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To test the hypothesis that the abundance of the apical urea transporter of the inner medullary collecting duct (IMCD) is regulated in vivo by factors associated with altered water balance, immunoblots of rat inner medullary membrane fractions were probed with rabbit polyclonal antibodies against the renal urea transporter (RUT) gene product. In inner medullas of Brattleboro rats, which manifest severe chronic water diuresis, a 117-kD band was seen, in addition to the previously described 97-kD band. These two bands were detectable by antibodies directed against two different regions of the RUT sequence. When Brattleboro rats were treated with a 5-d infusion of arginine vasopressin (AVP) by osmotic minipump, the 117-kD band was markedly diminished, whereas the 97-kD band was unchanged. Simultaneous infusion of the diuretic agent furosemide prevented the AVP-induced decrease in the 117-kD band. In AVP-infused Sprague Dawley rats, the 117-kD band was barely perceptible. However, when AVP-treated rats were infused with furosemide for 5 d, the 117-kD band was markedly accentuated, whereas the 97-kD band was unchanged. The abundance of the 117-kD RUT protein in the renal papilla was inversely correlated with dietary protein intake. Further immunoblotting studies revealed that the 117-kD protein is heavily expressed in IMCD cells and not in non-collecting duct components of the inner medulla, and is present in low-density microsome fractions from inner medulla. From this study, the following conclusions can be made: (1) The collecting duct urea transporter is present in at least two forms (97 and 117 kD) in the IMCD. (2) The expression level of the 117-kD urea transporter protein is regulated and is inversely correlated with medullary osmolality and urea concentration, but does not correlate with circulating AVP level. (3) Although AVP regulates RUT function on a short-term basis, long-term changes in AVP levels do not increase RUT abundance.

Providing glucocorticoids to adrenalectomized (Adx) rats results in downregulation of the vasopressin (AVP)-regulated urea transporter (VRUT) in the renal inner medullary (IM) tip. To examine the physiological relevance of this response, we studied rats with uncontrolled diabetes mellitus induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We measured VRUT protein in extracts from the IM tip or base of pair-fed control and diabetic rats by Western analysis using an antibody to rat VRUT. In the IM tip, VRUT was significantly reduced by 39% in diabetic compared with control rats. In the IM base, there was no significant difference between diabetic and control rats. To determine whether the decrease in VRUT in the IM tip was mediated by glucocorticoids, the experiment was repeated using the following three groups of rats: 1) Adx alone, 2) Adx + STZ, and 3) Adx + STZ + replacement with a physiological dose of glucocorticoid. There was no significant difference in VRUT between Adx and Adx + STZ rats. However, VRUT was significantly reduced by 32% in the IM tip of glucocorticoid-treated Adx + STZ rats compared with control Adx + STZ rats. We conclude that glucocorticoids regulate the abundance of VRUT protein independently of insulin in diabetic rats.

Testosterone has been implicated in the dehydration-induced increase in vasopressin (VP) mRNA observed in young rats (1). Since testosterone is diminished in aged rats, and since prior studies have demonstrated a deficit in the dehydration-induced increase in VPmRNA in aged male Fischer 344 rats (2), the hypothesis that aging-associated deficits in testosterone result in a diminished VP response to dehydration was evaluated.

Physiological and molecular data demonstrate that urea transport in kidney and erythrocytes is regulated by specific urea transporter proteins. The urea transporter in the terminal inner medullary collecting duct permits very high rates of regulated 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. The urea transporter in erythrocytes permits these cells to lose urea rapidly as they ascend through the ascending vasa recta, thereby preventing loss of urea from the medulla. Urea lost from the medulla would decrease concentrating ability by decreasing the efficiency of countercurrent exchange, as occurs in individuals who lack the Kidd antigen. The recent cloning of cDNAs for these two urea transporters has begun to yield new insights into the mechanisms underlying acute and long-term regulation of urea transport and should permit exciting new insights in the future. This review focuses on the physiological and biophysical evidence that established the concept of urea transporters, the subsequent cloning of cDNAs for urea transporters, and the recent integrative studies into the regulation of urea transport. We also propose a new systematic nomenclature and a new structural model for urea transporters.

