Renal tubular dysfunction in patients with cystic disease of the kidneys.

OBJECTIVE: To define the renal tubular functional abnormalities in patients with cystic disease of the kidneys. METHODS: Patients with autosomal dominant polycystic kidney disease (ADPKD) (n = 4) and medullary sponge kidneys (MSK) (n = 3) with normal glomerular filtration rate (GFR), determined by inulin clearance, and effective renal plasma flow (ERPF), measured by p-aminohippurate clearance, underwent measurement of proximal and distal tubular functions. Proximal tubular functions were determined by the maximum reabsorption of glucose (TmGlucose) and the maximum secretion of p-aminohippurate (TmPAH). Distal tubular functions were measured by the maximum urinary concentrating and diluting mechanisms, and the urinary acidification response to acid load. RESULTS: TmGlucose was low in both groups (209 +/- 25 mg/min/1.73 m2 in the ADPKD group and 110 +/- 28 mg/min/1.73 m2 in the MSK, compared with 375 +/- 40 mg/min/1.73 m2 in healthy controls; P < 0.05). Likewise, TmPAH was significantly diminished in patients with ADPKD (72 +/- 6 mg/min/1.73 m2) and MSK (63 +/- 5 mg/min/1.73 m2) when compared with healthy controls (89 +/- 4 mg/min/1.73 m2; P < 0.05). Urinary maximum concentration after fluid deprivation was impaired in both ADPKD and MSK patients, but the diluting mechanism was intact. Finally, the ability to excrete urinary ammonium and titratable acids following an oral acid load was inadequate in both the ADPKD and MSK groups. CONCLUSIONS: Proximal and distal tubular functions are impaired in patients with ADPKD and MSK when GFR and ERPF are normal, indicating tubular disruption by the cysts and the alteration of the tubulo-interstitial vascular relationship.

Exploration of the importance of the P2-P3-NHCO-moiety in a potent di- or tripeptide inhibitor of calpain I: insights into the development of nonpeptidic inhibitors of calpain I.

Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of cerebral ischemia. In this paper, we report on a series of peptidomimetic ketomethylene and carbamethylene inhibitors of recombinant human calpain I (rh calpain I). Our study reveals that the -NHCO-moiety (possible hydrogen-bonding site) at the P2-P3 region of a potent tripeptide or a dipeptide inhibitor of calpain I is not a strict requirement for enzyme recognition. Compounds 7d ((R)-2-isobutyl-4-oxo-4-(9-xanthenyl)butanoic acid ((S)-1-formyl-3-methyl)butyl amide), 31 ((R)-2-isobutyl-4-(2-sulfonylnaphthyl)butyric acid ((S)1-formyl-3-methyl)butyl amide) and 34 ((R)-2-isobutyl-4-(2-sulfoxylnaphthyl)butyric acid ((S)-1-formyl-3-methyl)butyl amide) which exhibited good activity in the enzyme assay, also inhibited calpain I in a human cell line.

Synthesis and biological activity of a series of potent fluoromethyl ketone inhibitors of recombinant human calpain I.

Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of stroke. In this paper, we report on a series of potent dipeptide fluoromethyl ketone inhibitors of recombinant human calpain I (rh calpain I). SAR studies revealed that while calpain I tolerates a variety of hydrophobic groups at the P1 site, Leu at P2 is preferred. However, the nature of the N-terminal capping group has a significant effect on the inhibitory activity of this series of compounds. Compound 4e [(1,2,3,4-tetrahydroisoquinolin-2-yl)carbonyl-Leu-D,L-Phe-CH2F+ ++], having a tetrahydroisoquinoline containing urea as the N-terminal capping group, is the most potent dipeptide fluoromethyl ketone inhibitor of calpain I (with a second-order rate constant for inactivation of 276,000 M-1 s-1) yet reported; tripeptide 4k (Cbz-Leu-Leu-D,L-Phe-CH2F) is equipotent. A number of compounds presented in this study displayed excellent selectivity for calpain I over cathepsins B and L, two related cysteine proteases. Compounds which exhibited good inhibitory activity in the assay against isolated rh calpain I also inhibited intracellular calpain I in a human cell line. Thus, in an intact cell assay, compounds 4e and 4k inhibited calpain I with IC50 values of 0.2 and 0.1 microM, respectively. Finally, we also disclose the first example of fluorination of a dipeptide enol silyl ether to generate the corresponding dipeptide fluoromethyl ketone.

