinated contrast media cause endothelial damage leading to vasoconstriction of human and rat vasa recta.

Contrast-induced acute kidney injury is an important clinical event with a worldwide increasing number of cases. Medullary hypoperfusion and hypoxia due to constriction of vasa recta are main factors in the pathophysiology of acute kidney injury. However, the mechanism of contrast media (CM)-induced vessel constriction is not known. We tested the hypothesis that vasa recta constriction is a consequence of endothelial dysfunction due to the cytotoxicity of CM. Human and rat descending vasa recta (DVR) were isolated and perfused with CM, and the luminal diameter was analyzed. For morphological analysis of the endothelium, renal arteries were CM perfused and then processed for electron microscopy. Transcellular electrical resistance was used to estimate CM-induced changes in the permeability of human umbilical vein endothelial cell (HUVEC) layers. Perfusion with CM constricted human and rat DRV (to 54.3 and 50.9% of initial diameter, respectively). This was blunted by adrenomedullin (77.7 and 77.1%, respectively). The ANG II response was enhanced by CM in rat DVR (reduction to 15.6 and 35.0% of initial diameter, respectively). Adrenomedullin blunted this effect (67.5%). CM led to endothelial damage of renal arteries characterized by a ragged surface, with sharply protruding intimal folds, spindle-like shape, and bulging in the lumen. These phenomena were reduced by adrenomedullin. The permeability of HUVEC cell layers was increased by CM, and this went along with increased myosin light chain phosporylation. Again, adremonedullin reduced the CM effect. Our study suggests that the constrictor effect of CM on the renal medullary microvasculature is a consequence of endothelial cell damage and the resulting endothelial dysfunction.

The mammalian urine concentrating mechanism: hypotheses and uncertainties.

The urine concentrating mechanism of the mammalian kidney, which can produce a urine that is substantially more concentrated than blood plasma during periods of water deprivation, is one of the enduring mysteries in traditional physiology. Owing to the complex lateral and axial relationships of tubules and vessels, in both the outer and inner medulla, the urine concentrating mechanism may only be fully understood in terms of the kidney's three-dimensional functional architecture and its implications for preferential interactions among tubules and vessels.

Nitric oxide reduces flow-induced superoxide production via cGMP-dependent protein kinase in thick ascending limbs.

We have shown that increased luminal flow induces O(2)(-) and nitric oxide (NO) production in thick ascending limbs (TALs). However, the interaction of flow-stimulated NO and O(2)(-) in TALs is unclear. We hypothesized that NO inhibits flow-induced O(2)(-) production in TALs via cGMP-dependent protein kinase (PKG). We measured flow-stimulated O(2)(-) production in rat TALs using dihydroethidium in the absence and presence of L-arginine (0.3 mM), the substrate for NO synthase. The addition of L-arginine reduced flow-induced net O(2)(-) production from 68 +/- 9 to 17 +/- 4 AU/s (P < 0.002). The addition of the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 5 mM) in the presence of L-arginine stimulated production (L-arginine: 15 +/- 4 AU/s vs. L-arginine + L-NAME: 63 +/- 7 AU/s; P < 0.002). The guanylate cyclase inhibitor LY-83583 (10 microM) also enhanced flow-induced net O(2)(-) production in the presence of L-arginine (L-arginine: 7 +/- 4 AU/s vs. L-arginine + LY-83583: 53 +/- 7 AU/s; P < 0.01). In the presence of LY-83583, L-arginine only reduced flow-induced net O(2)(-) by 36% (LY-83583: 80 +/- 7 AU/s vs. LY-83583 + L-arginine: 51 +/- 3 AU/s; P < 0.006). The cGMP analog dibutyryl (db)-cGMP reduced flow-induced net O(2)(-) from 39 +/- 9 to 7 +/- 3 AU/s (P < 0.03). The PKG inhibitor KT-5823 (5 microM) partially restored flow-induced net O(2)(-) in the presence of L-arginine (L-arginine: 4 +/- 4 AU/s vs. L-arginine + KT-5823: 32 +/- 9 AU/s; P < 0.03) and db-cGMP (db-cGMP: 9 +/- 7 AU/s vs. db-cGMP + KT-5823: 54 +/- 5 AU/s; P < 0.01). Phosphodiesterase II inhibition had no effect on arginine-inhibited O(2)(-) production. We conclude that 1) NO reduces flow-stimulated O(2)(-) production, 2) this occurs primarily via the cGMP/PKG pathway, and 3) O(2)(-) scavenging by NO plays a minor role.

