UNLABELLED:Rats with chronic partial unilateral ureteral obstruction (UUO) have a paradoxical resetting of the tubuloglomerular feedback (TGF) mechanism. During extracellular fluid volume expansion (VE) in control animals, a decrease in TGF sensitivity is normally noted. In rats with partial UUO, however, TGF sensitivity in the obstructed kidney was increased, associated with a relative reduction in single nephron glomerular filtration rate. In the present study, we examined the tubular and interstitial pressures, whole kidney function and the TGF system in rats with chronic partial bilateral ureteral obstruction. The rats were divided into preferentially ipsilaterally and preferentially contralaterally obstructed, as judged by pelvic volume. Measurements were performed both during hydropenia and during VE. During hydropenia TGF characteristics were the same in the 2 groups. During VE, however, TGF sensitivity was unchanged in the most obstructed kidneys (ipsilateral), while if the obstruction was preferentially contralateral, TGF-sensitivity decreased. This opposite change in TGF sensitivity resulted in higher electrolyte and water excretion from the least obstructed kidney. The change in TGF sensitivity was not correlated to renal interstitial pressure. IN CONCLUSION:rats with moderate chronic partial bilateral ureteral obstruction have an almost preserved function with regard to renal hemodynamics and excretion during hydropenia. During volume expansion, however, different responses were found in the least and most obstructed kidneys, with a decreased sensitivity of TGF in the least obstructed kidney, while TGF sensitivity was unchanged in the most obstructed kidney.
The in vitro effect of recombinant human granulocyte-macrophage colony stimulating factor (rh-GMCSF) and recombinant human granulocyte colony stimulating factor (rh-GCSF) on oxygen free radical (OFR) generation by human neutrophils and blood monocytes derived human macrophages stimulated with phorbol myristate acetate was investigated and compared. The production of OFR by neutrophils and macrophages was time dependent, and the maximum release of OFR by neutrophils and macrophages was measured 90 and 180 min after stimulation with phorbol myristate acetate, respectively. The priming effects or rh-GMCSF and rh-GCSF on OFR production by human neutrophils and macrophages was dose dependent. The maximum generation of OFR by neutrophils occurred when primed with 1,000 U/ml of rh-GMCSF and reached 2.383 +/- 0.191 nmol/10(5) neutrophils/90 min as compared with 1.072 +/- 0.113 nmol/10(5) neutrophils/90 min in the unprimed controls. This represents a 122.20% increase in OFR generation (p < 0.001). However, the percentage of maximum increase in OFR production was 57.84 when neutrophils were primed with a concentration of 5,000 U of rh-GCSF/ml. In 72-hour-old human macrophages, much higher levels of OFR production as compared with neutrophils were measured following stimulation with phorbol myristate acetate. The maximum generation of OFR was measured in macrophages primed for 45 min with 500 U/ml of rh-GMCSF. These cells produced 8.960 +/- 2.075 nmol/5 x 10(4) macrophage/180 min as compared with 4.563 +/- 1.773 nmol/5 x 10(4) unprimed macrophages/180 min (p < 0.001). In macrophages primed with rh-GCSF, however, the maximum OFR production was induced by a dose of 5,000 U/ml and reached 6.902 +/- 1.463 nmol/5 x 10(4) macrophages/180 min as compared with 4.563 +/- 1.773 nmol/5 x 10(4) macrophages/180 min in the unprimed controls (p < 0.05). In conclusion, the priming effect of rh-GMCSF on OFR generation by human macrophages and neutrophils was more potent than that of rh-GCSF, both in the extent of augmentation and in the dose required to produce maximum OFR generation.
