Walter H. Hörl

Release of Granulocyte Proteinases during Hemodialysis

Neutral proteinases of neutrophilic polymorphonuclear leukocytes were followed up cytochemically in blood smears of 12 patients submitted to regular hemodialysis treatment (RDT). Halo formation (ring-shaped area around each neutrophil due to protein degradation) was reduced in all patients with end-stage renal disease under RDT. Concomitant to the development of leukopenia, a maximal increase of the plasma levels of the granulocytic elastase in complex with alpha 1-proteinase inhibitor was observed 3 h after starting hemodialysis (+409%; p less than 0.001). On the other hand, the proteolytic activity of the plasma samples against azocasein as substrate, being significantly higher (+244%; p less than 0.001) in RDT patients compared with healthy controls, decreased permanently during therapy (-71%; p less than 0.001; 3 h after initiation of the treatment). The mechanisms of release as well as of elimination of proteolytic activity due to RDT are discussed.

Evidence for the participation of proteases on protein catabolism during hypercatabolic renal failure

SummaryIn ultrafiltrated plasma (molecular weight <50,000) obtained from four patients with multiple muscular trauma and acute post-traumatic renal failure, it was possible to verify a subcomponential specific digestion of the subunits alpha and gamma of phosphorylase kinase isolated from rabbit skeletal muscle. The activity of free proteolytic enzymes in ultrafiltrated plasma as well as an increase of plasma alpha1-antitrypsin values were correlated with the severity and unfavourable course of the illness. In contrast, the plasma levels of alpha2-macroglobulin were drastically lowered. The mean total protein concentration in the sera of patients with post-traumatic ARF was lowered, whereas the mean ultrafiltrate protein concentration was significantly enhanced. In ultrafiltrated plasma of two patients with hyperuricaemic ARF, three patients with ARF after drug over-dosage, one patient with acute pancreatic necrosis combined with acute renal failure and one patient with chronic pancreatitis, no proteolytic activity could be detected using phosphorylase kinase as substrate. Studies on the trypsin binding capacity of the plasma protease inhibitors revealed a significantly lowered level in patients with post-traumatic acute renal failure as compared to healthy controls, patients with chronic renal insufficiency and patients on regular dialysis treatment. Proteolytic activity was found in ca. 100-fold concentrated diafiltrates (molecular weight >10,000) of patients on regular dialysis treatment. Our data suggest a participation of proteases on protein catabolism in hypercatabolic states. Whilst the blood coagulation system can largely be excluded as a source of proteases, it is possible that proteolytic enzymes may be released from muscle lysosomes and/or macrophages after multiple muscular trauma.ZusammenfassungIm ultrafiltrierten Plasma (Molekulargewicht <50 000) von vier Patienten mit Polytrauma und akutem posttraumatischen Nierenversagen gelang der Nachweis einer proteolytischen Verdauung der Untereinheiten alpha und gamma von Phosphorylase-Kinase, isoliert aus Skelettmuskulatur von Kaninchen. Es bestand eine Beziehung zwischen der Aktivität der freien proteolytischen Enzyme im ultrafiltrierten Plasma und dem Anstieg der Plasma-Alpha1-Antitrypsin-Werte mit der Schwere und dem ungünstigen Verlauf der Erkrankung. Die Plasma-Alpha2-Macroglobulin-Spiegel waren bei Patienten mit posttraumatischem akuten Nierenversagen deutlich erniedrigt. Im Serum von Patienten mit posttraumatischem akuten Nierenversagen war die Gesamtproteinkonzentration erniedrigt, im Plasmaultrafiltrat signifikant erhöht. Bei zwei Patienten mit akuter hyperurikämischer Nephropathie und drei Patienten mit medikamentös induziertem akuten Nierenversagen, einem Patienten mit akuter Pankreasnekrose und akutem postoperativen Nierenversagen sowie einem Patienten mit chronischer Pankreatitis und Zustand nach Whipple-Operation konnten dagegen im ultrafiltrierten Plasma keine freien proteolytischen Enzyme mit Phosphorylase-Kinase als Substrat entdeckt werden. Die Titration der Plasmaproteaseninhibitoren mit Trypsin ergab eine signifikant verminderte Bindungskapazität bei Patienten mit posttraumatischem akuten Nierenversagen im Vergleich zu Patienten mit chronischer Niereninsuffizienz oder regelmäßiger Hämodialyse und gesunden Kontrollen. Proteolytische Aktivität fanden wir bei chronisch urämischen Dauerdialysepatienten im 100fach ankonzentrierten Diafiltrat (Molekulargewicht >10 000). Unsere Daten lassen an eine Beteiligung von Proteasen am Eiweißkatabolismus denken. Während das Blutgerinnungssystem als mögliche Quelle von Proteasen weitgehend ausgeschlossen werden konnte, ist es möglich, daß proteolytische Enzyme nach Polytrauma aus Lysosomen und/oder Makrophagen der Skelettmuskulatur freigesetzt werden.

