On multistability behavior of unstable dissipative systems.

We present dissipative systems with unstable dynamics called the unstable dissipative systems which are capable of generating a multi-stable behavior, i.e., depending on its initial condition, the trajectory of the system converges to a specific attractor. Piecewise linear (PWL) systems are generated based on unstable dissipative systems, whose main attribute when they are switched is the generation of chaotic trajectories with multiple wings or scrolls. For this PWL system, a structure is proposed where both the linear part and the switching function depend on two parameters. We show the range of values of such parameters where the PWL system presents a multistable behavior and trajectories with multiscrolls.

Podocyte vascular endothelial growth factor (Vegf164) overexpression causes severe nodular glomerulosclerosis in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: The pathogenic role of excessive vascular endothelial growth factor (VEGF)-A in diabetic nephropathy has not been defined. We sought to test whether increased podocyte VEGF-A signalling determines the severity of diabetic glomerulopathy. METHODS: Podocyte-specific, doxycycline-inducible Vegf164 (the most abundant Vegfa isoform) overexpressing adult transgenic mice were made diabetic with low doses of streptozotocin and examined 12 weeks after onset of diabetes. We studied diabetic and non-diabetic transgenic mice fed a standard or doxycycline-containing diet. VEGF-A and albuminuria were measured by ELISA, creatinine was measured by HPLC, renal morphology was examined by light and electron microscopy, and gene expression was assessed by quantitative PCR, immunoblotting and immunohistochemistry. RESULTS: Podocyte Vegf164 overexpression in our mouse model of diabetes resulted in advanced diabetic glomerulopathy, characterised by Kimmelstiel-Wilson-like nodular glomerulosclerosis, microaneurysms, mesangiolysis, glomerular basement membrane thickening, podocyte effacement and massive proteinuria associated with hyperfiltration. It also led to increased VEGF receptor 2 and semaphorin3a levels, as well as nephrin and matrix metalloproteinase-2 downregulation, whereas circulating VEGF-A levels were similar to those in control diabetic mice. CONCLUSIONS/INTERPRETATION: Collectively, these data demonstrate that increased podocyte Vegf164 signalling dramatically worsens diabetic nephropathy in a streptozotocin-induced mouse model of diabetes, resulting in nodular glomerulosclerosis and massive proteinuria. This suggests that local rather than systemic VEGF-A levels determine the severity of diabetic nephropathy and that semaphorin3a signalling and matrix metalloproteinase-2 dysregulation are mechanistically involved in severe diabetic glomerulopathy.

Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption.

Epithelia permit selective and regulated flux from apical to basolateral surfaces by transcellular passage through cells or paracellular flux between cells. Tight junctions constitute the barrier to paracellular conductance; however, little is known about the specific molecules that mediate paracellular permeabilities. Renal magnesium ion (Mg2+) resorption occurs predominantly through a paracellular conductance in the thick ascending limb of Henle (TAL). Here, positional cloning has identified a human gene, paracellin-1 (PCLN-1), mutations in which cause renal Mg2+ wasting. PCLN-1 is located in tight junctions of the TAL and is related to the claudin family of tight junction proteins. These findings provide insight into Mg2+ homeostasis, demonstrate the role of a tight junction protein in human disease, and identify an essential component of a selective paracellular conductance.

Torasemide significantly reduces thiazide-induced potassium and magnesium loss despite supra-additive natriuresis.

BACKGROUND: Resistance to high-dose loop diuretics can be overcome either by co-administration with thiazides or by treatment with medium-dose loop diuretics combined with thiazides. Combination therapy has been proven to be superior to high-dose loop diuretic monotherapy for cardiac and renal edema. However, such a strongly efficacious short-term regimen is often complicated by undesired effects, including circulatory collapse and electrolyte disturbances. The question of whether the loop diuretic/thiazide combinations are efficacious and safe when conventional doses are combined has not yet been answered. METHODS: The effects of hydrochlorothiazide (HCT) and torasemide (TO) given alone on the excretion of Na+, Cl-, K+, Mg2+, and Ca2+ were compared with the effects of combined administration of the diuretics in 12 healthy volunteers. RESULTS: The co-administration of HCT (25 mg) with TO (5 or 10 mg) strongly increased Na+ excretion. However, the combination significantly reduced K+ and Mg2+ excretion. The K+-sparing effect of the HCT/TO combination was shown to be due to a significant reduction in the HCT-induced increase in fractional K+ excretion by the loop diuretic. Total excretion of Ca2+ relative to Na+ excretion was less with the HCT/TO combination than with TO given alone. CONCLUSION: The enhancement of desired NaCl excretion by the HCT/TO combination with significant reduction of undesired loss of K+ and Mg2+ meets clinical requirements but has to be validated in long-term clinical trials.