Low-protein diets cause a urinary concentrating defect in rats and humans. Previously, we showed that feeding rats a low (8%) protein diet induces a change in urea transport in initial inner medullary collecting ducts (IMCDs) which could contribute to the concentrating defect. Now, we test whether decreased osmotic water permeability (Pf) contributes to the concentrating defect by measuring Pf in perfused initial and terminal IMCDs from rats fed 18 or 8% protein for 2 wk. In terminal IMCDs, arginine vasopressin (AVP)-stimulated osmotic water permeability was significantly reduced in rats fed 8% protein compared to rats fed 18% protein. In initial IMCDs, AVP-stimulated osmotic water permeability was unaffected by dietary protein. Thus, AVP-stimulated osmotic water permeability is significantly reduced in terminal IMCDs but not in initial IMCDs. Next, we determined if the amount of immunoreactive aquaporin-2 (AQP2, the AVP-regulated water channel) or AQP3 protein was altered. Protein was isolated from base or tip regions of rat inner medulla and Western analysis performed using polyclonal antibodies to rat AQP2 or AQP3 (courtesy of Dr. M.A. Knepper, National Institutes of Health, Bethesda, MD). In rats fed 8% protein (compared to rats fed 18% protein): (a) AQP2 decreases significantly in both membrane and vesicle fractions from the tip; (b) AQP2 is unchanged in the base; and (c) AQP3 is unchanged. Together, the results suggest that the decrease in AVP-stimulated osmotic water permeability results, at least in part, in the decrease in AQP2 protein. We conclude that water reabsorption, like urea reabsorption, responds to dietary protein restriction in a manner that would limit urine concentrating capacity.

Nephrotic syndrome is associated with up-regulation of the heparin-binding epidermal growth factor (HB-EGF). Erlotinib blocks the activation of the epidermal growth factor receptor (EGFR) in response to HB-EGF. This study investigates the effect of Erlotinib on the progression of proteinuria, renal dysfunction, and salt retention in doxorubicin treated nephrotic rats. Male rats were divided into 3 pair-fed groups (n = 13/group) as follows: Control rats (Ctrl); rats receiving intravenous doxorubicin (Dox); and rats receiving intravenous doxorubicin followed by daily oral Erlotinib (Dox + Erl). Upon establishment of high grade proteinuria, urine sodium and creatinine clearance were measured. Kidney tissue was dissected and analyzed for γ-epithelial sodium channel (γENaC), sodium-potassium -chloride co-transporter 2 (NKCC2), sodium chloride co-transporter (NCC), aquaporin 2 (AQP2), and EGFR abundances using western blot. Creatinine clearance was preserved in the Dox + Erl rats as compared to the Dox group (in ml/min: Ctrl: 5.2±.5, Dox: 1.9±0.3, Dox + Erl: 3.6±0.5). Despite a minimal effect on the degree of proteinuria, Erlotinib prevented salt retention (Urinary Na in mEq/d: Ctrl: 2.2±0.2, Dox: 1.8±0.3, Dox + Erl: 2.2±0.2). The cleaved/uncleaved γENaC ratio was increased by 41±16% in the Dox group but unchanged in the Dox + Erl group when compared to Ctrl. The phosphorylated EGFR/total EGFR ratio was reduced by 74±7% in the Dox group and by 77±4% in the Dox + Erl group. In conclusion, Erlotinib preserved renal function and prevented salt retention in nephrotic rats. The observed effects do not appear to be mediated by direct blockade of EGFR.

