Video-imaging microfluorometry identifies alpha- and beta-like cell types in Madin-Darby canine kidney monolayers.

On the basis of intracellular, accumulation of c-SNAFL-2, we have identified three cell subtypes in Madin-Darby canine kidney (MDCK) monolayers. Highly fluorescent cells (HFC) have a high intracellular pH (pHi, whereas cells with medium fluorescence (MFC) have low pHi when perfused with buffer containing 125 mM Cl-. HFC express a Cl-/HCO3- exchanger on the apical but not the basolateral membrane. MFC express a Cl-/HCO3- exchanger on the basolateral but not the apical membrane. We have termed these cells beta- and alpha-MDCK cells, respectively. Cells with low fluorescence (LFC) probably extrude c-SNAFL-2 through a monocarboxylate transporter, because p-4-(chloromercuri)phenylsulfonic acid (PCMBS), an inhibitor of this transporter, leads to homogeneous fluorescence.

Reverse transcriptase-polymerase chain reaction study of anion exchanger-2 in canine tissues and different Madin-Darby canine kidney cell types.

Reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA from canine large intestine, skeletal muscle, pancreas, kidney, and spleen and from cultured wild-type and C7 and C11 Madin-Darby canine kidney (MDCK) cells revealed considerable variation in anion exchanger (AE)1 and AE2 mRNA levels between the tissues. Similar high levels of AE2 mRNA were detected in all the MDCK cell populations. AE2 in MDCK cells is probably the basolateral Cl-/HCO3- exchanger common to the principal and beta-intercalated cells.

Influence of extracellular pH on intracellular pH in arterioles and skeletal muscle of spontaneously hypertensive and Wistar-Kyoto rats.

The effect of changing extracellular pH (pHo) on intracellular pH (pHi) in mesenteric arterioles of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats was investigated using the fluorescent indicator seminaphthorhodafluor c-SNARF-1 to measure pHi. Although the pHi of arterioles from WKY varied directly with pHo, that of SHR arterioles varied inversely. This abnormal pHi regulation in SHR was corrected by 50-microM 5-(N-ethylisopropyl)amiloride (EIPA). Vmax of H+ transport in response to changing extracellular Na+ was higher in SHR than in WKY, but Km did not differ. The Hill coefficient for H+ transport with respect to pH; was 1.69 in prehypertensive and 1.56 in hypertensive SHR. These results indicate that the Na+/H+ exchanger is particularly hyperactive in mesenteric arterioles of SHR.

Effect of glandular kallikrein on distal nephron HCO3- secretion in rats and on HCO3- secretion in MDCK cells.

Renal kallikrein is localized in the connecting tubule cells and secreted into the tubular fluid at late distal nephron segments. The present experiments were performed to further test the hypothesis that renal kallikrein reduces bicarbonate secretion of cortical collecting duct (CCD). The effect of orthograde injections of pig pancreatic kallikrein (1 or 3 micrograms/ml) into the renal tubular system was investigated. Urine fractions (Fr) were collected after a 2-min stop flow. Changes in the urine fraction with respect to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/ Fr:Ff polyfructosan) increased significantly in the first two urine fractions collected after glandular kallikrein administration (kallikrein, 1 microgram/ml, P < 0.05; kallikrein, 3 micrograms/ml, P < 0.01). HCO3- secretion of collecting ducts was significantly reduced dose dependently by orthograde and also reduced by retrograde pig pancreatic kallikrein administration. Release of kinins into the fractions was not affected by the retrograde kallikrein injection, even though the kallikrein activity increased considerably (2.26 +/- 0.2 vs. 1.55 +/- 0.2, P < 0.05). Adequacy of retrograde injections for delivering substances to the CCD was demonstrated by injecting colloidal mercury and detecting the appearance of this mercury in the renal cortex by transmission electron microscopy. The integrity of the renal tissue after a retrograde ureteral injection was confirmed by scanning electron microscopy. These results confirm and extend previous data (M. Marin-Grez and P. Vallés. Renal Physiol. Biochem. 17: 301-306, 1994; and M. Marin-Grez, P. Vallés, and P. Odigie. J. Physiol. 488: 163-170, 1995) showing that renal kallikrein reduces bicarbonate secretion at the CCD, probably by inhibiting HCO3- transported by a mechanism unrelated to its kininogenase activity. Support for this assessment was obtained in experiments testing the effect of kallikrein on the luminal bicarbonate secretion of a subpopulation of Madin-Darby canine kidney cells capable of extruding the anion. Kallikrein inhibited HCO3-/Cl- exchange, and the degree of inhibition was dose dependent. This inhibition occurred in the absence of kininogen in the bathing solution.