Adaptation of the distal convoluted tubule of the rat. Structural and functional effects of dietary salt intake and chronic diuretic infusion.

We studied the effects of dietary NaCl intake on the renal distal tubule by feeding rats high or low NaCl chow or by chronically infusing furosemide. Furosemide-treated animals were offered saline as drinking fluid to replace urinary losses. Effects of naCl intake were evaluated using free-flow micropuncture, in vivo microperfusion, and morphometric techniques. Dietary NaCl restriction did not affect NaCl delivery to the early distal tubule but markedly increased the capacity of the distal convoluted tubule to transport Na and Cl. Chronic furosemide infusion increased NaCl delivery to the early distal tubule and also increased the rates of Na and Cl transport above the rates observed in low NaCl diet rats. When compared with high NaCl intake alone, chronic furosemide infusion with saline ingestion increased the fractional volume of distal convoluted tubule cells by nearly 100%, whereas dietary NaCl restriction had no effect. The results are consistent with the hypotheses that (a) chronic NaCl restriction increases the transport ability of the distal convoluted tubule independent of changes in tubule structure, (b) high rates of ion delivery to the distal nephron cause tubule hypertrophy, and (c) tubule hypertrophy is associated with increases in ion transport capacity. They indicate that the distal tubule adapts functionally and structurally to perturbations in dietary Na and Cl intake.

Expression of the thiazide-sensitive Na-Cl cotransporter by rabbit distal convoluted tubule cells.

A thiazide-sensitive Na-Cl cotransporter contributes importantly to mammalian salt homeostasis by mediating Na-Cl transport along the renal distal tubule. Although it has been accepted that thiazide-sensitive Na-Cl cotransport occurs predominantly along the distal convoluted tubule in rats and mice, sites of expression in the rabbit have been controversial. A commonly accepted model of rabbit distal nephron transport pathways identifies the connecting tubule, not the distal convoluted tubule, as the predominant site of thiazide-sensitive Na-Cl cotransport. The thiazide-sensitive Na-Cl cotransporter has been cloned recently. The present experiments were designed to localize sites of thiazide-sensitive Na-Cl cotransporter mRNA expression along the rabbit distal nephron. Nonradioactive in situ hybridization with a thiazide-sensitive Na-Cl cotransporter probe was combined with immunocytochemistry with an antibody that recognizes distal convoluted tubule cells and with a Na+/Ca2+ exchanger antibody that recognizes only connecting tubule cells. The results indicate that thiazide-sensitive Na-Cl cotransporter mRNA is highly expressed by cells of the distal convoluted tubule and not by connecting tubule cells. Segments that stain with the Na+/Ca2+ exchanger antibody (connecting tubules) do not demonstrate thiazide-sensitive Na-Cl cotransporter mRNA expression. We conclude that the predominant site of thiazide-sensitive Na-Cl cotransporter mRNA expression in rabbit distal nephron is the distal convoluted tubule and that sites of mRNA expression of electroneutral Na and Cl transport are similar in rabbits, rats, and mice.

[Concordance between preoperatory diagnosis and arthroscopy findings of meniscal lesions associated to anterior cruciate ligament injury]. / Estudio del nivel de concordancia entre los diagnósticos prequirúrgico y artroscópico de lesiones meniscales asociadas a lesiones del ligamento cruzado anterior.