In vitro Inhibition of Protein Catabolism by Alpha2-Macroglobulin in Plasma from a Patient with Posttraumatic Acute Renal Failure

Rapid proteolytic digestion of three subunits of phosphorylase kinase was shown in plasma ultrafiltrates from a patient who was admitted with multiple traumatic injuries, respiratory insufficiency, hemorrhagic shock and acute renal failure. The observed cleavage of phosphorylase kinase may be a consequence of protease-antiprotease imbalance. After an initial determination of 80 mg/dl the alpha 2-macroglobulin values were too low to be detected. Addition of purified alpha 2-macroglobulin to the ultrafiltrates resulted in complete inhibition of phosphorylase kinase digestion in vitro. Aprotinin did not inhibit proteolytic digestion. Daily dialysis did not ameliorate the observed protein catabolism of this patient. These findings may have clinical application in hypercatabolic states, if alpha 2-macroglobulin becomes available in a form suitable for human use.

Carbohydrate metabolism and uraemia — Mechanisms for glycogenolysis and gluconeogenesis

ZusammenfassungStörungen des Kohlenhydratstoffwechsels bei akuter Urämie sind charakterisiert durch den Abbau von Leber- und Muskelglykogen bei gleichzeitiger Aktivierung der hepatischen Glukoneogenese. Die Substitution essentieller Aminosäuren und Ketosäuren führt nach bilateraler Nephrektomie in der Leber zu einer Stimulierung der Glykogensynthese, ein Effekt, der an der Skelettmuskulatur ausbleibt. Serin erweist sich unter den Bedingungen einer akuten Urämie als optimales Substrat für die Glukoneogenese der Leber und zeigt eine anabole Wirkung auf den Muskelglykogenstoffwechsel. Propranolol läßt die Glykogenolyse der Skelettmuskulatur bei akut urämischen Ratten unbeeinflußt.Unter Nüchternbedingungen kommt es bei akuter Urämie im Herzmuskel zu einem Anstieg des Gesamtkohlenhydratgehaltes, insbesondere von Glykogenund Glukosekonzentration. Änderungen der Kohlenhydratgehalte sind in der Niere nach Ureterligatur nicht nachweisbar. Als Ursachen der erhöhten Glykogenolyse der Skelettmuskulatur und Leber bei akuter Urämie kommt die Aktivierung von Phosphorylase-Kinase durch erhöhte Serumkonzentrationen verschiedener Hormone (Glukagon, Katecholamine, Parathormon) sowie durch freie proteolytische Enzyme, einen Anstieg der intrazellulären Ca2+-Konzentration und Strukturänderungen kontraktiler Proteine in Betracht.SummaryDisturbances of carbohydrate metabolism during acute uraemia are characterized by the degradation of liver and muscle glycogen with a simultaneous activation of hepatic gluconeogenesis. After binephrectomy, the substitution of essential amino acids and keto analogues stimulate liver, but not skeletal muscle glycogen synthesis. Serine proves to be an optimal substrate for liver gluconeogenesis and muscle glycogen generation under acute uraemic conditions. Propranolol does not influence glycogenolysis of skeletal muscle in acutely uraemic rats. During starvation, acute uraemia leads to an increase of total carbohydrate content as well as of glycogen and glucose concentrations in heart muscle Alterations in carbohydrate contents are not observed in the kidney after ureter ligation.Enhanced glycogenolysis of skeletal muscle and liver during acute uraemia may be due to activation of phosphorylase kinase caused by the increased serum concentrations of various hormones (glucagon, catecholamines, parathormone) as well as free proteolytic activity, an increase of intracellular Ca2+-concentration and finally by alterations in the structure of contractile proteins.

Long-term effects of nifedipine on carbohydrate and lipid metabolism in hypertensive hemodialyzed patients.

SummaryTo evaluate long-term effects of nifedipine on carbohydrate and lipid metabolism, 15 hypertensive patients undergoing regular hemodialysis treatment were investigated before nifedipine therapy, after 3 and 9 weeks, and 2 weeks after stopping nifedipine therapy. Three weeks following the administration of nifedipine, both glucose and insulin concentrations decreased significantly from 102.1±2.6 to 94.9±2.2 mg/dl and from 19.9±2.9 to 13.9±1.7 µU/ml and also remained significantly lower after 9 weeks of nifedipine therapy. This effect was paralleled by a fall of noradrenaline and dopamine. Glucagon levels remained constant. Glucose tolerance tests performed during nifedipine medication and 2 weeks after stopping of nifedipine therapy did not differ significantly. An increase of pyruvate, citric acid cycle intermediates, and ketone bodies — but not of lactate — was registered during nifedipine medication. The observed effects were not completely abolished after the 2-week placebo phase. Our data indicate that nifedipine lowers serum glucose values despite decreased insulin and constant glucagon levels in hypertensive hemodialyzed patients. Considering additionally the behavior of catecholamines and organic acids, the effects could be explained by the improvement of peripheral glucose utilization.

Inactivation of urinary kallikrein by alpha 1-antitrypsin.