Reduced glutathione inhibits rabbit and rat skeletal muscle lactate dehydrogenase and prevents dinitrophenol induced extracellular acidification by an epithelial cell line.

Glutathione (GSH) is a tripeptide synthesised enzymatically from its components amino-acids by unicellular and multicellular organisms. GSH acts as a cellular anti-oxidant, protects the structural configuration of some enzymes, is involved in erythrocyte function and plays a role as co-enzyme in several reactions. We have found that GSH inhibits purified lactate dehydrogenase (1.56 U LDH/ml) from rabbit skeletal muscle after 6 min pre-incubation with an ED50 of about 5.4 microM. The inhibition is time dependent with a maximum after 45 minutes pre-incubation. Buffer (5 x 10(-2) M TRIZMA hydrochloride, pH 7.4) and a chelator (2 x 10(-3) M EDTA) in the pre-incubation solution did not prevent the inhibition. Prolonged dialysis was almost without effect on GSH-inhibited LDH activity solution, indicating either an irreversible or a very tight binding inhibition. Kinetic analysis showed that this inhibition is of a very tight binding and at the same time of the uncompetitive type. GSH also inhibits LDH activity of rat M. soleus and M. gastrocnemius homogenates. This effect is probably unrelated to the reducing property of GSH since dithioerythritol (0.17-1.34 mM) does not mimic it. Loading of MDCK cells with glutathione ethylester completely prevented the acidification induced by 2,4-dinitrophenol, suggesting that GSH may influence the glycolytic pathway in vivo.

Role of perfusate hydrogen ion activity in kallikrein release from isolated rat kidneys.

The present experiments were performed to investigate whether renal kallikrein release by isolated perfused rat kidneys correlates with acid-base-related parameters. Kallikrein excretion per millilitre of glomerular filtrate was inversely correlated with perfusate pH (r = -0.49, P < 0.001) and HCO3- concentration (r = -0.46, P < 0.005). A direct relationship between kallikrein excretion per millilitre of glomerular filtrate and urinary Na+/K+ ratio was found (r = 0.59, P < 0.001). Some 86% of the variability (F ratio 110, P < 0.00001) of urinary kallikrein activity was attributable to the perfusate pH and the urinary cation ratio. Therefore, urinary kallikrein activity was highly correlated with perfusate H+ activity corrected by the urinary Na+/K+ ratio (r = 0.92, P < 0.0001). Kallikrein secretion into the distal tubular fluid appears to be regulated by blood H+ activity, and modulated by factors that affect the distal Na+ and K+ handling. The HCO3 - excretion rate was inversely correlated with the urinary kallikrein activity (r = -0.62, P < 0.001). This finding confirms previous data from the author's laboratory showing a kallikrein involvement in the regulation of HCO3- secretion in rats and rabbits. Kallikrein probably transduces the sensing of interstitial fluid H+ activity by the connecting tubule cells into an appropriate translocation of HCO3- or H+ to the tubular lumen by the intercalated cells.

Effect of metabolic alkalosis and metabolic acidosis on urinary kallikrein excretion of anaesthetized rats: evidence for a role of blood pH as regulator of renal kallikrein secretion.

The effect of altering the acid-base status on urinary kallikrein excretion of barbiturate-anaesthetized rats was investigated. Alkalosis was induced in a group of rats by intravenous (i.v.) infusion of NaOH at 0.45 mmol x h(-1) for 30 min. Acidosis was induced in two groups of rats by i.v. infusion of HCl at 1.5 mmol x h(-1) for 30 min (uncompensated acidosis) or 0.15 mmol x h(-1) for 3 h (compensated acidosis), respectively. Time controls received 0.45 mmol x h(-1) NaCl. Rats with alkalosis excreted less kallikrein than their controls (P < 0.05). Rats with uncompensated acidosis excreted more active kallikrein (P < 0.05), whereas rats with compensated acidosis excreted similar amounts when compared with their respective controls. In rats with uncompensated acid-base derangements, the urinary kallikrein excreted per millilitre of glomerular filtrate was correlated with blood H+ activity (r = 0.99, P < 0.01). Arterial blood pressure, haematocrit, glomerular filtration rate, urine flow rate and Na+ and K+ excretions of experimental and control animals did not differ. Thus, renal kallikrein secretion into the tubular fluid appears to be regulated by blood proton activity. This, along with our previous demonstration that kallikrein inhibits HCO3- secretion into the tubular lumen (Renal Physiol 17:301-306, 1994; J Physiol (Lond) 488:163-170, 1995), indicates that this enzyme is part of a feedback loop regulating acid-base balance.