INTRODUCTION: Ligament injuries of the knee joint are common during the second and third decades of life, clinical and radiological diagnosis presents difficulties since the surgical findings may differ significantly. The objective is to determine correlation between clinical-radiologic and arthroscopic diagnosis in patients with injury of the anterior cruciate ligament (ACL) and meniscal lesions. MATERIAL AND METHODS: Cross-sectional study held in 29 patients with ACL injury associated to meniscal lesions treated arthroscopically. Variables were age, gender, affected side, preoperative and postoperative diagnosis, type and location of meniscal injury; descriptive statistics and Cohen Kappa for concordance were used. RESULTS: There were 29 patients, 23 (79.3%) men and 6 (20.7%) women, mean age 39.04 (15-50) ± 13.19 years; right side was affected in 69% and left in 31%; 19% had not meniscal lesion, 17.2% had injury in anterior horn, 10.3% in posterior horn and 6.9% in the body. The concordance between preoperative and artroscopic diagnosis was: Kappa 0.2; intraboservador was Kappa 1.0; between suspected meniscal injury and arthroscopic findings was: Kappa 0.2. CONCLUSIONS: The concordance between the clinical-radiological and arthroscopic diagnosis in patients with ACL injuries and meniscal injury associated is low, which has to be considered in the initial review and the arthroscopic treatment of patients.

INTRODUCCIÓN: Las lesiones ligamentarias de la rodilla son comunes durante la segunda y tercera décadas de vida, su diagnóstico clínico es difícil ya que puede diferir con el diagnóstico definitivo. El objetivo es determinar la concordancia entre el diagnóstico preoperatorio-diagnóstico postoperatorio y entre la lesión meniscal sospechada-lesión meniscal encontrada, en pacientes con lesión del ligamento cruzado anterior. MATERIAL Y MÉTODOS: Estudio transversal. En 29 pacientes con lesión del ligamento cruzado anterior a los cuales se realizó artroscopía. Las variables fueron edad, género, lado afectado, diagnóstico preoperatorio, diagnóstico postoperatorio, lesión meniscal sospechada, lesión meniscal encontrada. La estadística utilizada fue descriptiva, para la concordancia se usó índice de Kappa de Cohen. RESULTADOS: Fueron 29 pacientes, 23 (79.3%) hombres y 6 (20.7%) mujeres, la edad promedio: 39.04 (15-50) ± 13.19 años, lado afectado derecho 69% e izquierdo 31%; el diagnóstico preoperatorio en 22 (75%) pacientes fue LLCA aislada, LLCA + menisco medial 6 (20.7%), LLCA + menisco lateral 1 (3.4%) y el diagnóstico artroscópico fue en 20 (68.96%) sin lesión meniscal, 7 (24.1%) lesión en menisco medial, 2 (6.9%) en menisco lateral, con una concordancia baja (Kappa 0.2), la concordancia diagnóstica preoperatoria intraobservador fue perfecta (Kappa 1.0); y la concordancia entre el diagnóstico de la lesión meniscal preoperatoria y de la lesión meniscal artroscópica fue baja (Kappa 0.2). CONCLUSIONES: La concordancia entre el diagnóstico clínico-radiológico y el diagnóstico artroscópico en lesiones del LCA con asociación de lesión meniscal es baja, lo cual se tiene que tener en cuenta en la realización del diagnóstico inicial y en el tratamiento artroscópico de los pacientes.

Metabolism of a potential 8-aminoquinoline antileishmanial drug in rat liver microsomes.

The metabolism of the 8-aminoquinoline, 8-(6-diethylaminohexylamino)-6-methoxy-lepidine dihydrochloride (WR 6026 X 2HCl), was studied in a rat hepatic microsomal system. The results show that WR 6026 X 2HCl was metabolized into two more polar compounds. The structures of these metabolites as proven by gas chromatography-mass spectrometry, ultraviolet absorption, and high performance liquid chromatography were: 8-(6-ethylaminohexylamino)-6-methoxy-lepidine (metabolite 1) and 8-(6-diethylaminohexylamino)-6-methoxy-4-hydroxymethyl quinoline (metabolite 2). The formation of both metabolites was NADPH dependent and also linearly dependent on incubation time and microsomal protein concentration at 0.24 mM WR 6026 X 2 HCl. Studies on the effects of pretreatment of animals with either phenobarbital or Aroclor 1254 suggest that cytochrome P-450 isozymes catalyzed both N-deethylation and hydroxylation reactions. N-deethylase activity was induced by either pretreatment: however, hydroxylase activity was unaffected by phenobarbital pretreatment and significantly elevated by Aroclor 1254 pretreatment. These results suggest that these two reactions are catalyzed by different cytochrome P-450 isozymes. The formation of these two metabolites in vivo may play an important role in the antileishmanial activity of WR 6026 X 2HCl.

