Tubular Fluid Research Articles

Mechanisms of intercellular hypertonicity and isotonic fluid absorption in proximal tubules of mammalian kidneys.

The main purpose of this theoretical analysis (second of two articles) is to examine whether transjunctional diffusion of NaCl causes intercellular hypertonicity, which permits transcellular water transport across solute-impermeable lateral cell membranes until osmotic equilibration. In the S2 segment with tubular NaCl concentration 140 mM, the calculated apical intercellular NaCl concentration is c0 approximately 132 mM, which exceeds peritubular NaCl concentration by 12 mM or 22 mOsm kg-1. Variations in volume flow, junctional reflection coefficient (sigmaNaCl = 0.25-0.50), gap distance (g = 6-8 A), junctional depth (d = 18-100 A), intercellular diffusion coefficient (DLIS=500-1500 microm2 s-1) and hypothetical active NaCl transport alter c0 only by a fraction of 1 mM. However, dilution and back-leakage of NaHCO3 lower apical intercellular hyperosmolality to approximately 18 mOsm kg-1. Water transport through solute-impermeable lateral cell membranes continues until intercellular and cellular osmolalities are equal. Transcellular and transjunctional volume flow are of similar magnitude (2 nL min-1 mm-1 tubule length) in the S2 segment. Thus, diffusion ensures isotonic absorption of NaCl. Two-thirds of NaHCO3 and other actively transported sodium salts are extruded into the last third of the exponentially widening intercellular space where the exposure time is only 0.9 s. Osmotic equilibration is dependent on aquaporins in the cell membranes. If permeability to water is low, transcellular water transport stops; tubular fluid becomes hypotonic; NaCl diffusion diminishes, but transjunctional water transport remains unaltered as long as transcellular transport of NaHCO3 and other solutes provides the osmotic force.

Nitric oxide modulation of neurally induced proximal tubular fluid reabsorption in the rat.

This study investigated the role of NO in mediating the renal sympathetic nerve-mediated increases in proximal tubular fluid reabsorption (Jva). In inactin-anesthetized Wistar rats, renal sympathetic nerve stimulation (15 V, 2 ms) at 0.75 and 1.0 Hz did not change blood pressure or glomerular filtration rate but did decrease urine flow and sodium excretion in a frequency-related fashion by 40% to 50% at 1.0 Hz (both, P<0.01). Renal nerve stimulation in control animals increased Jva by 11% at 0.75 Hz (P<0.05) and 31% at 1.0 Hz (P<0.01). Intraluminal N(omega)-nitro-L-arginine methyl ester (L-NAME) resulted in a higher basal Jva (19%, P<0.05), and renal nerve stimulation had no effect on Jva. When L-NAME plus sodium nitroprusside was present intraluminally, however, there were frequency-dependent increases in Jva that were similar in pattern and magnitude to the control rats. Introduction of the relatively selective nNOS blocker 7-nitroindazole intraluminally, at 10(-6) and 10(-4) M, raised basal Jva by 18% and 24%, respectively (P<0.01), and renal nerve stimulation did not change Jva. Intraluminal aminoguanidine (10(-4) M), a relatively selective iNOS blocker, did not affect basal Jva, which remained unchanged during renal nerve stimulation. These data are consistent with NO exerting a tonic inhibitory action on the basal levels of Jva, which, in part, is caused by NO generated by the nNOS isoform. Moreover, the findings have revealed that the presence of NO is necessary to ensure that renal nerves can stimulate fluid reabsorption by the proximal tubules, requiring NO generated from both nNOS and iNOS.

Mechanism by which superoxide potentiates tubuloglomerular feedback.