We have previously shown that smooth muscle contains three types of myosin heavy chains: SM1, SM2, and SMemb. The present study was designed to assess how glomerular expression of mRNA for these isoforms is regulated and whether their expression is affected by enalapril treatment in diabetic rats. Animals were divided into 4 groups: (1) untreated diabetic rats; (2) enalapril-treated diabetic rats; (3) untreated control rats, and (4) enalapril-treated control rats. Enalapril treatment was continued for 24 weeks. The glomerular mRNA levels for SM1 and SM2 showed little change in all groups throughout the experimental period. In contrast, SMemb mRNA in group 1 increased significantly with age compared to levels found in untreated controls (4.6-fold higher at 4 weeks, p < 0.01; 6.8-fold higher at 12 weeks, p < 0.01, and 10.6-fold higher at 24 weeks, p < 0.001). Enalapril reduced both creatinine clearance (p < 0.01) and urinary protein excretion (p < 0.01) in diabetic rats. Moreover, enalapril significantly attenuated the increase in the glomerular SMemb mRNA level in diabetic rats (the difference between treated and untreated rats was significant at p < 0.01 from week 4 to 24). However, enalapril had no effect on SMemb mRNA levels in controls. These data suggest that SMemb is a molecular marker for phenotypic alteration and that the beneficial effect of enalapril on proteinuria and renal function may be, at least in part, associated with reducing SMemb mRNA expression in diabetic glomeruli.
Acute and chronic proteinuria were studied in rats, using lysosomal cathepsin B and L as marker enzymes for tubular protein degradation. The activity of cathepsin B and L has been determined in microdissected segments S1, S2 and S3 of the proximal tubule by an ultramicroassay. Z-Phenylalanyl-arginine-7-amido-4-methylcoumarin served as a substrate. In normoproteinuric Sprague-Dawley rats, induction of acute unselective glomerular proteinuria with Adriamycin (5 mg/kg body weight) revealed a moderate activity increase of cathepsin B and L in the S2 segment, reaching 12.6 +/- 5.6 versus 8.6 +/- 4.2 pmol.mm-1.min-1 in controls. In contrast, Munich Wistar Frömter (MWF) rats, that are characterized by a genetically determined, chronically elevated glomerular protein excretion, showed a very high activity of cathepsin selectively in S2 of 25.0 +/- 12.1 pmol.mm-1.min-1. Acute proteinuria induced by Adriamycin in chronic proteinuric MWF rats could increase cathepsin activity in the S3 segment only, showing 12.0 +/- 8.3 versus 6.8 +/- 4.0 pmol.mm-1.min-1 in MWF control rats. In conclusion, chronically increased protein filtration changes the functional reserve capacity of the proximal tubule. While acutely induced glomerular proteinuria in normoproteinuric rats stimulates lysosomal proteolytic activity mainly in S2 segment, chronic proteinuric MWF rats may display already a maximally stimulated cathepsin activity in this segment probably due to long-term increased tubular protein load. In case of acute elevation of chronic proteinuria, the consecutive S3 segment shows increased lysosomal function for protein conservation.
It was shown that atrial natriuretic factor (ANF)-induced increase in glomerular filtration rate (GFR) is blunted in low sodium diet. We investigated whether saralasin, as an angiotensin II receptor antagonist, or verapamil, as a calcium entry blocker, or theophylline and zaprinast, as inhibitors of cGMP-phosphodiesterase activity, could restore the effect of ANF on GFR increase in low sodium diet rats. ANF alone (5 micrograms/kg bolus then 0.5 micrograms/min/kg BW maintenance) increased diuresis and natriuresis to the same extend in low and normal sodium diet rats but had no GFR-increasing effect in low sodium diet rats. Infusion of ANF in the presence of verapamil, saralasin, theophylline or zaprinast induced a significant increase in GFR and filtration fraction (FF). Administration of verapamil or saralasin alone did not alter GFR and FF whereas theophylline or zaprinast alone resulted in moderate but significant increases in both parameters. The increase of nephrogenous cGMP excretion in response to ANF infusion in low sodium diet rats was markedly lower as compared to normal sodium diet rats (243.4 +/- 43.3 vs. 444.0 +/- 35.6 pmol/min, respectively, p < 0.01). Administration of a selective inhibitor of cGMP-phosphodiesterase activity (zaprinast) abolished the differences in ANF-stimulated nephrogenous cGMP excretion in low and normal sodium diet rats. Basal and peak ANF (0.5 microM)-stimulated cGMP formation by isolated glomeruli was significantly lower in low than normal sodium diet rats (1.4 +/- 0.1 vs. 2.9 +/- 0.2 and 7.1 +/- 0.5 vs. 12.5 +/- 1.1 pmol/mg protein, for basal and ANF-stimulated cGMP formation, respectively; both p < 0.05). Zaprinast both alone and in combination with ANF, potentiated cGMP formation by glomeruli isolated from both groups of rats. In the presence of zaprinast, there were no differences in both basal and peak ANF-stimulated cGMP formation by glomeruli isolated from low and normal sodium diet rats. cGMP-phosphodiesterase activity was the same in the medulla of both groups of rats but markedly higher in the renal cortex of low sodium diet rats as compared to normal sodium diet rats (82.6 +/- 6.0 vs. 59.8 +/- 4.3, respectively; p < 0.05). These data suggest that the lack of GFR-increasing response to ANF in low sodium diet rats is primarily due to the increase of cGMP hydrolysis in glomeruli.