Mechanism of glomerular hyperfiltration after a protein meal in humans. Role of hormones and amino acids.

OBJECTIVES: Previous studies demonstrated that protein meals and amino acid (AA) infusions increase glomerular filtration rate (GFR) and renal plasma flow (RPF) and that somatostatin (SRIH) infusion inhibits these increments. We tested whether a single AA such as alanine could increase GFR and RPF and whether the changes in GFR and RPF could be explained on the basis of changes in glucagon, growth hormone (GH), and insulin. RESEARCH DESIGN AND METHODS: In the first experiment, alanine was infused with or without SRIH in five normal subjects. In the second experiment, five other subjects were infused with SRIH on three separate occasions. In a control study, insulin, glucagon, and GH were given at replacement doses; in a hyperglucagonemia study, glucagon was given at a rate of 0.2 microgram.kg-1.h-1 (hypoglucagonemia); and in a high GH study, GH was given at a rate of 2 micrograms.kg-1.h-1. GFR and RPF were measured using insulin and para-aminohippurate, respectively. RESULTS: Alanine increased GFR and RPF, whereas SRIH inhibited these changes (P < 0.05). Hyperglucagonemia or high GH with or without insulin failed to increase RPF or GFR. CONCLUSIONS: A single AA such as alanine increases GFR and RPF, and this increase is dependent on a factor inhibited by SRIH. Although GH, glucagon, and insulin are factors inhibited by SRIH, none of these factors explains the changes in RPF and GFR in our acute studies.

Effect of partial urinary outlet obstruction in the rabbit on the incorporation of adenine into adenine nucleotides in bladder smooth muscle.

Bladder outlet obstruction induces marked morphological, functional, and metabolic changes within the urinary bladder. Recent studies indicate that there is a close correlation between the contractile dysfunction induced by partial outlet obstruction and a marked decrease in mitochondrial oxidative activity of the hypertrophied bladder tissue. The current study investigates the effect of partial outlet obstruction on adenine metabolism within the bladder tissue. After transport into the cell, adenine becomes available as a substrate for adenine phosphoribosyl transferase (APRT), the enzyme that catalyses the non-mitochondrial conversion of adenine into AMP. Subsequently, AMP is phosphorylated to ADP, the phosphate acceptor in mitochondrial oxidative phosphorylation. The results of these studies demonstrate that partial outlet obstruction induces a significant increase in 14C-adenine uptake into the urinary bladder smooth muscle which in turn provides substrate for APRT and results in an increase in 14C-AMP synthesis. In contrast, the rate of incorporation of adenine into ATP+ADP was similar for both control and obstructed tissue. The activity of APRT was not significantly different in control and obstructed tissue.

Effect of outlet obstruction on 3H-thymidine uptake: a biochemical and radioautographic study.

Experimental outlet obstruction in the rabbit is characterized by a rapid and substantial increase in urinary bladder mass. Although it is clear that both the smooth muscle and connective tissue compartments are increasing in mass, there is little information on the mechanisms by which this increase in mass occurs. As an initial investigation in this process, urinary bladders from normal and obstructed NZW rabbits were exposed in vitro to tritiated thymidine (3H-TdR) in order to determine which populations of cells are induced to synthesize DNA following outlet obstruction, and when, after obstruction, such synthesis occurs. Biochemical analysis of nucleic acids was performed on each specimen to determine total and radioactive DNA. These analyses showed a marked increase in DNA synthesis at 24 hours following obstruction which remained relatively high through seven days after obstruction. There was a decline in labelling at 14 days. Incorporation of radioactive label peaked at three days and declined to control levels by 14 days. Samples of tissue were taken from each subject and processed for radioautography. At 24 hours after obstruction, significant numbers of cells of the basal cell layer of the urothelium are observed to be actively involved in DNA synthesis, while the other two tissue compartments (muscularis and connective tissue) show no significant changes when compared to normal specimens. Connective tissue, on the other hand, showed significantly increased levels of labelling above control level from three to 14 days after obstruction. Smooth muscle cells were observed to be frequently labelled in only one of the experimental bladders observed three days after obstruction.