Quantitative analysis of functional reconstructions reveals lateral and axial zonation in the renal inner medulla.

Three-dimensional functional reconstructions of descending thin limbs (DTLs) and ascending thin limbs (ATLs) of loops of Henle, descending vasa recta (DVR), ascending vasa recta (AVR), and collecting ducts (CDs) permit quantitative definition of lateral and axial zones of probable functional significance in rat inner medulla (IM). CD clusters form the organizing motif for loops of Henle and vasa recta in the initial 3.0-3.5 mm of the IM. Using Euclidean distance mapping, we defined the lateral boundary of each cluster by pixels lying maximally distant from any CD. DTLs and DVR lie almost precisely on this independently defined boundary, placing them in the intercluster interstitium maximally distant from any CD. ATLs and AVR lie in a nearly uniform pattern throughout intercluster and intracluster regions, which we further differentiated by a polygon around CDs in each cluster. Loops associated with individual CD clusters show a similar axial exponential decrease as all loops together in the IM. Because approximately 3.0-3.5 mm below the IM base CD clusters cease to form the organizing motif, all DTLs lack aquaporin 1 (AQP1), and all vasa recta are fenestrated, we have designated the first 3.0-3.5 mm of the IM the "outer zone" (OZ) and the final 1.5-2.0 mm the "inner zone" (IZ). We further subdivided these into OZ-1, OZ-2, IZ-1, and IZ-2 on the basis of the presence of completely AQP1-null DTLs only in the first 1 mm and on broad transverse loop bends only in the final 0.5 mm. These transverse segments expand surface area for probable NaCl efflux around loop bends from approximately 40% to approximately 140% of CD surface area in the final 100 microm of the papilla.

Flow increases superoxide production by NADPH oxidase via activation of Na-K-2Cl cotransport and mechanical stress in thick ascending limbs.

Superoxide (O(2)(-)) regulates renal function and is implicated in hypertension. O(2)(-) production increases in response to increased ion delivery in thick ascending limbs (TALs) and macula densa and mechanical strain in other cell types. Tubular flow in the kidney acutely varies causing changes in ion delivery and mechanical stress. We hypothesized that increasing luminal flow stimulates O(2)(-) production by NADPH oxidase in TALs via activation of Na-K-2Cl cotransport. We measured intracellular O(2)(-) in isolated rat TALs using dihydroethidium in the presence and absence of luminal flow and inhibitors of NADPH oxidase, Na-K-2Cl cotransport, and Na/H exchange. In the absence of flow, the rate of O(2)(-) production was 5.8 +/- 1.4 AU/s. After flow was initiated, it increased to 29.7 +/- 4.3 AU/s (P < 0.001). O(2)(-) production was linearly related to flow. Tempol alone and apocynin alone blocked the flow-induced increase in O(2)(-) production (3.5 +/- 1.7 vs. 4.5 +/- 2.8 AU/s and 8.2 +/- 2.1 vs. 10.6 +/- 2.8 AU/s, respectively). Furosemide decreased flow-induced O(2)(-) production by 55% (37.3 +/- 5.2 to 16.8 +/- 2.8 AU/s; P < 0.002); however, dimethylamiloride had no effect. Finally, we examined whether changes in mechanical forces are involved in flow-induced O(2)(-) production by using a Na-free solution to perfuse TALs. In the absence of NaCl, luminal flow enhanced O(2)(-) production (1.5 +/- 0.5 to 13.5 +/- 1.1 AU/s; P < 0.001), approximately 50% less stimulation than when flow was increased in the presence of luminal NaCl. We conclude that flow stimulates O(2)(-) production in TALs via activation of NADPH oxidase and that NaCl absorption due to Na-K-2Cl cotransport and flow-associated mechanical factors contribute equally to this process.

Enhanced superoxide production in renal outer medulla of Dahl salt-sensitive rats reduces nitric oxide tubular-vascular cross-talk.