Feeding rats a low-protein (8%) diet (LPD) for 2 wk induces a facilitated urea transporter in rat initial inner medullary collecting ducts (IMCDs). To determine whether this is preceded by an increase in mRNA abundance, we designed degenerate polymerase chain reaction primers to the rabbit facilitated urea transporter (UT2; G. You, C. P. Smith, Y. Kanai, W.-S. Lee, M. Stelzner, and M. A. Hediger. Nature Lond. 365: 844–847, 1993) and amplified a 716-bp cDNA fragment to perform Northern analysis of the base or tip of rat inner medulla. In the base, the predominant transcript was a 2.9-kb band, which increased 55% after 1 wk on an LPD; there was no change in a 4-kb band. In the tip, the 4-kb band predominated, but neither band varied with an LPD. Next, we functionally characterized the induced urea transporter using microperfused initial IMCDs from rats fed an LPD for 2 wk. First, 100 pM arginine vasopressin (AVP) stimulated urea permeability (Purea); 10 nM AVP increased Purea further. Second, raising perfusate and bath osmolality to 690 mosmol/kgH2O (NaCl added) stimulated Purea; adding AVP (10 nM) increased Purea further. Third, thiourea reversibly inhibited AVP-stimulated Purea.(ABSTRACT TRUNCATED AT 250 WORDS)

International audience; The regulation of mRNA for aldose reductase, sorbitol dehydrogenase, and the Na+/Cl-/taurine cotransporter was studied with three in vivo models in which urinary concentration is reduced: Sprague-Dawley rats undergoing a water diuresis or fed a low-protein diet or Brattleboro rats. In Sprague-Dawley rats, 3 days of water diuresis reduced inner medullary aldose reductase mRNA abundance 6.5-fold compared with untreated rats, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. When water diuretic rats were acutely deprived of water, urine osmolality increased significantly after 4 h but aldose reductase mRNA did not increase until 12 h. Heat shock protein-70 mRNA was not increased by water deprivation. Second, in rats fed a low-protein diet for 3 wk, aldose reductase mRNA increased two-fold, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. Finally, in Brattleboro rats, urine osmolality and levels of aldose reductase and taurine cotransporter mRNA increased in response to 1 day of water deprivation, whereas sorbitol dehydrogenase mRNA was unchanged. Administering vasopressin (1 U/day) to Brattleboro rats for 8 days also increased urine osmolality and aldose reductase mRNA but did not alter sorbitol dehydrogenase or taurine cotransporter mRNA. This result is consistent with the hypothesis that changes in urine osmolality induce changes in aldose reductase mRNA abundance that are independent of vasopressin. It was concluded that, in rat inner medulla: (1) aldose reductase mRNA abundance varies with changes in water balance or dietary protein, whereas sorbitol dehydrogenase and taurine cotransporter mRNA do not; and (2) heat shock protein-70 mRNA abundance is not increased during acute osmotic stress.

Na-K-ATPase is a heterodimeric complex composed of an alpha-catalytic and a glycosylated beta-subunit. Previous studies using in situ hybridization and Northern blot analysis to determine alpha- and beta-subunit mRNA isoform expression in the rat kidney have given conflicting results. This heterogeneity may be due to detection of alpha 2- or alpha 3-isoforms arising from nonrenal epithelial sources such as peripheral nerves or vascular smooth muscle. To address this possibility, we investigated alpha-subunit mRNA isoform expression in different nephron segments using tubule microdissection and reverse transcription-polymerase chain reaction amplification. Southern blot analysis of polymerase chain reaction products using isoform-specific primers and probes detected the expression of alpha 1- and alpha 3-mRNA isoforms in whole kidney, cortical collecting ducts (CCD), and proximal tubule S2 subsegments (S2). No evidence for alpha 2 was found in kidney or microdissected CCD or S2. To determine whether alpha 1- or alpha 3-mRNA in the CCD or S2 is translated into protein, Western blot analysis of total protein from microdissected S2, CCD, medullary thick ascending limb (MTAL), or outer medullary collecting duct outer stripe (OMCDos) was performed. Total protein was separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then probed using polyclonal antibodies specific for the alpha 1-, alpha 2-, alpha 3-, beta 1-, and beta 2-protein isoforms. Rat brain was used as a positive control and demonstrated that the antibodies could detect a single 97- and 35-kDa band for the alpha- and beta-isoforms, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Low protein diets reverse the urea concentration gradient in the renal inner medulla. To investigate the mechanism(s) for this change, we studied urea transport and cell ultrastructure in initial and terminal inner medullary collecting ducts (IMCD) from rats fed 18% protein or an isocaloric, 8% protein diet for 4 wk. Serum urea, aldosterone, and albumin were significantly lower in rats fed 8% protein, but total protein and potassium were unchanged. Vasopressin stimulated passive urea permeability (Purea) threefold (P < 0.05) in initial IMCDs from rats fed 8% protein, but not from rats fed 18% protein. Luminal phloretin reversibly inhibited vasopressin-stimulated Purea. However, in terminal IMCDs from rats fed either diet, vasopressin stimulated Purea. Net transepithelial urea flux (measured with identical perfusate and bath solutions) was found only in initial IMCDs from rats fed 8% protein. Reducing the temperature reversibly inhibited it, but phloretin did not. Electron microscopy of initial IMCD principal cells from rats fed 8% protein showed expanded Golgi bodies and prominent autophagic vacuoles, and morphometric analysis demonstrated a marked increase in the surface density and boundary length of the basolateral plasma membrane. These ultrastructural changes were not observed in the terminal IMCD. Thus, 8% dietary protein causes two new urea transport processes to appear in initial but not terminal IMCDs. This is the first demonstration that "active" urea transport can be induced in a mammalian collecting duct segment.