Renal clearance, microperfusion and patch-clamp techniques were used to investigate the effects of the K-channel blocker glyburide on electrolyte excretion, the transport properties of the thick ascending limb (TAL) of Henle and K-channel activity in the apical membrane of the TAL and of the cortical collecting tubule. Our data suggest that the K-channel blocker glyburide can inhibit transport of Na and K in the TAL by blocking K recycling across the apical membrane. Additionally, inhibition of K secretion in the collecting ducts occurs by decreasing the activity of apical K channels and prevents kaliuresis.
The role of nitric oxide (NO) synthesis in the peripheral vasodilation and sodium retention that occurs after partial portal vein ligation (PVL) was investigated. Hemodynamic studies in PVL rats with sodium retention and in sham-operated controls were conducted on the day when PVL rats developed transient and maximal sodium retention. Measurements were obtained before and during two consecutive periods after NO synthesis inhibition with NG-monomethyl-L-arginine (L-NMMA). Under baseline conditions, PVL rats with sodium retention were hypotensive, with equivalent decreases in total peripheral resistance and glomerular filtration rate in comparison to the control group. After L-NMMA, peripheral resistance and arterial pressure increased by similar extent in both groups. As compared with controls, PVL rats with sodium retention remained hypotensive and vasodilated. Furthermore, L-NMMA-induced natriuresis was attenuated in the PVL group. Additionally, serum and urinary levels of nitrate and nitrite did not vary before surgery and at the time of sodium retention. These results suggest that in PVL rats (1) vasodilation is not NO mediated; (2) vasodilation is not a sufficient explanation for sodium retention, and (3) a sodium-retaining factor acting on the renal tubules is responsible for sodium retention.