Studies were conducted to determine whether the diffusion of NO from the renal medullary thick ascending limb (mTAL) to the contractile pericytes of surrounding vasa recta was reduced and, conversely, whether diffusion of oxygen free radicals was enhanced in the salt-sensitive Dahl S rat (SS/Mcwi). Angiotensin II ([Ang II] 1 micromol/L)-stimulated NO and superoxide (O(2)(*-)) production were imaged by fluorescence microscopy in thin tissue strips from the inner stripe of the outer medulla. In prehypertensive SS/Mcwi rats and a genetically designed salt-resistant control strain (consomic SS-13(BN)), Ang II failed to increase either NO or O(2)(*-) in pericytes of isolated vasa recta. Ang II stimulation resulted in production of NO in epithelial cells of the mTAL that diffused to vasa recta pericytes of SS-13(BN) rats but not in SS/Mcwi rats except when tissues were preincubated with the superoxide scavenger TIRON (1 mmol/L). Ang II resulted in a greater increase of O(2)(*-) in the mTAL of SS/Mcwi compared with SS.13(BN) mTAL. The O(2)(*-) diffused to adjoining pericytes in tissue strips only in SS/Mcwi rats but not in control SS-13(BN) rats. Diffusion of Ang II-stimulated O(2)(*-) from mTAL to vasa recta pericytes was absent when tissue strips from SS/Mcwi rats were treated with the NO donor DETA-NONOate (20 micromol/L). We conclude that the SS/Mcwi rat exhibits increased production of O(2)(*-) in mTAL that diffuses to surrounding vasa recta and attenuates NO cross-talk. Diffusion of O(2)(*-) from mTAL to surrounding tissue could contribute to reduced bioavailability of NO, reductions of medullary blood flow, and interstitial fibrosis in the outer medulla of SS/Mcwi rats.

Computer analysis of the significance of the effective osmolality for urea across the inner medullary collecting duct in the operation of a single effect for the counter-current multiplication system.

BACKGROUND: Although urea and water are transported across separate pathways in the apical membrane of the inner medullary collecting duct (IMCD), the existence of a cellular diffusion barrier as an unstirred layer makes it possible to use coefficients of effective osmotic force (sigma*) as equivalent to reflection coefficients. The difference in effective osmolality between urea and NaCl across the IMCD becomes a driving force for water if the compositions of solutes are different between tubular lumen and interstitium. Since reported values for sigma*(urea) are discrepant, we compared the efficiency of a single effect in the counter-current system between an ascending thin limb (ATL) and the IMCD, with the interposition of capillary networks (CNW), between two models with sigma(urea)* = 0.7 (model 1) and sigma(urea)* = 1.0 (model 2). METHODS: The time courses (within 3 s) of solute and the water transport profiles among ATL, CNW, and IMCD were simulated with a computer in the absence of flow in each compartment. RESULTS: In spite of small differences in the profiles of urea and NaCl concentrations between the two models, model 1 displayed a larger volume flux in the IMCD than model 2, resulting in an increase of osmolality in the IMCD and a decrease of osmolality in the ATL. These findings are vital for the operation of the counter-current multiplication system. CONCLUSIONS: The concept of coefficients for effective osmotic force can be applied to the counter-current model between the IMCD and the ATL with the interposition of CNW. The model of sigma(urea)* = 0.7 is more efficient than that of sigma(urea)* = 1.0.

Unraveling the relationship between macula densa cell volume and luminal solute concentration/osmolality.

At the macula densa, flow-dependent changes in luminal composition lead to tubuloglomerular feedback and renin release. Apical entry of sodium chloride in both macula densa and cortical thick ascending limb (cTAL) cells occurs via furosemide-sensitive sodium-chloride-potassium cotransport. In macula densa, apical entry of sodium chloride leads to changes in cell volume, although there are conflicting data regarding the directional change in macula densa cell volume with increases in luminal sodium chloride concentration. To further assess volume changes in macula densa cells, cTAL-glomerular preparations were isolated and perfused from rabbits, and macula densa cells were loaded with fluorescent dyes calcein and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate. Cell volume was determined with wide-field and multiphoton fluorescence microscopy. Increases in luminal sodium chloride concentration from 0 to 80 mmol/l at constant osmolality led to cell swelling in macula densa and cTAL cells, an effect that was blocked by luminal application of furosemide. However, increases in luminal sodium chloride concentration from 0 to 80 mmol/l with concomitant increases in osmolality caused sustained decreases in macula densa cell volume but transient increases in cTAL cell volume. Increases in luminal osmolality with urea also resulted in macula densa cell shrinkage. These studies suggest that, under physiologically relevant conditions of concurrent increases in luminal sodium chloride concentration and osmolality, there is macula densa cell shrinkage, which may play a role in the macula densa cell signaling process.