We showed previously that both increasing osmolality by adding NaCl or manitol (hyperosmolarity) or adding vasopressin can stimulate urea permeability in rat terminal inner medullary collecting ducts (IMCD). Vasopressin acts via adenosine 3',5'-cyclic monophosphate (cAMP), but the mechanism by which hyperosmolarity acts is unknown. To study the mechanism, we determined the effect of varying osmolality (with NaCl) on two potential second messenger systems, i.e., cAMP and intracellular calcium. There was no significant difference in cAMP production among tubules incubated at 290, 490, 690, or 890 mosmol/kg. In contrast, cAMP did increase significantly after vasopressin (10(-8) M) addition. Intracellular calcium increased significantly when osmolality was increased from 290 to 490 mosmol/kg in the absence of vasopressin. To examine whether changes in intracellular calcium affect urea permeability, we added thapsigargin (and removed bath calcium) while maintaining osmolality at 290 mosmol/kg. Both intracellular calcium and urea permeability increased significantly. Next, we buffered intracellular calcium by pretreatment with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM). BAPTA completely blocked the increase in urea permeability occurring when osmolality was increased from 290 to 490 mosmol/kg, but did not block the increase in urea permeability occurring when vasopressin (10(-8) M) was added. In summary, 1) hyperosmolarity increases intracellular calcium, but has no effect on cAMP accumulation; 2) thapsigargin increases intracellular calcium and urea permeability; and 3) BAPTA blocks the hyperosmolarity-stimulated increase in urea permeability, but not vasopressin-stimulated urea permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Water homeostasis is regulated in large part by the proper operation of the urinary concentrating mechanism. In the renal inner medulla, urea recycling from the inner medullary collecting duct to the inner medullary interstitium is thought to be essential for the production of a concentrated urine; however, it has not been possible to test this hypothesis in humans. Recently, a unique combination of genetic abnormalities has been described: absence of Kidd blood group antigens and absence of carrier-mediated urea transport in erythrocytes. Because animal studies indicate a similarity between urea transport in red blood cells and the nephron, it was postulated that patients without the Kidd antigen might lack facilitated urea transport in their kidneys. Hence, their ability to concentrate urine maximally was measured. Current models of nephron function would predict that in the complete absence of urea transport, the maximal concentrating ability would be around 800 to 900 mosM/kg H2O. Two homozygous patients had a moderate decrease in maximal concentrating ability (UosM,max = 819 mosM/kg H2O); a heterozygote also had some limitation. These studies raise the possibility that the erythrocyte urea transporter and the kidney urea transporter are encoded by a single gene (detected by the mutational loss of the Kidd antigen) and that a lack of facilitated urea transport impairs urea recycling in the kidney and, hence, maximal urinary concentrating ability.