Since it became evident that organ dysfunctions after acute hemolysis are not induced by hemoglobin per se, but by stroma-contaminated hemoglobin, solutions of ultrapure stroma-free hemoglobins were regarded to be possible substitutes for blood in transfusion medicine. We tested one of the recently developed modified bovine hemoglobins (Ultrapure polymerized bovine hemoglobin 1; UPPBHb1) in the isolated perfused rat kidney (IPRK) model, using a recirculating system. Control kidneys were perfused with a substrate-enriched Ringer solution containing hydroxyethyl starch (HES) to produce isoncotic conditions. In the experimental group HES was substituted in part by UPPBHb1 (34 g/l). For determination of functional parameters, the kidneys were perfused for 180 min. A separate set of kidneys of both groups was perfusion fixed after 80 min of perfusion which is the period of optimal function. Light and electron microscopic analysis revealed major alterations only for the outer medulla of HES kidneys. Only these suffered from a considerable extent of proximal tubular S3 damage, exhibiting condensed tubular epithelia. In the inner stripe of the outer medulla, which is the zone of greatest sensitivity to damage in the isolated perfused kidney, severe hydropic degeneration, cell detachment, and necrotic destruction of the medullary thick ascending limb were seen in the HES-perfused group, too. In the UPPBHb1 group, the medullary thick ascending limb was well preserved, and S3 showed only a minor degree of damage. UPPBHB1 kidneys were further characterized by the occurrence of intracapillary and interstitial precipitates of UPPBHb1 in inner stripe of the outer medulla and inner medulla. The glomerular filtration rate was significantly higher in UPPBHb1-perfused kidneys (870 +/- 80 vs. 630 +/- 55 microliters/min/g kidney weight for HES). Absolute reabsorption of sodium paralleled the behavior of the glomerular filtration rate. The values for renal perfusate flow and urinary flow rate did not differ significantly between both groups. Renal autoregulation was better preserved in UPPBHb1-perfused kidneys (74 +/- 6% of full autoregulatory response) than in HES-perfused controls (42 +/- 4%). Our results suggest that perfusion of isolated rat kidneys with UPPBHb1 improves kidney function and morphology, providing better oxygenation than in control kidneys. UPPBHb1 does not exert additional nephrotoxic effects on the IPRK that will exceed the noxious potential of the method itself. Thus, it must be concluded that UPPBHb1 may be an oxyphoretic blood substitute with nephroprotective characteristics when compared with nonoxyphoretic substitutes. At least, UPPBHb1 seems to be a promising candidate as oxyphoretic additive to perfusates for the IPRK model.
Ifosfamide (IF) is an alkylating cytostatic with urotoxic and tubulotoxic side effects which may result in the development of Fanconi syndrome in children. While the urotoxicity of IF is effectively prevented by the uroprotective thiol compound sodium-2-mercaptoethanesulfonate (Mesna), tubulo-toxicity of IF may occur even in the presence of Mesna and in the absence of any signs of urotoxicity. Using the renal tubular cell line LLC-PK1, we investigated whether there is a protective effect of Mesna or of its major dimeric metabolite Dimesna against metabolites of IF with respect to the Na/H exchanger activity. We tested the major IF metabolites 4-hydroperoxy-IF (4-OOH-IF), chloroacetaldehyde (CAA), and acrolein. All metabolites significantly inhibit the Na/H exchanger activity. Half-maximal inhibition of transport occurs at concentrations of 120 mumol/l (4-OOH-IF), 80 (CAA), and 60 mumol/l (acrolein) after 2 h of incubation. The onset of the inhibitory effect of all three metabolites is rapid. Complete inhibition of Na/H exchange by acrolein and CAA is present after a 6-hour exposure to 100 mumol/l of the respective metabolite, while 100 mumol/l 4-OOH-IF causes only 50% inhibition after 24 h of incubation. Dimesna, which the proximal tubular cell has to reduce to Mesna at the expense of intracellular glutathione before it exerts a uroprotective effect, has no protective effect in LLC-PK1 cells. Dimesna (0.3 mmol/l) displaces the dose-response curve for acrolein to the left, indicating an increased toxicity of the combination of acrolein plus Dimesna. Mesna (0.3 mmol/l) has a complete protective effect with respect to acrolein and CAA, while the protective effect versus 100 mumol/l of 4-OOH-IF is incomplete. We conclude that the function of the Na/H exchanger in LLC-PK1 cells is altered by metabolites of IF. The incomplete protection against the toxic effect of 4-OOH-IF by Mesna may explain the pathomechanism by which IF causes tubulotoxicity in the absence of urotoxicity.
The effects of acute ipsilateral denervation (AID) and contralateral ureteral obstruction on tubuloglomerular feedback (TGF) was studied by micropuncture in rats. Denervation alone was associated with an increase in the TGF turning point, the tubular flow rate required for a half-maximal TGF response, but other TGF characteristics were not affected. Contralateral ureteral obstruction alone was also associated with an increased turning point, but contralateral obstruction had no effect on turning point, when AID was done prior to the obstruction. We conclude that renal nerves have a tonic modulating effect on TGF in anesthetized rats, and that renorenal reflexes are involved in the TGF resetting associated with acute contralateral ureteral obstruction.