[Nutritional status of vitamin D in mothers and neonates of Ushuaia and Buenos Aires]. / Estado nutricional de vitamina D en madres y neonatos de Ushuaia y Buenos Aires.

Serum levels of calcium, phosphorus, total alkaline phosphatase (AP) and 25 hydroxyvitamin D (250HD) were measured at the end of the winter in Group 1 (Ushuaia, latitude 55 degrees S): 16 women (24-48 hs postpartum serum blood) and 20 neonates (cord blood) and in Group 2 (Buenos Aires, latitude 34 degrees S) 21 women (24-48 hs postpartum serum blood) and their 21 neonates (cord blood). The neonatal serum calcium and phosphorus were higher and the neonatal serum AP and 250HD level were lower than maternal levels in both groups (Table 1 and 2). Serum levels of 250HD were diminished (< 8 ng/ml) in 62% of the mothers and 81% of the neonates of Ushuaia and in 24% of the mothers and 16% of the neonates of Buenos Aires (figure 1). Neonatal serum 250HD levels correlate with maternal serum 250HD levels in the paired group of Buenos Aires (r = 0.65, p < 0.003) (Figure 2). In Ushuaia the serum 250HD levels (X +/- SD) in neonates (3.9 +/- 2.7 ng/ml) and in mothers (6.3 +/- 4.8 ng/ml) were lower than in Buenos Aires (neonates: 11.3 +/- 6.0 ng/ml and mothers: 14.4 +/- 8.4 ng/ml, p < 0.001). Maternal serum calcium levels were lower in Ushuaia (8.7 +/- 0.8 mg/dl) than in Buenos Aires (9.2 +/- 0.4 mg/dl) (p < 0.05). In conclusion, 1) In Ushuaia pregnant women and their neonates had a deficient nutritional state of vitamin D. Preventive administration of vitamin D would probably be beneficial.(ABSTRACT TRUNCATED AT 250 WORDS)

Reducing exposure to long days from 75 to 30 days of extra-light treatment does not decrease the capacity of male goats to stimulate ovulatory activity in seasonally anovulatory females.

The response of male goats exposed to different durations of long days (LD) during an extra-light treatment in autumn-winter, and their ability to induce ovulations in seasonally anovulatory goats were investigated in 2 experiments. In experiment 1, control males were exposed to natural photoperiod (n = 5), whereas 4 additional groups (n = 5/group) were exposed to 16 h of light per d during 75, 45, 30, or 15 d of LD. In the 4 groups, photoperiodic treatments ended on January 15th. Plasma concentrations of testosterone were determined in blood samples obtained once a week from October 15th to May 30th. The rise of testosterone levels occurred earlier in males from the 75-LD and 45-LD groups than in those from the 30-LD, 15-LD, and control groups (P < 0.05). In addition, the time during which levels of testosterone remained >5 ng/mL was longer in males from the 75-LD and 45-LD than in those from the 30-LD and 15-LD groups (P < 0.05). In experiment 2, a group of anovulatory goats (n = 13) was isolated from males, while 3 additional groups were put in contact during 15 d with males previously exposed to 75, 45, or 30 days of LD (n = 25, 27, and 26 females/group, respectively and n = 3 males per group). The proportion of goats that ovulated was higher in the 3 groups in contact with the photo-stimulated males (range: 88%-92%) than in the group isolated from them (0%; P < 0.05). The proportion of pregnant females did not differ between the 3 groups of does in contact with photo-stimulated males (range: 78%-92%; P > 0.05). We conclude that, in our experimental conditions, a photoperiodic treatment as short as 30 d of LD during autumn-winter, stimulated testosterone secretion of bucks during their period of sexual rest and rendered them able to induce ovulations in seasonal anestrous goats and to obtain pregnancies in these females.