Video-imaging micro-fluorometric assessment of luminal chloride/bicarbonate exchange activity in Madin-Darby canine kidney cells: influence of cell density, 4,4'-diisothiocyano-2,2'-disulfonic stilbene and acetazolamide.

To investigate whether or not MDCK cells may be used as a model for beta-intercalated cells, we studied: (1) The effect of luminal [Cl-]0 changes on pHi measured by video-imaging micro-fluorometry, (2) the influence of the inhibitor 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) on anion-exchange activity, and (3) the effect of acetazolamide on intracellular pH-indicator (c-SNAFL-2) accumulation and anion-exchange activity. At least three different modes of fluorescence accumulation were found in confluent monolayers: cells with high, low or undetectable fluorescence. Highly fluorescent cells responded to a rise of [Cl-]0 (30-140 mM) with a proportional decrease of pHi (7.6-6.4). Acetazolamide (10(-4) M) completely blocked the acidifying effects of the increased [Cl-]0, indicating that HCO3- is the intracellular ion exchanged for extra-cellular Cl-. Acetazolamide caused a reduction of SNAFL-2 fluorescence suggesting that carbonic anhydrase activity contributes to indicator accumulation. The high DIDS concentration (50 microM) required to prevent intracellular acidification suggests that the exchanger involved is identical to that present in beta-intercalated cells. All cells of non-confluent monolayers were highly fluorescent and expressed Cl-/ HCO3(-)-exchanger activity. In conclusion, highly fluorescent MDCK cells in confluent monolayers have a luminal DIDS inhibitable, carbonic anhydrase dependent Cl-/HCO3(-)-exchanger, and may therefore be used as a model for beta-intercalated cells.

Involvement of renal kallikrein in the regulation of bicarbonate excretion in rats.

1. The experiments reported here were performed to test the hypothesis that renal kallikrein is involved in the regulation of acid-base balance. 2. The bicarbonate concentration and the kallikrein activity in the spontaneously voided urine of conscious rats (experiment 1) were inversely correlated (correlation coefficient (r) = -0.63, P < 0.0001). The correlation was even greater when the urinary bicarbonate concentration was expressed per milligram excreted creatinine (r = -0.74, P < 0.00002). 3. Intravenous injection of the kallikrein inhibitor aprotinin in barbiturate-anaesthetized rats (experiment 2) reduced urinary kallikrein activity (P < 0.05) and increased bicarbonate excretion rate (P < 0.012). 4. Renal arterial infusion of aprotinin in barbiturate-anaesthetized rats (experiment 3) reduced urinary kallikrein activity (120 min, P < 0.01), and increased bicarbonate excretion rate (120 min, P < 0.01). Animals infused with the inhibitor developed a moderate metabolic acidosis (base excess: control, 2.9 +/- 0.7 mM (mean +/- S.E.M.); experimental, -8.1 +/- 0.7 mM; P < 0.05). 5. The bicarbonate concentration of urine fractions obtained after retrograde injection of kallikrein through the ureter into the collecting duct system of barbiturate-anaesthetized rats was lower than that from kidneys administered the vehicle (experiment 4; P < 0.001). A retrograde injection of bradykinin was without effect (experiment 5). 6. We conclude that renal kallikrein is involved in the regulation of urinary bicarbonate excretion. Increased intraluminal activity of the enzyme reduces, and decreased kallikrein activity increases, bicarbonate excretion. The enzyme may be a component of a negative feedback loop controlling the hydrogen ion activity of the extracellular space.

Atrial-natriuretic-peptide receptors in glomerular cryosections of renal malignant and spontaneously hypertensive rats.