To examine a physiological role of 11 beta-hydroxysteroid dehydrogenase (11OHSD) in the aldosterone target tissue, we measured Na(+)-K(+)-ATPase activity in the cortical collecting ducts (CCD) from adrenalectomized rats, which were treated with a physiological dose of corticosterone and/or carbenoxolone (an inhibitor of 11OHSD). The Na(+)-K(+)-ATPase activity in adrenalectomized rats was not significantly changed by either corticosterone alone or carbenoxolone alone, whereas its activity showed a significant increase only in the rats that received both corticosterone and carbenoxolone. In these rats, the plasma concentration of corticosterone was within the physiological range (10(-6) M) and the plasma carbenoxolone concentration was about 10(-7) M. Furthermore, the direct effect of carbenoxolone was examined in the microdissected CCD because it has been reported that carbenoxolone per se had an affinity for the aldosterone receptor. Na+K(+)-ATPase activity in the microdissected CCD was increased in a dose-dependent manner after a 3-hour incubation with carbenoxolone, and this effect was completely inhibited by canrenoic acid (an aldosterone antagonist). However, the minimal carbenoxolone concentration exerting a stimulatory effect was 10(-6) M, which is about 10 times higher than the plasma concentration of carbenoxolone in the in vivo treated rats. These results indicate that an inhibition of 11OHSD but not a direct action of carbenoxolone induces an increase in Na(+)-K(+)-ATPase activity, which is a well-known aldosterone effect in the CCD.
In previous investigations, it was found that rats depleted in parathyroid hormone (TPTX rats) had reduced rates of proximal bicarbonate reabsorption independent on blood calcium levels. In the present work, the role of calcium (Ca2+) in rat proximal tubule bicarbonate reabsorption was studied by in vivo stationary microperfusion. Tubules were perfused at different lumen Ca2+ concentrations in the presence and absence of the calcium ionophore A23187. Bicarbonate reabsorption was not affected by Ca2+ in the range of 0 to 1 mM, but was significantly reduced when 0.5 mM EGTA was added to the 0 Ca2+ perfusates, indicating that only at very low luminal Ca2+ levels, bicarbonate reabsorption ( = H+ secretion) was impaired. These observations indicate that Ca2+ in the tubule lumen is important for the maintenance of normal proximal bicarbonate transport, but the low Ca2+ level necessary to impair this transport mechanism is achieved only in the presence of EGTA, a condition that simulates the absence of parathyroid hormone.
The DBA/2FG-pcy mouse has a form of slowly progressive kidney disease that appears similar in many respects to that seen in the autosomal dominant form of human polycystic kidney disease. This study was designed to assess how the expression of extracellular matrix component genes is regulated in a model of murine polycystic kidney disease and control DBA/2 mice at 8, 16, and 30 weeks of age. The mRNA levels encoding for collagen IV, the B1 and B2 chains of laminin, heparan sulfate proteoglycan, fibronectin, and collagens I and III increased with the progression of cystic lesions in the kidney of DBA/2FG-pcy mice. At 30 weeks of age, mRNA levels for collagen IV, laminin B1 and B2, heparan sulfate proteoglycan, fibronectin, and collagens I and III were increased 8.1-fold, 7.0-fold, 7.0-fold, 9.8-fold, 7.0-fold, 5.5-fold, and 5.4-fold, respectively, compared to those of control DBA/2 mice. An immunofluorescence study revealed the irregular staining for collagen IV, laminin, heparan sulfate proteoglycan, and collagens I and III around the cysts. These data suggest that changes in the expression of basement membrane components and interstitial collagens are associated with the development of polycystic kidney disease.