Urea transport across the terminal inner medullary collecting duct (IMCD) is mediated by a urea transporter that is stimulated by vasopressin (AVP) or hyperosmolarity. To determine whether hyperosmolarity stimulates urea transport by an adenylyl cyclase-dependent or -independent mechanism, terminal IMCDs were perfused with 10 microM forskolin followed by an increase in osmolality or with increasing osmolality followed by 10 nM AVP. In both protocols, stimulating adenylyl cyclase caused an additive increase in urea permeability (Purea) to that stimulated by hyperosmolarity. Next, we investigated whether hyperosmolarity stimulates the same urea transporter as AVP by studying the inhibitor profile and IMCD subsegment response of hyperosmolarity-stimulated urea transport and comparing it to properties already demonstrated for AVP-stimulated urea transport. In terminal IMCDs, luminal phloretin (250 microM) reversibly inhibited Purea by 63%. Thiourea (100 mM) inhibited Purea by 73% at two different levels of osmolality, 690 and 290 mosmol/kgH2O. The half-maximal inhibitory concentration (K1/2) for thiourea at 690 mosmol/kgH2O was not significantly different from the K1/2 value at 290 mosmol/kgH2O, suggesting that stimulation by hyperosmolarity is related to an increase in the Vmax for the urea transporter. Finally, we found that hyperosmolarity did not stimulate Purea in the initial IMCD. In summary, the data suggests that hyperosmolarity stimulates urea transport by an adenylyl cyclase-independent mechanism. However, the inhibitor profile and the IMCD subsegment response for hyperosmolarity-stimulated and AVP-stimulated Purea are similar, suggesting that both hyperosmolarity and AVP stimulate the same urea transporter.

We have shown that urea transport across the terminal inner medullary collecting duct (terminal IMCD) is mediated by a vasopressin-stimulated, facilitated diffusion process exhibiting properties consistent with a transporter. To investigate whether hypertonic NaCl, as exists in vivo in the inner medulla, affects urea permeability, we studied isolated perfused rat terminal IMCD segments. Perfusate and bath osmolality were varied symmetrically by adding or removing NaCl or mannitol. Urea permeability rose progressively when osmolality was increased with NaCl or mannitol from 290 to 690 mOsm/kg H2O in the absence of vasopressin; there was no further increase at 890 mOsm/kg H2O. In the presence of 10(-8) M arginine vasopressin, urea permeability increased when NaCl was added to raise osmolality from 290 to 490 mOsm/kg H2O but there was no further increase at 690 mOsm/kg H2O. When 1 mM 8-bromo cyclic AMP was added to the bath, raising NaCl still increased urea permeability. These results suggest that urea transport across the rat terminal IMCD is regulated both by vasopressin and by osmolality at values present in the renal inner medulla. Osmolality seems to activate urea transport across the rat terminal IMCD by mechanisms distinct from those of vasopressin or cyclic AMP.

Atrial natriuretic factor (ANF) ameliorates renal damage in animal models of acute ischemic renal failure. Consequently, ANF could blunt acute tubular necrosis related to ischemia that occurs frequently in cadaveric renal transplants. Ten pairs of cadaveric kidneys were transplanted into 20 recipients. Paired recipients received either alpha-human ANF (hANF) or vehicle alone in a prospective, double-blind protocol. Upon revascularization of the allograft, either hANF or vehicle was administered intravenously as a 50-micrograms bolus, followed by a 4-h infusion (0.1 microgram/kg/min). Glomerular filtration rate ([125I]iothalamate clearance) was measured between 4 and 7 days posttransplant and again between 14 and 21 days posttransplant. Serum creatinine was measured daily when patients were in the hospital, then twice weekly as patients were examined in the outpatient clinic. Between the groups, there was no significant difference in age of the recipients or donors, cold ischemia time, or histocompatibility leukocyte antigen match. Infusion of hANF had no adverse effects. When subjects receiving hANF were compared with those treated with vehicle alone, there were no significant differences in serum creatinine or glomerular filtration rate. Three hANF and four vehicle recipients required dialysis postoperatively. At 1 month posttransplant, 19 of 20 patients had functioning allografts; an allograft from one hANF recipient never functioned. It was concluded that hANF, when given by the protocol of this study, had no beneficial effect on the outcome of cadaveric renal transplantation in humans.