The macula densa detects changes in NaCl concentration in tubular fluid and transmits a feedback signal, known as tubuloglomerular feedback (TGF), which helps to control glomerular afferent arteriole resistance. We and other investigators have reported that synthesis of NO in the macula densa inhibits TGF. NO can be scavenged by superoxide (O(-)(2)) to form peroxynitrite, effectively reducing the bioavailability of NO; there is growing evidence that O(-)(2) regulates vascular tone in the kidney. We hypothesized that O(-)(2) produced in the macula densa enhances TGF and this effect acts only in an autocrine manner within the cells of the macula densa. Afferent arterioles and attached macula densas from Sprague-Dawley rats were simultaneously microperfused in vitro and TGF response examined before and after perfusing the tubular lumen, bath, or vascular lumen with a superoxide scavenger. The macula densa was perfused with solutions containing either 5 mmol/L Na(+) and 3 mmol/L Cl(-) (low NaCl) or 80 mmol/L Na(+) and 77 mmol/L Cl(-) (high NaCl) while keeping pressure in the afferent arteriole constant at 60 mm Hg. When 10(-4) M Tempol, a stable membrane-permeant superoxide dismutase (SOD) mimetic, was added to the tubular lumen, it inhibited TGF by 56% (before Tempol: TGF, 3.2 +/- 0.3 microm; after Tempol: TGF, 1.4 +/- 0.2 microm; n=6; P<0.05, control versus Tempol). Adding Tempol to the bath inhibited TGF by 48% (before Tempol: TGF, 2.5 +/- 0.25 microm; after Tempol: TGF, 1.3 +/- 0.18 microm; n=6; P<0.05). However, adding Tempol to the vessel lumen did not change TGF response significantly (before Tempol: TGF, 2.7 +/- 0.37 microm; after Tempol: TGF, 3.2 +/- 0.25 microm; n=7; P=0.25). When 300 U/mL of the enzyme SOD, which is not membrane-permeant, was added to either the tubular lumen or bath, it had no effect on TGF response. Finally, to determine whether the effect of O(-)(2) in the macula densa is mediated by its scavenging of NO, 7-nitroindazole (7-NI) was added to the macula densa to inhibit neuronal nitric oxide synthase (nNOS). In the presence of 7-NI, Tempol had no effect (7-NI only: TGF, 3.0 +/- 0.4 microm; 7-NI plus Tempol: TGF, 2.8 +/- 0.5 microm; n=6; P=0.343). Our findings suggest that (1) reducing O(-)(2) increases the bioavailability of NO, which inhibits TGF, (2) both O(-)(2) and NO act within the macula densa, and (3) O(-)(2) appears to have no effect on its own.

The clinical value of urinary N-acetyl-beta-D-glucosaminidase levels in childhood age group.

N-acetyl-beta-D-glucosaminidase is a high molecular-weight lysosomal enzyme found in many tissues of the body. It cannot pass into glomerular ultrafiltrate due to its high molecular weight. However, this enzyme shows high activity in renal proximal tubular cells, and leaks into the tubular fluid as the ultrafiltrate passes through proximal tubules. When proximal tubular cells are injured due to to any disease process including glomerular proteinuria, nephrolithiasis, hyperglycemia, interstitial nephritis, transplant rejection or nephrotoxic agents such as antibiotics, antiepileptics, or radiocontrast agents, its urine level increases and thus is used as a reflection of proximal tubular cell necrosis. However, the clinical use of urinary N-acetyl-beta-D-glucosaminidase determination is limited in childhood because of certain technical problems. In addition, the urinary level of this enzyme changes with the maturational level of proximal tubular cells. Thus, difficulties are involved in assessing normal urine levels of this enzyme for age. On the other hand, successive measurements of urinary N-acetyl-beta-D-glucosaminidase during the longitudinal follow-up of the patients may enhance its clinical use as an indicator of ongoing tubular injury.

Urinary excretion of angiotensinogen reflects intrarenal angiotensinogen production

In rats maintained on a high salt diet (H/S) to suppress basal renal angiotensinogen levels, angiotensin II (Ang II) infusion for 13 days increased renal angiotensinogen mRNA and protein, thus providing a mechanism for further augmentation of intrarenal Ang II levels. The present study tested the hypothesis that enhanced intrarenal angiotensinogen formation during Ang II infusion is reflected by secretion into the tubular fluid leading to increased urinary excretion of angiotensinogen (UAGT). The effects of chronic Ang II infusion were examined on kidney and plasma Ang II levels and UAGT in male Sprague-Dawley rats maintained on an 8% salt diet for three weeks (N=10). Following one week on the H/S diet, Ang II (40 ng/min) was administered for two weeks via an osmotic minipump to one group (H/S + Ang II, N=5), while the remaining rats were sham-operated (H/S + Sham, N=5). Additionally, a control group was prepared with normal salt diet and sham-operation (N/S + Sham, N=5). H/S alone did not alter systolic blood pressure (BP) (103 +/- 2 vs. 104 +/- 2 mm Hg), while Ang II infusion to H/S rats significantly increased systolic BP from 103 +/- 2 to 154 +/- 2 after two weeks. Intrarenal Ang II content in H/S + Ang II was significantly greater than H/S + Sham (435 +/- 153 vs. 65 +/- 14 fmol/g). Ang II infusion significantly increased UAGT (4.0 +/- 0.5 vs. 1.0 +/- 0.2 nmol Ang I/day by radioimmunoassay of generated Ang I; 57 +/- 15 vs. 14 +/- 2 densitometric units by Western blotting analysis) compared to Sham. UAGT by radioimmunoassay was highly correlated with kidney Ang II content (r=0.79); but not with plasma Ang II concentration (r=0.20). These data demonstrate that chronic Ang II infusion increases urinary excretion rate of angiotensinogen, and suggest that UAGT provides a specific index of intrarenal angiotensinogen production in Ang II-dependent hypertension.