Proximal tubular stop flow pressure: an index of glomerular capillary pressure?

Central to the assumption that glomerular capillary pressure (Pgc) can be equated with the sum of arterial oncotic pressure (pi art) and the pressure in a blocked proximal tubule ("stop flow" pressure, Psf) is that filtration ceases in the blocked nephron. Should filtration not cease, but continue at a rate equal to tubular reabsorption between the block and the glomerulus, Psf, for a given Pgc, will depend on the distance between block and glomerulus. This would have serious consequences for the interpretation of Psf, particularly in respect to its frequent use in analysis of the tubuloglomerular feedback (TGF) mechanism. Experiments were performed in anaesthetized Wistar rats to examine whether a length dependency of Psf exists and, if so, to what extent this relationship alters during maximal TGF stimulation by loop of Henle perfusion. A length dependency of Psf existed both in the absence and presence of loop flow. The regression coefficients were significantly different from 0 and from each other. Pgc cannot thus be equated with the sum of Psf and pi art. The length dependent error in Psf makes it unsuitable for the quantitative analysis of TGF and glomerular haemodynamics.

Tubulo-vascular relationships in the developing kidney.

The postnatal development of the tubulo-vascular relationships in the kidney was studied in 60 rats. The kidney is relatively immature at birth and only the juxtamedullary glomeruli are present. As their loops of Henle develop they grow down into the medulla around the previously developed collecting ducts, forming tubulovascular 'units' around whose periphery the shorter loops of the more peripheral glomeruli grow down. The bundles of vasa recta develop in loose interstitial tissue which lies between the tubulo-vascular units so that the peripheral vessels of the bundles, which are ascending vasa recta, lie in close relationship with the descending short loops of Henle. Thus, the vascular bundles do not form the central axis around which the medullary components develop but rather take up the available space left after the tubulo-vascular units have developed. Development is virtually complete by the fourth week of postnatal life.

Studies on the mechanism of "exaggerated natriuresis" in essential hypertension.

Prompt and exaggerated natriuresis and diuresis were seen one to two hours after the starting of an infusion of 300 ml of 3% saline for one hour in patients with essential hypertension on a high sodium chloride intake. There were no significant differences in urinary volume and sodium excretion after the saline load in patients with normal and low plasma renin activity. The inhibition of angiotensin converting enzyme with SQ 14225 in patients with normal plasma renin activity did not produce additional natriuresis and diuresis after the saline load. Mean arterial blood pressure and/or changes in mean arterial blood pressure after the saline load showed a positive correlation with urinary volume and sodium excretion in each collection period in hypertensive subjects. Free water reabsorption in hypertensives was lower at high levels of osmolar clearance than that in control subjects. These results suggest that "exaggerated natriuresis" in essential hypertension is due to a decrease in tubular sodium reabsorption, which may be the result of intrarenal hemodynamic changes related to high blood pressure per se. The decreased medullary osmolar gradient is a possible contributing factor in the enhanced sodium and water excretion, while the renin-angiotensin-aldosterone system does not seem to play an important role.

Nitric oxide inhibits sodium/hydrogen exchange activity in the thick ascending limb.

Nitric oxide (NO) inhibits transport in various nephron segments, and the thick ascending limb (TAL) expresses nitric oxide synthase (NOS). However, the effects of NO on TAL transport have not been extensively studied. We tested the hypothesis that NO inhibits apical and basolateral Na(+)/H(+) exchange by the TAL by measuring intracellular pH (pH(i)) of isolated, perfused rat TALs using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). The NO donor spermine NONOate (SPM, 10 microM) decreased steady-state pHi in medullary TALs from 7.18 +/- 0.13 to 7.13 +/- 0.14 (P < 0.02), whereas controls did not decrease significantly. We next measured the buffering capacity of medullary TALs and the rate at which they recovered from acid loads to investigate the mechanism whereby NO reduces steady-state pHi. SPM decreased H+ flux (JH) from 2.41 +/- 0.66 to 0.97 +/- 0.19 pmol. min(-1). mm(-1), 55%. To assure that the decrease in JH was due to NO, another donor, nitroglycerin (NTG; 10 microM), was used. NTG decreased J(H) from 1.65 +/- 0.11 to 1.07 +/- 0.24 pmol. min(-1). mm(-1), 37%. To determine the relative contributions of the apical and basolateral Na+/H+ exchangers, 5-(N,N-dimethyl)amiloride (DMA; 100 microM) was added to either bath or lumen. With DMA added to the bath, SPM decreased J(H) from 4.78 +/- 1.08 to 2.74 +/- 0.54 pmol. min(-1). mm(-1), an inhibition of 41%; and with DMA added to the lumen, SPM decreased J(H) from 2.31 +/- 0.29 to 1.74 +/- 0.27 pmol. min(-1). mm(-1), a reduction of 26%. Addition of DMA alone to both bath and lumen resulted in an 87% inhibition of JH. We conclude that NO inhibits both apical and basolateral Na+/H+ exchangers and consequently may play an important role in regulating pHi and may alter acid/base balance by directly affecting bicarbonate absorption in the TAL.