The diurnal time course of urinary flow rate (UV), urinary sodium (UNaV), and potassium (UKV) excretion, and of hormones such as atrial natriuretic peptide (ANP) and aldosterone, was investigated during 5 days of continuous captopril infusion (15 micrograms.kg body weight-1.min-1) in 4 conscious dogs on a high sodium diet (14.5 mmol Na.kg body weight-1.24 h-1). All food and water was given once daily at 8.30 a.m. On the control day and on days 1, 3, and 5 of captopril infusion, urine was collected by an automated system at 20-min intervals over 24 h, blood was taken every 4 h. Mean arterial blood pressure (MABP) and heart rate were evaluated as 5-min averages. Time courses of UNaV, UV, and UKV were compared with the individual control day without captopril. With captopril, 24-hour balances for Na and H2O were slightly negative, while the K balance was slightly positive for 2-3 days. Thereafter, all 24-hour balances were restored. MABP continued to decrease even after Na and water intake and output had come into balance again. Captopril treatment changed the diurnal excretion pattern for UNaV and UV characteristically. In the postprandial period until 5 p.m., less Na and urine were excreted than on the control day, whereas during the evening and night more Na and urine were excreted. The changes in the excretion pattern persisted for the entire observation period. The results indicate that disturbances in the regulating systems, induced by converting-enzyme blockade, bring about complex reactions of, e.g., MABP, ANP and aldosterone that finally restore Na and water 24-hour input/output balances.
We have purified a 17-kD puromycin aminonucleoside (PAN) binding protein from porcine kidney and identified it as the reported 17-kD protein kinase C inhibitor on the basis of its partial amino acid sequence. Of 54 determined amino acid sequences of the 17-kD porcine renal protein, 51 residues were identical to those of the 17-kD bovine brain protein kinase C inhibitor. However, our purified protein did not carry the inhibitory activity on protein kinase C. Immunohistochemical studies showed a unique intrarenal distribution of the 17-kD PAN-binding protein at the apical side of epithelial cells of Henle’s loops in the inner medulla and of distal convoluted tubules. Immunoblot analysis revealed that the 17-kD PAN-binding protein was extractable by an isotonic buffer without sodium deoxycholate extraction. These results suggest that this protein binds loosely to the apical membranes of epithelial cells of Henle’s loops and distal tubules and has specific functions related to tubular functions of these nephron segments at the apical side. Whether this protein is a real inhibitor of protein kinase C or not remains to be investigated.
The excimer-to-monomer (E/M) fluorescence ratio of pyrene (p) and its derivatives has been found to correlate with membrane fluidity. We explored the possible use of this method in living renal epithelial cells. In Modin-Darby canine kidney cells, successful fluorescent labeling was obtained with p-cholesterol, p-trimethylammonium (TMA), p-sulfonamidethyl-TMA, p-buty-rate, p-propanoate or p-dodecanoate. Among them, the labeling with p-cholesterol was most stable. The whole cell cholesterol content did not significantly increase after loading p-cholesterol and the probe incorporated was not degraded. The E/M ratio changed reproducibly and significantly by temperature change, a standard method to alter membrane fluidity. Thus, the E/M ratio of p-cholesterol fluorescence is suggested to be a potential parameter of membrane fluidity in living renal epithelial cells and might be useful in investigating the relationship between the physical state of the membrane and epithelial function.
Previous studies have demonstrated that it is possible to prevent postexercise proteinuria with angiotensin-converting enzyme inhibitors. To determine whether calcium antagonists have the same effect, 40 young healthy volunteers underwent maximal aerobic exercise with and without nifedipine 10 mg per os 1 h before the first or second trial. Urinary excretion of albumin (UAE), transferrin (UTE) and alpha 1-microglobulin (UME) were examined before and after each trial. UAE, UTE and UME were significantly increased after exercise. Nifedipine significantly decreased UAE (p = 0.001) and UTE (p = 0.02) after exercise, and slightly decreased the maximal work load and the basal excretion of albumin. UME was unchanged. Therefore, the results of this study demonstrate that nifedipine administration before exercise significantly reduces postexercise proteinuria.