The Dual AngII/AVP Receptor Gene N119S/C163R Variant Exhibits Sodium-Induced Dysfunction and Cosegregates With Salt-Sensitive Hypertension in the Dahl Salt-Sensitive Hypertensive Rat Model

Essential hypertension is a prevalent complex polygenic disease and a major risk factor for cardiovascular disease, the leading cause of death in developed countries. Because of its complex and multifactorial nature, its genetic determinants still remain largely unknown. The Dahl salt-sensitive hypertensive rat model exhibits impaired sodium handling, which is hypothesized to play a key role in the pathophysiology of polygenic hypertension. Thus, genes associated with renal regulation of salt and water balance are a priori likely candidates for a causative role in hypertension pathogenesis. The functional properties and renal-specific expression of the recently characterized AngII/AVP receptor suggest a putative modulator role in tubular sodium and fluid reabsorption. Based on these observations, we investigated the potential involvement of the AngII/AVP receptor in salt-sensitive hypertension. We performed cosegregation analysis of the AngII/AVP receptor locus with salt-sensitive hypertension in an F2 (Dahl S X Dahl salt-resistant [R]) hybrid male cohort characterized for blood pressure by radiotelemetry after 8 weeks of high salt challenge. Further molecular analysis was done to identify putative AngII/AVP receptor molecular variants that could account for the AngII/ AVP receptor involvement in salt-sensitive hypertension pathogenesis. The AngII/AVP receptor was mapped to rat chromosome 1, 1.7 cM centromeric to the D1Rat188 marker by radiation hybrid mapping analysis. Quantitative trait locus (QTL) analysis detected a highly significant linkage of the AngII/AVP receptor locus with high blood pressure (LRS = 13.8, p= 0.0002). Molecular characterization of the Dahl S and Dahl R AngII/AVP receptor cDNAs revealed two amino acid substitutions in the Dahl S AngII/AVP receptor (N119S, C163R) when compared to the Dahl R AngII/AVP receptor. These mutations are associated with an increased receptor affinity for both ligands (AVP and AngII) and an enhanced G(s)-coupling by the receptor resulting in increased activation of adenylate cyclase with concomitant increase in cAMP production. The observed molecular dysfunction in the Dahl S AngII/AVP receptor is consistent with increased tubular sodium and fluid reabsorption observed in Dahl S rats. Interestingly, the AngII/AVPr locus is within the narrowed chromosome 1 QTL region for blood pressure detected in different rat intercross linkage analyses. Altogether, the data strongly suggest that the AngII/AVP receptor is a hypertension susceptibility gene in the Dahl S rat model, as well as raises the hypothesis that it too underlies the chromosome 1 blood pressure QTL identified in other hypertension rat models.

Possible role of adenosine in macula densa control of glomerular hemodynamics

The macula densa (MD), a plaque of specialized tubular epithelial cells, senses changes in tubular NaCl concentration and sends a signal(s) that controls the resistance of the glomerular afferent arteriole (Af-Art). This mechanism, called tubuloglomerular feedback (TGF), is thought to be important in the homeostasis of body fluids and electrolytes. Our aim was to determine the range of NaCl concentrations in tubular fluid at the MD that would elicit the Af-Art response. In addition, we examined the possible involvement of adenosine in transmitting the signal from the MD to the Af-Art. Rabbit Af-Arts and attached MD were simultaneously microperfused in vitro, keeping pressure in the Af-Art at 60 mm Hg. Increasing the Na+/Cl- concentration of the MD perfusate from 26/7 to 41/22 mEq/L decreased the luminal diameter of the terminal Af-Art segment by 10 +/- 4% (N=9; P < 0.01). The response was maximal at 55/36 mEq/L (18 +/- 6%), so that further elevation of NaCl concentration had no additional effect (20 +/- 6% at 84/65 mEq/L). When FK838 (10(-6) mol/L), a specific adenosine A1 receptor antagonist, was added to both Af-Art perfusate and bath, Af-Art constriction was completely abolished. The maximum response was 20 +/- 3% before FK838 and 0.6 +/- 1% afterward (N=12). Adding adenosine at 10(-8) mol/L to both bath and perfusate significantly augmented Af-Art constriction induced by increased NaCl at the MD (P < 0.01); however, adding 10-8 to 10-6 mol/L adenosine to the MD perfusate had no effect regardless of the NaCl concentration at the MD. These results demonstrate that MD control of Af-Art resistance is induced by relatively low NaCl concentrations at the MD, and that activation of the adenosine A1 receptor in the vascular and interstitial space (but not the tubular lumen) may be essential for signal transmission from the MD to the Af-Art.