Sexually active bucks are able to stimulate three successive groups of females per day with a 4-hour period of contact.

Bucks rendered sexually active by a photoperiod treatment of long days can induce fertile ovulation in a group of goats with only 4 h of contact daily with a male:female ratio of 1:10. Here we tested whether such bucks could induce fertile ovulations when stimulating successively three different groups of anovulatory goats when interacting 4 h per day during 15 consecutive days. Control males (n=3) were introduced in the control group (n=25) of does at 8:00 h and were removed at 12:00 h. Experimental males (n=3) were in contact with the experimental groups of does: from 8:00 h to 12:00 h with a first group (n=27), from 12:00 h to 16:00 h with a second group (n=26) and with a third one (n=27) from 16:00 h to 20:00 h. Bucks were then placed until next day in another pen. Both in the control and the experimental groups, more than 85% of females ovulated, and the proportions did not differ between the control and experimental groups (P≥0.67) or between the three experimental groups (P≥0.67). Moreover, the ovulation rate did not differ significantly between the control and the experimental females nor between the three experimental groups. Bucks were able to fertilize more than 72% of does independently of the number of females they were exposed to (P≥0.17). Finally, more than 58% of females kidded and fertility did not differ between the control and experimental groups (P=1) nor among experimental groups (P≥0.77). We conclude that sexually active bucks are able to induce fertile ovulation in three successive groups of anovulatory goats even when the period of contact between sexes is reduced to 4 h per day.

Estudio del nivel de concordancia entre los diagnósticos prequirúrgico y artroscópico de lesiones meniscales asociadas a lesiones del ligamento cruzado anterior / Concordance between preoperatory diagnosis and arthroscopy findings of meniscal lesions associated to anterior cruciate ligament injury

Introducción: Las lesiones ligamentarias de la rodilla son comunes durante la segunda y tercera décadas de vida, su diagnóstico clínico es difícil ya que puede diferir con el diagnóstico definitivo. El objetivo es determinar la concordancia entre el diagnóstico preoperatorio-diagnóstico postoperatorio y entre la lesión meniscal sospechada-lesión meniscal encontrada, en pacientes con lesión del ligamento cruzado anterior. Material y métodos: Estudio transversal. En 29 pacientes con lesión del ligamento cruzado anterior a los cuales se realizó artroscopía. Las variables fueron edad, género, lado afectado, diagnóstico preoperatorio, diagnóstico postoperatorio, lesión meniscal sospechada, lesión meniscal encontrada. La estadística utilizada fue descriptiva, para la concordancia se usó índice de Kappa de Cohen. Resultados: Fueron 29 pacientes, 23 (79.3%) hombres y 6 (20.7%) mujeres, la edad promedio: 39.04 (15-50) ± 13.19 años, lado afectado derecho 69% e izquierdo 31%; el diagnóstico preoperatorio en 22 (75%) pacientes fue LLCA aislada, LLCA + menisco medial 6 (20.7%), LLCA + menisco lateral 1 (3.4%) y el diagnóstico artroscópico fue en 20 (68.96%) sin lesión meniscal, 7 (24.1%) lesión en menisco medial, 2 (6.9%) en menisco lateral, con una concordancia baja (Kappa 0.2), la concordancia diagnóstica preoperatoria intraobservador fue perfecta (Kappa 1.0); y la concordancia entre el diagnóstico de la lesión meniscal preoperatoria y de la lesión meniscal artroscópica fue baja (Kappa 0.2). Conclusiones: La concordancia entre el diagnóstico clínico-radiológico y el diagnóstico artroscópico en lesiones del LCA con asociación de lesión meniscal es baja, lo cual se tiene que tener en cuenta en la realización del diagnóstico inicial y en el tratamiento artroscópico de los pacientes.