Indigocarmine as a quantitative indicator of urothelial integrity.

There is increasing evidence that the urothelium of the bladder mucosa prevents the penetration of solutes from the urine into the bladder wall. In the current study, in vivo treatments of rabbit urinary bladder with DMSO, acetone and overdistension resulted in damage to the physical integrity of the bladder mucosa as quantitated by the penetration of the dye indigocarmine (1% in saline) into submucosal tissues. Penetration of the dye can be quantitated, because the dye can be extracted from the tissue and measured spectrophotometrically. Indigocarmine does not penetrate normal, control, bladder mucosae. Bladders treated with gentle 20, 30 and 50% acetone washes for one minute permit dye penetration which is proportional to the acetone concentration utilized. Intravesical 50% DMSO ("RIMSO 50") administration permits modest dye penetration. Distension by slow filling with saline to volumes 90% of capacity and greater causes a marked increase in dye penetration which is proportional to the magnitude of overdistension. Although pretreatment of the bladder with heparin did not reduce the dye penetration following acetone administration, it completely abolished penetration of the dye following overdistension. Indigocarmine is potentially useful as both a quantitative and qualitative indicator of bladder mucosal integrity.

Renal handling and physiologic effects of the paramagnetic contrast medium Gd-DOTA.

Gadolinium DOTA (Gd-DOTA) is a magnetic resonance (MR) contrast agent similar to Gd-DTPA but with greater stability in vitro. The effects of a high intravenous dose (0.5 mmol/kg) of Gd-DOTA (1360 mOsm/kg) on renal excretory function and its general systemic effects are examined in this animal study. This dose was selected to accentuate and better define the qualitative nature of these effects. A decrease in arterial pressure of 8% (131.9 +/- 6.8 at 120 minutes versus a control of 142.8 +/- 3.7 mm Hg, mean +/- standard error of mean, no significant change in electrocardiogram (ECG) lead II, a 16% increase in renal blood flow (106.0 +/- 5.4 at 7.5 minutes versus 91.2 +/- 3.2 ml/min), and a decrease in arterial hematocrit of 9% (38.9 +/- 1.5 at 120 minutes versus 41.9% +/- 1.7%) were noted. In general, qualitatively similar effects have been noted as a nonspecific effect of other hyperosmolar solutions. The filtration fraction decreased (0.23 +/- 0.01 at 7.5 minutes versus 0.28 +/- 0.02) followed by a rapid return to baseline values. No significant change was noted in glomerular filtration rate throughout the experimental protocol. Urine flow increased nearly 1.5-fold and osmolal clearance (Cosm) increased approximately 1.5 times. A natriuresis occurred as the fractional excretion of sodium (FENa+) increased from a control value of 3.5 +/- 0.3 to 5.2 +/- 0.5 at 7.5 minutes. The systemic and renal physiologic effects of high-dose intravenous Gd-DOTA on the kidney reflects a nonspecific, osmotically induced alteration. These data suggest that the main systemic and renal physiologic actions of Gd-DOTA are a nonspecific response to agent osmolality that is similar qualitatively to conventional, water-soluble contrast media.

Potassium homeostasis during angiotensin-converting enzyme inhibition with enalapril.

The effect of angiotensin-converting enzyme (ACE) inhibition on renal and extrarenal potassium (K) regulation was examined. Six healthy men were studied in double-blinded crossover fashion on placebo or enalapril, 80 mg/day. On day 4, the subjects were given an intravenous infusion of KCl and on day 5 an oral dose of 10% NH4Cl. Treatment with enalapril decreased plasma aldosterone and increased plasma renin activity (PRA), epinephrine and norepinephrine, but did not affect serum glucose, plasma insulin or basal plasma K. Maximal increases in plasma K during K infusion or NH4Cl ingestion were similar during enalapril and placebo treatment. With enalapril treatment urinary K excretion was unchanged following K loading but moderately reduced following NH4Cl loading. We conclude that ACE inhibition does not acutely impair K homeostasis in men with normal renal function.

Renal tubular dysfunctions in patients with idiopathic calcium nephrolithiasis.