Effects of a nucleoside transporter inhibitor, dilazep, on renal microcirculation in rats.

Adenosine, one of the endogenous modulators in renal hemodynamics, has recently been shown to be a mediator of tubuloglomerular feedback (TGF). Dilazep augments endogenous adenosine actions by blocking its cellular uptake. Our purpose in the present study was to clarify the effects of dilazep on renal microcirculation and the TGF mechanism. Clearance and micropuncture experiments were performed in anesthetized rats. TGF responsiveness was assessed in superficial nephrons by measuring the changes of early proximal flow rate (EPFR) in response to loop perfusion at 0-40 nl/min with artificial tubular fluid (ATF). Under dilazep administration (0.3 mg/kg+0.3 mg/kg/h i.v.) systemic BP and GFR were decreased and renal plasma flow was unaltered; as a result, the filtration fraction tended to decrease (p=0.076). Renal vascular resistance was reduced, but not to a significant degree. The reduction in EPFR by loop perfusion was similar between controls (47 +/- 2%) and rats administered dilazep i.v. (44 +/- 5%). Intraluminal application of dilazep in ATF suppressed TGF-mediated EPFR reduction to by 46 +/- 4%, 43 +/- 7%, and 37 +/- 3% at dilazep concentrations of 10(-6), 10(-5), and 10(-4) mol/l, respectively. TGF suppression with 10(-4) mol/l dilazep was reversed by co-perfusion of 10(-5) mol/l DMPX, a selective adenosine A2 receptor antagonist. DMPX alone did not affect TGF response. In conclusion, these results indicate that systemic dilazep dilates postglomerular arterioles and does not affect TGF, and thus reduces GFR. A pharmacological concentration of dilazep applied to single nephrons clearly attenuates TGF, indicating afferent arteriolar vasodilatation. Extracellular adenosine augmented by dilazep dilates glomerular vessels at both afferent and efferent sites, probably via the activation of A2 receptors.

Long-term effects of irradiation before adulthood on reproductive function in the male rhesus monkey.

Today, many patients, who are often young, undergo total body irradiation (TBI) followed by bone marrow transplantation. This procedure can have serious consequences for fertility, but the long-term intratesticular effects of this treatment in primates have not yet been studied. Testes and epididymides of rhesus monkeys that received doses of 4-8.5 Gy of TBI at 2-4 yr of age were studied 3-8 yr after irradiation. In all irradiated monkeys, at least some seminiferous tubule cross-sections lacked germ cells, indicating extensive stem cell killing that was not completely repaired by enhanced stem cell renewal, even after many years. Testes totally devoid of germ cells were only found in monkeys receiving doses of 8 Gy or higher and in both monkeys that received two fractions of 6 Gy each. By correlating the percentage of repopulated tubules (repopulation index) with testicular weight, it could be deduced that considerable numbers of proliferating immature Sertoli cells were killed by the irradiation. Because of their finite period of proliferation, Sertoli cell numbers did not recover, and potential adult testis size decreased from approximately 23 to 13 g. Most testes showed some dilated seminiferous tubules, indicating obstructed flow of the tubular fluid at some time after irradiation. Also, in 8 of the 29 irradiated monkeys, aberrant, densely packed Sertoli cells were found. The irradiation did not induce stable chromosomal translocations in spermatogonial stem cells. No apparent changes were seen in the epididymides of the irradiated monkeys, and the size of the epididymis adjusted itself to the size of the testis. In the irradiated monkeys, testosterone and estradiol levels were normal, whereas FSH levels were higher and inhibin levels lower when testicular weight and spermatogenic repopulation were low. It is concluded that irradiation before adulthood has considerable long-term effects on the testis. Potential testis size is reduced, repopulation of the seminiferous epithelium is generally not complete, and aberrant Sertoli cells and dilated tubules are formed. The latter two phenomena may have further consequences at still longer intervals after irradiation.