ANP-receptors affinities (KD) and capacities (Bmax) were assayed in cryosections of glomeruli from 'malignant' hypertensive rats (2K-1C) and spontaneously hypertensive rats (PHR). Plasma ANP concentration was twofold higher in 2K-1C (P < 0.05) and PHR (P < 0.02) than in the respective controls, KD and Bmax for rANP99-126 and ANP103-123 did not differ. ANP mediated cGAMP release in 2K-1C rats was also unaffected. ANP-C glomerular receptors (i.e. displacement of tracer binding with ANP103-123) were not down-regulated and had unchanged peptide binding affinity in either kidney of rats with 'malignant' hypertension and in PHR. The difference between Bmax for rANP99-126 and Bmax for rANP103-123 (ANP-A receptor binding) indicates moderate up-regulation of ANP-A receptors in the clipped, and down-regulation in the contralateral kidney of 2K-1C (2K-1C, right vs. left, P < 0.05). Since [ANP]pl, and also Bmax and KD for ANP were similar in both hypertension models investigated, changes of the [ANP]pl/ANP-receptor system can not completely explain the marked natriuresis of rats with 'malignant' hypertension.

Evidence for an inhibitory effect of kallikrein on collecting duct bicarbonate secretion in rats and rabbits.

The luminal membrane of collecting duct cells, especially the intercalated cells, is normally exposed to active kallikrein. This is the consequence of the specific localization of this renal enzyme in the connecting tubule cells and its principal route of secretion being into the tubular lumen. It is conceivable that kallikrein acts downstream on a transporter involved in distal bicarbonate handling. To investigate this possibility, we estimated bicarbonate concentration and measured kallikrein amidolytic activity in urine fractions collected after a classical stop-flow experiment in rabbits. A highly significant inverse correlation was found between these parameters (r = -0.94, p < 0.001) in the peak kallikrein fractions. Neither sodium nor potassium concentration were correlated to kallikrein. This suggests that the physiological role of renal kallikrein may be to regulate extracellular fluid pH by inhibiting collecting duct bicarbonate secretion. To test the hypothesis that tubular fluid kallikrein activity and bicarbonate secretion are causally related, we developed a novel in vivo stop-flow injection model ('orthograde stop-flow'). A hog-kallikrein containing solution (0.5 microgram/ml) was injected through the abdominal aorta into the renal tubular system of one kidney of barbiturate-anesthetized rats, while the renal blood supply was interrupted. The ureter was then occluded and renal blood perfusion reinitiated. After a 2-min contact time five 125-microliters urine fractions were collected. Bicarbonate secretion was clearly detected in the second and third fractions (i.e. those coming from the collecting ducts) of the control animals, which had received only the vehicle. There was no bicarbonate secretion peak in the corresponding urine fractions collected from kallikrein-injected animals.(ABSTRACT TRUNCATED AT 250 WORDS)

[Congenital lung hypoplasia in adulthood]. / Kongenitale Lungenhypoplasie im Erwachsenenalter.

This is a report on a largely asymptomatic patient with a unilaterally non-functional lung. The differential diagnosis between an acquired, "destroyed" lung and congenital hypoplasia of the lung with secondary inflammatory changes proves difficult. The various imaging procedures that are available, used in conjunction with the histological findings, permit a classification.

Lithium treatment reduces the renal kallikrein excretion rate.

Lithium salts are widely used agents for the prophylactic treatment of affective disorders. Lithium salts may be associated with distal nephron dysfunction. Kallikrein is a protease which is generated by the distal nephron. We used an amidolytic assay of chromatographically purified enzyme to determine the urinary excretion rate of active kallikrein in relation to lithium treatment. All plasma lithium concentrations were within the therapeutic range (0.4 to 0.9 mmol/liter). In 15 patients the urinary excretion rate of active kallikrein was 267.4 +/- 65.6 mU/24 hrs before lithium treatment, and fell to 117.8 +/- 39.6 mU/24 hrs (P less than 0.05) on day 14 of lithium treatment. This reduction was associated with a decrease of immunoreactive kallikrein in the same urines by 66%. In another 15 patients who had undergone lithium therapy for an average period of 5.6 years, the urinary excretion rate of active kallikrein was 86.1 +/- 14.5 mU/24 hrs, while 21 age-matched healthy controls had an excretion rate of 364.1 +/- 58.4 mU/24 hrs (P less than 0.05). Measurements of immunoreactive kallikrein in the same urine samples demonstrated a reduction of kallikrein after long-term lithium treatment by 78%. These observations could not be attributed to changes in creatinine clearance, renal sodium or potassium excretion rates or plasma concentrations of aldosterone and vasopressin. Addition of lithium to the urine in vitro had no demonstrable effect on kallikrein measurement by amidolytic assay. We conclude that lithium in therapeutic plasma concentrations may directly suppress the secretion of kallikrein by renal connecting tubule cells.