In this section we feature some of the latest and most striking new findings in physiology, interpreting the term “physiology” in its broadest sense. In each instance, an effort will be made to place the new findings in perspective. Heinz Valtin Editor, trendsetters Urea transport has always

It has been one year since the present team of associate editors, Drs. Sharon Anderson, Pam Carmines, Douglas Eaton, Tom Kleyman, and Susan Wall, and I took the helm of the American Journal of Physiology-Renal Physiology . Our goal was to make the best even better. With your help, we believe that

This focussed issue of Cardiovascular Research summarizes several review articles describing our up-to-date understanding of the physiology and pathophysiology of diuretic and antidiuretic hormones together with new findings in several original reports. These diuretic and antidiuretic hormones comprise vasopressin, different members of the natriuretic peptide family, nitric oxide, and endothelin. The collection of reviews and original articles attempts to address questions from the molecular structure of these hormones and their receptors, their physiological and pathophysiological function, clinical implications, therapeutic use of mimetics and inhibitors to mutations in these molecules found in patients. The majority, especially of the review articles, focuses on the renal actions of these hormones and their importance for fluid homeostasis. A number of additional papers discusses … *Corresponding author. Tel.: +49-251-83-56991; +49-251-83-56973

the new associate editors and I are tremendously excited and honored to have this opportunity to serve the American Journal of Physiology-Renal Physiology . Dr. Steven Hebert and his Associate Editors have done an outstanding job during the previous six years, and we are confident that the renal

Atrial natriuretic factor (ANF) is a peptide hormone that increases renal NaCl and water excretion. Several renal sites of ANF action have been identified, but general agreement has not been reached concerning the quantitative contribution of each action to the natriuresis and diuresis. Using a five-nephron central core model of NaCl, urea, KCl, and water transport in the rat kidney, we have quantitatively evaluated the hypothetical effects on whole kidney function of three experimentally observed ANF actions: 1) inhibition of active NaCl absorption in the collecting duct, 2) inhibition of osmotic water permeability in the collecting duct, and 3) increased NaCl and water delivery out of the proximal convoluted tubule simulating an increase in glomerular filtration rate. The simulations show that inhibition of collecting duct active NaCl absorption by greater than or equal to 50% can increase NaCl and water excretion to levels that match experimental values. In addition, the model predicted that the urinary sodium concentration will increase to greater than plasma levels as observed experimentally. Simulated decreases in collecting duct water permeability predicted an increase in water excretion with little change in NaCl excretion. Simulated 2.5–5% increases in glomerular filtration rate also increased simulated NaCl and water excretion rates to experimentally observed levels in response to ANF. However, this action was less effective than inhibition of collecting duct active NaCl absorption in increasing the urinary NaCl concentration. We conclude that a combination of several actions are likely to account for the overall renal effect of ANF.

The inner medullary collecting duct (IMCD) has been proposed to be a site of atrial natriuretic factor (ANF) action. We carried out experiments in isolated perfused terminal IMCDs to determine whether ANF (rat ANF 1-28) affects either osmotic water permeability (Pf) or urea permeability. In the presence of a submaximally stimulating concentration of vasopressin (10(-11) M), ANF (100 nM) significantly reduced Pf by an average of 46%. Lower concentrations of ANF also significantly inhibited vasopressin-stimulated Pf by the following percentages: 0.01 nM ANF, 18%; 0.1 nM, 46%; 1 nM, 48%. Addition of exogenous cyclic GMP (0.1 mM) mimicked the effect of ANF, decreasing Pf by an average of 48%. ANF also inhibited cyclic AMP-stimulated Pf by an average of 31%. ANF did not affect urea permeability, nor did it alter vasopressin-stimulated cyclic AMP accumulation. We conclude that ANF at physiological concentrations causes a large inhibition of vasopressin-stimulated Pf in the rat terminal IMCD, and that cyclic GMP is the second messenger mediating the effect. ANF appears to act at a site distal to cyclic AMP generation in the chain of events linking vasopressin receptor binding to an increase in osmotic water permeability.