REPRODUCTIVE AND REPEATED DOSE TOXICITY OF CYCLODODECATRIENE (CDDT) IN RATS FOLLOWING ORAL (GAVAGE) TREATMENT

ABSTRACTCyclododecatriene (CDDT, CAS No. 4904-61-4) was administered daily by oral gavage to groups of Crl:CD®(SD)IGS BR rats at dose levels of 0 (control), 30, 100, or 300 mg/kg/day. Female rats were dosed for four weeks premating, through mating, gestation, and lactation (a total of 55 to 63 days of treatment). Male rats were treated for 55 days (four weeks premating and through mating). Premating, body weights, food consumption, and clinical signs were recorded. Hematology, clinical chemistry, and urine analyses were conducted at the end of the premating period. A neurobehavioral test battery was conducted prior to and after four weeks of treatment. After the premating period, females were paired with males from the same groups for 1–2 weeks. Litters were delivered, pups were evaluated for structural integrity, and pup body weights were recorded on days 0 and 4 postpartum. Lactating females and their offspring were sacrificed on postpartum day 4. Selected organs were weighed and the tissues were examined microscopically from the lactating females. Offspring were examined for clinical abnormalities. A test substance-related reduction in body weight gain occurred in male rats administered 300 mg/kg/day. Decreased body weight gain in the 300 mg/kg/day males was accompanied by increased food consumption and decreased food efficiency. Females administered 100 or 300 mg/kg/day had test substance-related, significantly decreased body weight and body weight gain during gestation, that was accompanied by a significant increase in food consumption (300 mg/kg/day group only), and significantly decreased food efficiency. There were no test-substance related effects on clinical observations in males or females during the premating phase, or in females during gestation or lactation. Neurobehavioral parameters and motor activity were unaffected by CDDT-treatment. During this study, statistically significant treatment-related changes were observed in several clinical pathology parameters. The decreases in red cell mass (RBC, HGB, HCT) were minimal and, due to the magnitude, were not expected to result in biological effects. Similarly, minimally increased potassium and mildly decreased triglycerides were not of a magnitude to be biologically significant. Finally, changes in serum enzymes (AST, ALT, ALP), urea nitrogen, and serum protein occurred in directions that are not associated with toxicity. The changes in urine volume, urine concentration, and urea nitrogen may be the result of elevated glomerular filtration rate and altered tubular fluid flow, in the absence of any histopathological change. No effects on reproduction in parental males or females were produced by CDDT. Body weights of pups in the 300 mg/kg group were significantly decreased on postpartum days 0 and 4. There were no test-substance related effects on clinical observations, number of pups born, and the number of pups born alive, or the number of pups surviving through lactation day 4. The no-observed-adverse-effect level (NOAEL) for CDDT was 30 mg/kg/day based on decreased body weight and body weight gain, increased food consumption, and decreased food efficiency in females administered 100 or 300 mg/kg/day. The NOEL in pups was 100 mg/kg/day, based on decreased body weights of pups in the 300 mg/kg/day group during lactation.

Intrarenal angiotensin II: Interstitial and cellular levels and site of production

Both local production and angiotensin II subtype 1 (AT1) receptor-mediated uptake from the circulation contribute to the high levels of angiotensin (Ang) II in the kidney. It is largely unknown where Ang II is produced in the kidney and how much of it originates from the circulation. The concentrations of endogenous and 125I-labeled Ang I and II were measured in renal tissue and in blood from pigs receiving systemic infusions of 125I-Ang I. Pigs were either untreated or treated with the angiotensin converting enzyme (ACE) inhibitor captopril or the AT1 receptor antagonist eprosartan. 125I-Ang I was undetectable in renal tissue but the steady-state concentrations of 125I-Ang II in cortical and medullary tissue were four and two times the concentration in arterial blood plasma, respectively. The tissue concentrations of endogenous Ang II were 100 and 60 times higher than in arterial plasma. Eprosartan reduced 125I-Ang II accumulation by 90%, but did not lower tissue Ang II. Captopril did not alter either 125I-Ang II accumulation or tissue Ang II. The bulk of Ang II in the kidney is cell-associated. The high tissue/blood concentration ratio of endogenous Ang II may depend on the same mechanism as demonstrated for 125I-Ang II, that is, AT1 receptor-mediated binding to cells and endocytosis. If so, the results indicate that most renal AT1 receptors are exposed to locally generated Ang II rather than Ang II from the circulation. We propose the existence of a low-Ang II vascular system-related interstitial compartment that is separate from tubular fluid, where, according to micropuncture studies, Ang II levels might be high.