To define the degree of renal tubular involvement in idiopathic calcium nephrolithiasis, 18 patients (aged 23-60 years, 15 men and 3 women, with 1-30 years of renal stone history) with normal glomerular filtration rate (GFR) and effective renal plasma flow with no history of urinary tract infection and on no dietary or drug therapy underwent the following studies: measurement of proximal tubular maximum reabsorption of glucose (Tmglucose) and secretion of para-aminohippurate (TmPAH), urinary concentrating ability after 14 h of fluid deprivation, and urinary net acid excretion following an oral dose of ammonium chloride, 0.1 g/kg of body weight. Seventeen healthy subjects in the same age range served as control. Patients with calcium nephrolithiasis, with normal renal hemodynamic functions, have significantly lower proximal tubular maximum reabsorptive and secretory functions, diminished urinary concentrating mechanism, and reduced urinary net acid excretion following an oral acid load. These tubular functional abnormalities were observed in patients with or without hypercalciuria.

A comparison of clearance and arteriovenous extraction techniques for measurements of renal hemodynamic functions.

No previous studies have directly compared timed urine collections (UV/P) vs. arteriovenous (A-V) extraction methods for determination of renal function in whole kidney preparations. We examined different markers and techniques for assessing renal plasma flow (RPF), filtration fraction (FF), and glomerular filtration rate (GFR) in both steady-state and rapidly changing conditions following 2 ml/kg bolus intravenous injections of either Renografin 76% (meglumine/sodium diatrizoate-76%) or hypertonic mannitol 25%. During steady-state conditions, excellent correlations were obtained when comparing markers and techniques. Thus, timed urinary clearances of inulin vs. 99m-technetium DTPA (Tc) had a correlation coefficient (R) of .96 (P less than .01; n = 16), and the A-V extraction technique of inulin vs. Tc as determinants of GFR showed a correlation of R = .98 (P less than .01; n = 15). The timed urinary clearance of inulin vs. the A-V extraction of inulin for glomerular filtration gave a correlation of R = .93 (P less than .01; n = 15). The clearance of para-aminohippurate (PAH) divided by the extraction of PAH vs. flow determinations using the electromagnetic flowmeter gave a correlation of R = .92 (P less than .01; n = 16). The anticipated decrease in GFR following contrast medium and hypertonic mannitol was observed using the A-V extraction technique, whereas an artifactual, exaggerated increase in GFR was observed using the timed urine collection technique. Similarly, we noted an exaggerated increase in RPF using CPAH/EPAH as the methodology. We conclude that rapid changes in renal hemodynamics may be measured accurately using the A-V extraction technique but not with clearance techniques requiring timed urine collections.

Acute systemic and renal hemodynamic effects of meglumine/sodium diatrizoate 76% and iopamidol in euvolemic and dehydrated dogs.

We examined the acute systemic and renal hemodynamic effects of intravenous meglumine/sodium diatrizoate-76% and iopamidol in euvolemic and dehydrated dogs. The physiologic responses were compared with acute changes in the level of an endogenous heparin-like material (EHM). One of eight dehydrated dogs receiving diatrizoate (2 ml/kg) had an immediate vomiting reflex associated with a very significant decline in all measured renal hemodynamic parameters; none of eight dehydrated dogs receiving iopamidol experienced a similar reaction. EHM levels did not correspond to the magnitude of the physiologic responses following either iopamidol or diatrizoate. Significant differences between iopamidol and diatrizoate were noted when comparing the magnitude of the decrease in systemic pressure (- delta 3.8 +/- 3.02, iopamidol, n = 8; vs. - delta 19.4 +/- 7.3 mm Hg, diatrizoate, n = 8; P less than .03), increased renal plasma flow (+ delta 6.2 +/- 4.9, iopamidol, n = 8; vs. + delta 33.7 +/- 8.0 ml/min, diatrizoate, n = 8; P less than .05), and decreased filtration fraction (- delta 0.09 +/- 0.01, iopamidol, n = 8; vs. - delta 0.14 +/- 0.02, diatrizoate, n = 8; P less than .03). There was no significant difference in the decrease in glomerular filtration rate (- delta 7.4 +/- 1.0, iopamidol, n = 8; vs. - delta 9.3 +/- 1.3, diatrizoate, n = 8; P greater than .05), since the marked drop in filtration fraction occurring with diatrizoate was counterbalanced by the marked increase in renal plasma flow. Acute systemic and renal hemodynamic effects are significantly lessened when comparing iopamidol with diatrizoate.