Introduction: Ligament injuries of the knee joint are common during the second and third decades of life, clinical and radiological diagnosis presents difficulties since the surgical findings may differ significantly. The objective is to determine correlation between clinical-radiologic and arthroscopic diagnosis in patients with injury of the anterior cruciate ligament (ACL) and meniscal lesions. Material and methods: Cross-sectional study held in 29 patients with ACL injury associated to meniscal lesions treated arthroscopically. Variables were age, gender, affected side, preoperative and postoperative diagnosis, type and location of meniscal injury; descriptive statistics and Cohen Kappa for concordance were used. Results: There were 29 patients, 23 (79.3%) men and 6 (20.7%) women, mean age 39.04 (15-50) ± 13.19 years; right side was affected in 69% and left in 31%; 19% had not meniscal lesion, 17.2% had injury in anterior horn, 10.3% in posterior horn and 6.9% in the body. The concordance between preoperative and artroscopic diagnosis was: Kappa 0.2; intraboservador was Kappa 1.0; between suspected meniscal injury and arthroscopic findings was: Kappa 0.2. Conclusions: The concordance between the clinical-radiological and arthroscopic diagnosis in patients with ACL injuries and meniscal injury associated is low, which has to be considered in the initial review and the arthroscopic treatment of patients.

Thiazide diuretics.

Thiazide diuretics increase salt and water excretion primarily by inhibiting mechanisms for electroneutral sodium and chloride transport by distal convoluted tubule cells. This might be termed the 'specific' effect of this class of diuretics and accounts for the 'chlorouretic' effectiveness of the drug. Secondary to this inhibition of sodium and chloride absorption, potassium secretion is stimulated most likely because of the resultant increase in distal tubule fluid flow rate, and calcium absorption is stimulated possibly via a decrease in distal convoluted tubule cell sodium activity and an increase in basolateral sodium/calcium exchange. To a varying degree, thiazides also inhibit carbonic anhydrase. This effect can contribute to the diuresis, but is largely buffered by the reserve transport capacity of the loop of Henle. To the extent that the effects of transport inhibition in the proximal tubule are transmitted to the distal tubule, tubuloglomerular feedback may be activated and effect a reduction in the glomerular filtration rate.

Effects of diuretic drugs on Na, Cl, and K transport by rat renal distal tubule.

Diuretic drugs were used to characterize mechanisms involved in transporting sodium, chloride, and potassium across the wall of surface distal tubules of the rat kidney using in vivo microperfusion techniques. Both furosemide and chlorothiazide inhibited sodium and chloride absorption but did not affect the rate of potassium secretion or the transepithelial voltage. However, chlorothiazide inhibited sodium and chloride absorption more completely than furosemide and was additive to the effect of furosemide; furosemide was ineffective if chlorothiazide was already present. In contrast to the effect of furosemide, bumetanide did not affect sodium and chloride absorption but did increase potassium secretion. Amiloride reduced sodium absorption and potassium secretion without affecting net chloride absorption. These effects were additive to those of chlorothiazide. In the loop of Henle bumetanide was more effective than furosemide in inhibiting net sodium potassium and chloride absorption. It appears that cells of the distal tubule in the rat possess an Na-Cl cotransport mechanism that differs from the Na-K-2Cl cotransport mechanism found in the thick ascending limb. Sodium transport also proceeds via a conductive pathway that is inhibited by amiloride. The two modes of sodium transport, conductive and coupled to chloride, may occur in different cell types along the distal tubule.

Control by drugs of renal potassium handling.

This review has focused on the influence of several diuretic drugs on potassium handling by the kidney. One class of drugs (loop diuretics) acts by directly inhibiting a potassium absorptive mechanism in the luminal membrane of cells of the thick ascending limb of Henle's loop. Two other groups of diuretics affect potassium transport indirectly by inhibiting salt and water absorption upstream from the potassium secretory site in the late distal tubule: carbonic anhydrase inhibitors act in the proximal tubule; thiazides act in the early distal tubule. The subsequent increase in lumen flow rate then stimulates net potassium secretion by the distal tubule. A fourth class of drugs (spironolactone) acts by antagonizing the response of the distal tubule to aldosterone. These drugs decrease the ability of aldosterone to stimulate distal potassium secretion. Finally, a fifth group of drugs (potassium-sparing diuretics) decreases potassium secretion by increasing the luminal membrane voltage and thus decreasing the electrochemical gradient for potassium exit from the cell.