Glomerular protein sieving and implications for renal failure in Fanconi syndrome

Glomerular sieving coefficients (GSCs) of proteins have been measured extensively in animals but not humans. We have studied the proteinuria of Fanconi syndrome, a "knock-out" of renal tubular protein reabsorption, to estimate GSCs and detect potential contributors to development of renal failure. Immunoassay of proteins and polypeptides in serum and urine of patients with early Dent's disease (mean GFR = 83 mL/min, range 60 to 101, N = 5), Lowe's syndrome (N = 3), and ADIF (N = 2) were used. Twenty-one proteins, ranging in mass from insulin (5.1 kD) and parathyroid hormone (PTH; 9.4 kD) to transferrin (78 kD) and intact IgG (160 kD), were present in Fanconi urine at> 6 to 1000-fold normal. A simple model assuming complete "knock-out" of the reuptake of each protein filtered normally by the glomerulus was applied to protein excretion by Dent's patients. GSCs were estimated for 12 plasma proteins, including albumin (7.7 +/- 0.9 x 10-5) and IgG (4.2 +/- 0.28 x 10-5; mean +/- SEM). We calculated the albumin concentration in normal glomerular filtrate to be 3.5 +/- 0.41 mg/L (53 +/- 6.4 nmol/L), consistent with studies in rat and dog. To our knowledge, this study provides the first estimates of human in vivo GSCs. Our model explains why tubular proteinuria of Fanconi syndrome includes proteins of mass of albumin and above as well as low-molecular-weight proteins, and further characterizes the endocytic pathway(s) believed defective in these syndromes. High urinary concentrations of potentially bioactive hormones such as PTH, insulin, IGF-1 and the chemokine monocyte chemoattractant protein-1 (MCP-1), were found; their presence in tubular fluid may contribute to the hypercalciuria, interstitial fibrosis, and the progressive renal failure of Fanconi syndromes.

Sialic acid-containing glycoproteins on renal cells determine nucleation of calcium oxalate dihydrate crystals

The interaction between the surfaces of renal epithelial cells and calcium oxalate dihydrate (COD), the most common crystal in human urine, was studied to identify critical determinants of kidney stone formation. A novel technique utilizing vapor diffusion of oxalic acid was employed to nucleate COD crystals onto the apical surface of living cells. Confluent monolayers were grown in the inner 4 wells of 24-well culture plates. To identify cell surface molecules that regulate crystal nucleation, cells were pretreated with a protease (trypsin or proteinase K) to alter cell surface proteins, neuraminidase to alter cell surface sialoglycoconjugates, or buffer alone. COD crystals were nucleated on the surface of cells by diffusion of oxalic acid vapor into a calcium-containing buffer overlying the cells. Crystal face-specific nucleation was evaluated by scanning electron microscopy. Nucleation and growth of a COD crystal onto an untreated control cell occurred almost exclusively via its (001) face, an event rarely observed during COD crystallization. In contrast, when COD crystals were nucleated onto protease- or neuraminidase-treated cells, they did so via the (100) face of the crystal. Specific sialic acid-containing glycoproteins, and possibly glycolipids (sialoglycoconjugates), appear to be critical determinants of face-specific nucleation of COD crystals on the apical renal cell surface. We hypothesize that crystal retention within the nephron, and the subsequent development of a kidney stone, may result when the number or composition of these cell surface molecules is modified by genetic alterations, cell injury, or drugs in tubular fluid.

A 22-amino acid synthetic peptide corresponding to the second extracellular loop of rat occludin perturbs the blood-testis barrier and disrupts spermatogenesis reversibly in vivo.

When Sertoli cells were cultured in vitro on Matrigel-coated bicameral units, the assembly of the inter-Sertoli tight junction (TJ) permeability barrier correlated with an induction of occludin expression. Inclusion of a 22-amino acid peptide, NH(2)-GSQIYTICSQFYTPGGTGLYVD-COOH, corresponding to residues 209-230 in the second extracellular loop of rat occludin, at 0.2-4 microM into Sertoli cell cultures could perturb the assembly of Sertoli TJs dose-dependently and reversibly. This peptide apparently exerts its effects by interfering with the homotypic interactions of two occludin molecules between adjacent Sertoli cells at the sites of TJs, thereby disrupting TJs, which, in turn, causes a decline in transepithelial electrical resistance across the Sertoli cell epithelium. When similar experiments were performed using a 22-amino acid myotubularin peptide, NH(2)-TKVNERYELCDTYPALLAVPAN-COOH (residues 156-177), no effects on the assembly of inter-Sertoli TJs in vitro were noted. When a single dose of this synthetic occludin peptide was administered to adult rats intratesticularly at 1.5-10 mg/testis, germ cells began to deplete from the seminiferous epithelium within 8-16 days. By 27 days, virtually all tubules were devoid of germ cells. This antispermatogenic effect was reversible, because germ cells progressively repopulated the epithelium thereafter. Treated testes were indistinguishable from normal or control testes by 68 days post-occludin peptide treatment when assessed using histological analysis. In contrast, control rats receiving either no treatment, vehicle alone, or a 22-amino acid synthetic peptide of myotubularin displayed no changes in the testicular morphology at all time points. The occludin peptide-induced germ cell depletion was also accompanied by a disruption of the blood-testis barrier (BTB) when assessed by micropuncture techniques quantifying [(125)I]-BSA in rete testis fluid and seminiferous tubular fluid following i.v. administration of [(125)I]-BSA through the jugular vein. These results illustrate that the occludin peptide-induced disruption of the BTB may possibly affect the underlying adherens junctions, which causes premature release of germ cells from the epithelium and reversible infertility.