Effects of human calcitonin on water and electrolyte movements in rat juxtamedullary nephrons: inhibition of medullary K recycling.

The effects of synthetic human calcitonin (HCT) on water and electrolyte deliveries to the thin limbs of Henle's loop of juxtamedullary nephrons were investigated by micropuncture in the rat. To avoid undesirable interference with exogenous calcitonin, experiments were performed in hormone-deprived rats with reduced circulating calcitonin, antidiuretic hormone, parathyroid hormone and glucagon, all four of which stimulate the adenylate-cyclase activity in the thick ascending limb and the distal tubule. Administration of HCT (1.0 mU/min X 100 g body wt) to such rats significantly reduced the urinary fractional excretion rate of water, Mg, Ca and K. At the tip of the longlooped nephrons, the fractional delivery of water diminished in the presence of HCT, although the glomerular filtration rate of these nephrons was unaltered. Simultaneously, the loop fluid osmolality rose significantly. HCT, however, did not alter the fraction of total filtered solutes remaining in the thin limbs, nor the NaCl fractional delivery. As previously observed in this laboratory with dDAVP, the reduced fractional delivery of water at the hairpin turn was accompanied by a decrease in Mg and Ca deliveries in rats given HCT, indicating that the handling of these two ions along the descending limb may be linked in part to the water movements in this nephron segment. The fractional deliveries of K at the hairpin turn and in urine were significantly correlated, and both decreased in the presence of HCT. Since, as shown previously, HCT reduces the net addition of K along the superficial distal tubule, it is concluded that calcitonin inhibits the medullary recycling of K between the nephron terminal segments and the loop of Henle of juxtamedullary nephrons.

Distribution of cytochrome P-450 4A and 4F isoforms along the nephron in mice.

The production of 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney is thought to be involved in the control of renal vascular tone and tubular sodium and chloride reabsorption. 20-HETE production in the kidney has been extensively studied in rats and humans and occurs primarily via the actions of P-450 enzymes of the CYP4A and -4F families. Recent advancements in molecular genetics of the mouse have made it possible to disrupt genes in a cell-type-specific fashion. These advances could help in the creation of models that could distinguish between the vascular and tubular actions of 20-HETE. However, isoforms of the CYP4A and -4F families that may be responsible for the production of 20-HETE in the vascular and tubular segments in the kidney of the mouse are presently unknown. The goal of this study was to identify the isoforms of the CYP4A and -4F families along the nephron by RT-PCR of RNA isolated from microdissected renal blood vessels and nephron segments from 16- to 24-wk-old male and female C57BL/6J mice. CYP4A and -4F isoforms were detected in every segment analyzed, with sex differences only observed in the proximal tubule and glomeruli. In the proximal tubular segments from male mice, the 4A10 and -12 isoforms were present, whereas the 4A10 and -14 isoforms were detected in segments from female mice. In glomeruli, sex differences in the expression pattern of CYP4F isoforms were also observed, with male mice expressing the 4F13, -14, and -15 isoforms, whereas female mice expressed the 4F13, -16, and -18 isoforms. These results demonstrate that isolated nephron and renal vessel segments express multiple isoforms of the CYP4A and -4F families; therefore, elimination of a single CYP4A or -4F isoform may not decrease 20-HETE production in all nephron segments or the renal vasculature of male and female mice. However, the importance of CYP4A vs. -4F isoforms to the production of 20-HETE in each of these renal tubular and vascular segments of the mouse remains to be determined.