[A supratentorial hemangioblastoma]. / Supratentorielles Hämangioblastom.

A case of supratentorial hemangioblastoma combined with two hemangiomas of the liver without manifestation of von Hippel-Lindau disease is presented. The cerebral cystic hemangioblastoma was localized in the right parietal lobe and contained a calcified area but no visible mural solid nodules. The CT and MRT differential diagnosis (arachnoidal cyst, glioma, Echinococcosis, hamartoma and metastasis) is discussed, and the literature on the subject is reviewed.

[Oleothorax 40 years later]. / Oleothorax nach 40 Jahren.

Between 1930 and 1950 the induction of an oleothorax was widely accepted as treatment of patients with progressive tuberculosis. Now, the late complications of this therapeutic approach can occasionally be observed. A patient with an oleothorax induced forty years ago and extensive x-ray exposure through frequent fluoroscopie follow-up is presented. In addition, he has a carcinoma of the lung infiltrating the chest wall. The late complications of oleothorax and the possibility of a radiation-induced malignancy are discussed.

Imaging positron emitting radionuclides generated during radiation therapy.

In vivo generated positron emitting radioisotopes, primarily C-11 and N-13, have been documented following therapy with accelerators larger than 10 MeV. Six patients had positron emission tomography 15 to 25 minutes after radiation therapy with a 42 MeV accelerator. Five patients had recurrent colorectal malignancy, and one required therapy for a carcinoma of the common bile duct. We sought to determine whether state-of-the-art PET technology could be used to monitor the three-dimensional activity distribution of radiation-induced radioactivity. At the time of the examination all six patients had sufficient concentrations of C-11 and N-13 activity in the irradiated volume to permit the evaluation of the activity distribution. We found significant activity at the body surface, which permitted field delineation. We conclude that the in vivo generated radioactivity can be monitored with PET.

[Stereotactically targeted radiotherapy of cerebral arteriovenous malformations]. / Stereotaktisch gezielte Strahlentherapie zerebraler arteriovenöser Gefässmissbildungen.

A report is given about radiotherapy in 41 patients suffering from cerebral vessel anomalies. A modified linear accelerator was used in a moving field technique with multiple pendulum planes to apply single doses between 8 and 28 Gy by means of stereotaxis into the angiographically determined target volume. The medium follow-up is 23 months. The latency of radiogenic effects is between one and two years. Radiological controls with an interval of more than 18 months after therapy are available in 17 out of 41 patients. Angiographic investigation showed complete obliterations of pathological vessels in six out of these patients and partial obliterations in six patients; five patients remained unchanged. There were no acute complications. Seven patients presented neurological deficiencies with a latency of 6 to 12 months, however, in all cases but one they regressed completely. Even taking into consideration the small number of patients and the short time of observation, a comparison with the results of other radiotherapeutical proceedings allows to draw the conclusion that the presented technique of stereotaxic convergent-beam irradiation represents a relatively simple, reliable and, in case of precise indication, efficient method for the therapy of cerebral arteriovenous malformations.

Combined neutron-photon-therapy of locally recurrent rectosigmoidal tumors.

21 patients with unresectable recurrent adenocarcinoma of the rectum were treated with combined photon-neutron radiation therapy. 40 Gy photon were given to the whole pelvis followed by a boost field of 6.6 or 10 Gy utilizing 14 MeV monoenergetic neutrons. The latter was given with an arc therapy technique whereby the dose output fluctuations normally encountered during gantry rotation were compensated for by a computer guided system. All patients had severe pain symptoms before therapy. Twelve patients had a full remission of the symptoms and nine reported considerable relief of pain during follow-up examination. In three patients, further pain symptoms developed after six, seven, and nine months due to renewed tumor progression. In spite of the relative high neutron doses applied, side effects with the arc-technique remained minimal and did not exceed those encountered with photon therapy alone. Although the total follow-up time is relatively short at a maximum of 20 months, with a mean time of 8.5 months, the preliminary results so far are extremely optimistic leading us to further pursue the study.