Effects of the somatostatin analogue octreotide on renal function in conscious diabetic rats.

Studies performed during the last decade have indicated that growth hormone (GH) and insulin-like growth factors (IGFs) may mediate the early renal changes in diabetes mellitus, i.e. hypertrophy and hyperfiltration. This and other observations have led to the suggestion that GH/IGF inhibitors, such as long-acting somatostatin analogue (e.g. octreotide and lanreotide), may be useful in order to inhibit or prevent development of long-term diabetic complications. The present study examined the acute and chronic effects of octreotide on renal function following induction of streptozotocin (STZ)-diabetes in rats. The studies were carried out in conscious, non-fasted diabetic animals. Chronic administration of octreotide for 7 days, from onset of diabetes, prevented the decrease of effective renal vascular resistance (ERVR), and the increases in filtration fraction (FF), glomerular filtration rate (GFR), and absolute proximal tubular fluid reabsorption (APR) induced by diabetes. The renal hypertrophy was only partially prevented. In the acute study, similar changes were observed in effective renal plasma flow (ERPF) and ERVR but FF increased and GFR remained unaltered. Chronic but not acute treatment with octreotide prevented the renal hyperfiltration caused by diabetes. This effect is most likely due to an increase in afferent arteriolar resistance.

Variable toxicity of dyes used in micropuncture studies

SUMMARY: This study evaluated the potential toxicity of Fast green and Lissamine green, the two dyes commonly used for visualizing tubular fluid flow in micropuncture studies. In both the in vitro perfused kidney and cultured tubular epithelial cells, Fast green exhibited a profound, dose‐dependent toxicity. In contrast, Lissamine green was non‐toxic. In conclusion, we recommend that Fast green not be used in micropuncture studies.

Role of KCNE1-dependent K+ fluxes in mouse proximal tubule.

The electrochemical gradient for K+ across the luminal membrane of the proximal tubule favors K+ fluxes to the lumen. Here it was demonstrated by immunohistochemistry that KCNE1 and KCNQ1, which form together the slowly activated component of the delayed rectifying K+ current in the heart, also colocalize in the luminal membrane of proximal tubule in mouse kidney. Micropuncture experiments revealed a reduced K+ concentration in late proximal and early distal tubular fluid as well as a reduced K+ delivery to these sites in KCNE1 knockout (-/-), compared with wild-type (+/+) mice. These observations would be consistent with KCNE1-dependent K+ fluxes to the lumen in proximal tubule. Electrophysiological studies in isolated perfused proximal tubules indicated that this K+ flux is essential to counteract membrane depolarization due to electrogenic Na+-coupled transport of glucose or amino acids. Clearance studies revealed an enhanced fractional urinary excretion of fluid, Na+, Cl-, and glucose in KCNE1 -/- compared with KCNE1 +/+ mice that may relate to an attenuated transport in proximal tubule and contribute to volume depletion in these mice, as indicated by higher hematocrit values.

Antidiuretic action of vasopressin: quantitative aspects and interaction between V1a and V2 receptor-mediated effects.