Immunolocalization of phospholipase C isoforms in rat kidney. Background Phospholipase C (PLC) is an important factor in signal transduction because this enzyme is activated by several hormones and growth factors. Eight PLC isoforms have been described raising the possibility that different cells express a single isoform or activate specific isoforms in different cells. Therefore, the goal of this study was to determine which PLC isoforms are expressed in specific regions of rat kidney. Methods Western blot analysis was performed in microdissected nephron segments of rat kidney, while immunohistochemical analysis was performed on whole rat kidney slices using PLC isoform-specific antibodies. Results All three families of PLC isoforms (β, γ, and δ) were present throughout the cortical and medullary regions of the kidney. Only the PLC-β1 isoform was observed in the brush border of the proximal tubule, but all isoforms were present in glomeruli and in the cytoplasm of tubular epithelial cells. In addition, only the PLC-γ1 isoform was expressed in the internal elastic lamina of the renal artery, while vasa recta expressed PLC-β1 most intensely. Medullary thick ascending limbs showed an intense level of expression of all three isoforms. Conclusion Multiple PLC isoforms are present in glomeruli, renal tubules, and renal vasculature in vivo , but with some segment-specific differences. These findings suggest that the response of a specific cell is not determined by expression of only one PLC isoform, with the exception of the brush border of the proximal tubule and the renal arteries. Instead, the presence of multiple PLC isoforms in specific regions of the kidney suggests that hormonal regulation in vivo involves mechanisms beyond cell-specific isoforms of PLC.

Evidence that the inhibition of Na + /K + -ATPase activity by FK506 involves calcineurin. We reported that cyclosporin A (CsA) inhibits Na + /K + -ATPase activity in specific segments of the rat nephron. In this study, we tested the hypothesis that cyclosporin A reduces Na + /K + -ATPase activity through inhibition of calcineurin. In T cells, cyclosporin A and FK506 bind to immunophilins and inhibit the phosphatase activity of calcineurin; Rapamycin and SDZ 220-384 also bind to immunophilins but do not change calcineurin activity. Na + /K + -ATPase activity was measured in microdissected rat proximal tubule (S2 subsegment), medullary thick ascending limb (mTAL), and cortical collecting duct (CCD). First we found that two inhibitors of calcineurin, pentafluorophenol (PFP, 100 mM) and peptide 412 (1 mM), significantly reduced Na + /K + -ATPase activity in the CCD by 78% and 70%, respectively. In CCDs, FK506 inhibited Na+/K+-ATPase activity by 61 to 85% at concentrations of 1.5 to 6 ng/ml, but not at 0.5 ng/ml. FK506 (6 ng/ml) inhibited Na+/K+-ATPase activity in mTALs by 56% but did not inhibit it in S2s or glomeruli. In contrast, Rapamycin (12.5 ng/ml) did not change Na+/K+-ATPase activity in CCDs or mTALs, but at a concentration of 12.5 µg/ml did block the inhibitory effect of FK506 (6 ng/ml) in both segments. SDZ 220-384 (600 ng/ml) did not change Na + /K + -ATPase activity in CCDs. Thus, in CCDs and mTALs: (1) FK506, like cyclosporin A, inhibits Na + /K + -ATPase activity; (2) Rapamycin and SDZ 220-384 do not inhibit Na + /K + -ATPase activity; and (3) Rapamycin prevents FK506-induced inhibition of Na + /K + -ATPase activity. These responses may be explained by a direct inhibition of calcineurin activity yielding lower Na + /K + -ATPase activity in CCDs and mTALs.

Rare cases of acute mucormycosis in immunocompromised persons have been attributed to Cunninghamella bertholletiae. We report the first known case of this organism causing a chronic, indolent, systemic disease clinically similar to entomophthoramycosis. Cunninghamella bertholletiae is an opportunistic pathogen whose morbidity may be underappreciated because it is usually considered to be a laboratory contaminant.