Identification of a novel K-channel gene (KC22) that is highly expressed in distal tubule of rabbit kidney.

The Shaker gene family encodes voltage-gated K channels. Five partial-length Shaker-like cDNAs (KC2, 4, 10, 19, and 22) were previously isolated from rabbit kidney using polymerase chain reaction (PCR) [G. V. Desir, E. Hamlin, A.H. Puente, R.F. Reilly, F. Hiledebrandt, and P. Igarashi. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F151-F157, 1992]. We now report the cloning of another Shaker-like cDNA (KC6) from rabbit kidney and the identification of one isoform that is highly expressed in rabbit distal tubule cells grown in culture. A partial-length cDNA (859 bp) for KC6 was isolated by PCR amplification of rabbit kidney cDNA using Shaker-specific degenerate primers. KC6 was most similar to the rat brain clone RBK2 (77% amino acid identity) and to the rabbit clone KC19 (78% amino acid identity). Transcript levels for KC2, 4, 6, 10, 19, and 22 were quantified using the ribonuclease protection assay. Transcripts for all six isoforms were detected in renal tissues. KC22 was the most abundant isoform in kidney cortex and medulla (20- to 40-fold greater than the other isoforms). Furthermore, KC22 expression levels were fivefold higher in primary cultures of rabbit distal convoluted tubules and connecting tubules than in whole kidney cortex. Although the partial-length sequence for KC22 represents the most conserved regions in the Shaker gene family it only has 35-88% amino acid identity with other Shaker channels, suggesting that KC22 represents a novel isoform. In contrast, KC4 and KC19 (less abundant in kidney than KC22) are highly homologous to the rat brain clones RBK1 and RBK2, respectively (97% amino acid identity).(ABSTRACT TRUNCATED AT 250 WORDS)

Resetting of tubuloglomerular feedback by interrupting early distal flow.

Cell-cell contact between the macula densa and the glomerular arterioles is is thought to provide the information pathway for the tubuloglomerular feedback (TGF) mechanism. When concentrations of sodium and chloride in the macula densa segment are increased, a signal is transmitted through the extraglomerular mesangium to contract the afferent arteriole. In addition, some observers have described a second region of contact between a later part of the distal tubule and the afferent arteriole of the same nephron. In this region the connecting tubule (CNT), and sometimes nerves that make contact with the cells of this CNT, were found. This arrangement gives another potential tubular segment, besides the macula densa plaque, in which the composition of tubular fluid may regulate glomerular dynamics. The present study was designed to investigate whether interrupting flow in the distal tubule downstream from the macula densa would influence the TGF mechanism. TGF was examined in rats by orthograde microperfusion, before and after blockade of the distal nephron with castor oil. Two variables were measured: maximum decrease in stop-flow pressure (delta Psf), and perfusion rate which elicits half-maximal decrease in delta Psf (V1/2). The fluid arriving at the blocking point was collected into a micro-pipette. The results show a significant increase in V1/2 from 19 to 25 nl min-1 after 30 min of blockade. In conclusion the results support a role of the distal nephron in the control of the TGF mechanism.

Effect of Na-channel blockers and lumen Ca on K secretion by rat renal distal tubule.

In previous studies the effectiveness of amiloride (AML) in reducing K secretion has been variable. Based on studies by Cuthbert and Wong (Mol. Pharmacol. 8: 222-229, 1972) in which the Na-channel-blocking action of AML in frog skin was found to require the availability of Ca ions in extracellular fluid, we postulated that the ability of AML and its analogue, benzamil (BZA), to inhibit distal tubule K secretion depends on the presence of Ca in luminal fluid. We found that addition of Ca to a perfusion solution containing 50 microM BZA did reduce K secretion more than BZA alone. Maximal inhibition was observed with 2.5 mM free ionic Ca. Graded increases in luminal Ca in presence of AML or BZA reduced K transport in a dose-dependent manner. The decrease in K secretion with increasing luminal Ca was paralleled by a decrease in transepithelial voltage. These results support our hypothesis that the effectiveness of Na-channel blockers to reduce K secretion by the rat distal tubule depends on presence of luminal Ca and suggest an interaction between luminal Ca and Na-channel blockers on the Na channel.