(1) Vasopressin (VP), or antidiuretic hormone, is secreted in response to either increases in plasma osmolality (very sensitive stimulus) or to decreases in plasma volume (less sensitive stimulus). Its normal plasma level is very low (about 1 pg/ml, i.e. 10(-12) M), close to the detection limit of present immunoassays, and distinct antidiuretic effects are observed after infusion of small undetectable amounts of VP. (2) This antidiuretic action results from three main effects of VP on principal cells of the collecting duct (CD) mediated by occupancy of peritubular V2 receptors. (i) Increase in water permeability along the entire CD (via AQP2). (ii) Increase in urea permeability in only the terminal inner medullary CD (via UT-A1). (iii) Stimulation of sodium reabsorption, mainly in the cortical and outer medullary CD (via ENaC). VP also acts on medullary vasculature (V1a receptors) to reduce blood flow to inner medulla without affecting blood flow to outer medulla. Besides these actions, all concurring to increase urine osmolality in different and additive ways, other VP effects, probably exerted through V1a receptors located on luminal membrane, tend to limit the antidiuretic effects of the hormone. They induce the formation of prostaglandins which reduce V2-dependent cAMP accumulation in these cells and thus partially inhibit all three V2 effects. (3) Because urine is first diluted along the nephron before being concentrated in the medulla, VP is required, not only for urine concentration, but first for re-equilibration of tubular fluid osmolality with plasma osmolality, a step taking place in the renal cortex, and achieved through the reabsorption of large quantities of water (more than what is subsequently reabsorbed in the medulla to concentrate urine). Accordingly, VP effects on urine flow-rate are not linear. Small changes in plasma VP in the low range of urine osmolality will induce wide changes in urinary flow-rate, whereas in the upper range of urine osmolality larger changes in plasma VP induce much more limited further reduction in urine flow-rate. (4) Most likely, the different effects of VP require different levels of VP concentration to occur and are thus recruited successively with progressive rise in VP secretion.

A numerical model of acid-base transport in rat distal tubule.

The purpose of this study is to develop a numerical model that simulates acid-base transport in rat distal tubule. We have previously reported a model that deals with transport of Na(+), K(+), Cl(-), and water in this nephron segment (Chang H and Fujita T. Am J Physiol Renal Physiol 276: F931-F951, 1999). In this study, we extend our previous model by incorporating buffer systems, new cell types, and new transport mechanisms. Specifically, the model incorporates bicarbonate, ammonium, and phosphate buffer systems; has cell types corresponding to intercalated cells; and includes the Na/H exchanger, H-ATPase, and anion exchanger. Incorporation of buffer systems has required the following modifications of model equations: new model equations are introduced to represent chemical equilibria of buffer partners [e.g., pH = pK(a) + log(10) (NH(3)/NH(4))], and the formulation of mass conservation is extended to take into account interconversion of buffer partners. Furthermore, finite rates of H(2)CO(3)-CO(2) interconversion (i.e., H(2)CO(3) &rlharr; CO(2) + H(2)O) are taken into account in modeling the bicarbonate buffer system. Owing to this treatment, the model can simulate the development of disequilibrium pH in the distal tubular fluid. For each new transporter, a state diagram has been constructed to simulate its transport kinetics. With appropriate assignment of maximal transport rates for individual transporters, the model predictions are in agreement with free-flow micropuncture experiments in terms of HCO reabsorption rate in the normal state as well as under the high bicarbonate load. Although the model cannot simulate all of the microperfusion experiments, especially those that showed a flow-dependent increase in HCO reabsorption, the model is consistent with those microperfusion experiments that showed HCO reabsorption rates similar to those in the free-flow micropuncture experiments. We conclude that it is possible to develop a numerical model of the rat distal tubule that simulates acid-base transport, as well as basic solute and water transport, on the basis of tubular geometry, physical principles, and transporter kinetics. Such a model would provide a useful means of integrating detailed kinetic properties of transporters and predicting macroscopic transport characteristics of this nephron segment under physiological and pathophysiological settings.

RAPID COMMUNICATIONIn vivo inhibition of renal 11β-hydroxysteroid dehydrogenase in the rat stimulates collecting duct sodium reabsorption

In order to test the proposal that the aldosterone specificity of mineralocorticoid receptors in the collecting duct depends on inactivation of glucocorticoids by the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD), we have assessed the effect of pharmacological inhibition of 11beta-HSD on collecting duct Na+ reabsorption in vivo. Adrenalectomized rats (n=14) were infused intravenously with high-dose corticosterone, and late-distal tubules were perfused orthogradely with artificial tubular fluid containing [14C]inulin and 22Na; urinary recoveries of the radioisotopes were monitored. Half of the rats received intravenous carbenoxolone to inhibit renal 11beta-HSD activity. The urinary recovery of [14C]inulin was complete in both groups of animals (101+/-2% versus 101+/-3%), but the recovery of 22Na was lower in carbenoxolone-treated rats (34+/-5%) than in the corticosterone-alone group (54+/-4%, P<0.01). These data, which provide the first demonstration of enhanced Na+ reabsorption in the distal nephron during inhibition of renal 11beta-HSD in vivo, strongly support the proposal that 11beta-HSD normally prevents endogenous glucocorticoid from exerting mineralocorticoid-like effects.