SummaryProteolytic activity, with azocasein as substrate in the presence and absence of 0.4 IU kallikrein (Padutin) was measured in the 24 h urine fractions of 100 ambulatory patients with hypertension, proteinuria or haematuria. Urinary protein and alpha1-antitrypsin concentration have also been assayed. There was an inverse relationship between kallikrein activity and urinary alpha1-antitrypsin concentration (r=0.84;y=39.2e−0.009x). Furthermore, kallikrein activity and 24 h urinary alpha1-antitrypsin excretion were also inversely correlated (r=0.81;y=886.4e−0.011x). Our data suggest an inactivation of renal kallikrein by urinary alpha1-antitrypsin.ZusammenfassungBei 100 Patienten, die sich in unserer nephrologischen Ambulanz zur Abklärung einer Hypertonie, Proteinurie oder Erythrozyturie vorstellten, wurde im 24-h-Urin die proteolytische Aktivität vor und nach Zugabe von 0,4 IE Kallikrein (Padutin) ermittelt. Parallel wurden Protein- und alpha1-Antitrypsin-Konzentration im Urin gemessen. Dabei ließ sich eine inverse Beziehung zwischen Kallikrein-Aktivität und alpha1-Antitrypsin-Konzentration in den untersuchten Urinproben aufzeigen (r=0,84;y=39,2e−0,009x). Es bestand ferner eine inverse Korrelation zwischen Kallikrein-Aktivität und 24-h-Ausscheidung von alpha1-Antitrypsin (r=0,81;y=886,4e−0,011x). Unsere Daten sprechen für eine Inaktivierung von renalem Kallikrein durch alpha1-Antitrypsin im Urin.

Proteolytic Activity in Patients with Hypercatabolic Renal Failure

Proteolytic enzymes exist in ultrafiltrated plasma, concentrated dialysates and urine fractions of patients with posttraumatic renal failure. Differences in digestion pattern of phosphorylase kinase suggest the existence of different proteases in patients with hypercatabolic renal failure. Trypsin binding capacity is reduced in RDT patients and markedly lower in patients with posttraumatic ARF. Protein catabolism is inhibited in vitro by alpha 2-macroglobulin. From our in vitro studies we favour the application of fresh frozen plasma instead of the available plasma protein solutions to hypercatabolic patients. Hemodialysis may enhance proteinase inhibitory capacity of the plasma. Hemodialysis therapy induces the increase of plasma E-X1 PI. The continuous release of granulocyte elastase during hemodialysis therapy may enhance the risk for the development of destructive lung disease.

Calciumantagonisten in der Therapie der Hypertonie

Calcium antagonists (nifedipine, verapamil, diltiazem) are potent vascular smooth muscle relaxants. Experimental and clinical investigations provide growing evidence that they are effective in acute and (sub)chronic therapy of arterial hypertension by lowering peripheral vascular resistance and improvement of altered hemodynamics--independent from pathogenesis of hypertension. Due to its prompt and profound hypotensive action, sublingual or oral nifedipine has been used successfully in hypertensive crises. The hypotensive effect usually correlated closely with the severity of hypertension and is nearly absent in normotensive controls. Since the blood pressure drop may occasionally results in absolute or relative hypotension, the initial dose should be as low as possible. The activation of the adrenergic and renin angiotension systems seen after nifedipine administration is less pronounced after chronic administration of the drug and is nearly absent after verapamil and diltiazem. Plasma aldosterone concentrations remain constant or are slightly decreased. In contrast to classic vasodilators, the long-term administration of calcium antagonists usually does not result in tachycardia (nifedipine), but slight sinus bradycardia (verapamil, diltiazem). Peripheral edema may occasionally occur after nifedipine. A tolerance has been observed during long-term treatment of hypertension. Combining these drugs (verapamil, diltiazem) with betablockers is not recommended due to the negative inotropic and bathmotropic effects. Simultaneous administration of nifedipine and beta-blockers enhances the hypotensive action, but favours the development of peripheral edema and in rare cases (especially in severe coronary heart disease) results in a dramatic drop in blood pressure and/or congestive heart failure. Further clinical evaluation and long-term trials of calcium antagonists as antihypertensive agents will be needed before definite conclusions can be drawn.SummaryCalcium antagonists (nifedipine, verapamil, diltiazem) are potent vascular smooth muscle relaxants. Experimental and clinical investigations provide growing evidence that they are effective in acute and (sub)chronic therapy of arterial hypertension by lowering peripheral vascular resistance and improvement of altered hemodynamics — independent from pathogenesis of hypertension. Due to its prompt and profound hypotensive action, sublingual or oral nifedipine has been used successfully in hypertensive crises. The hypotensive effect usually correlates closely with the severity of hypertension and is nearly absent in normotensive controls. Since the blood pressure drop may occasionally result in absolute or relative hypotension, the initial dose should be as low as possible. The activation of the adrenergic and renin angiotensin systems seen after nifedipine administration is less pronounced after chronic administration of the drug and is nearly absent after verapamil and diltiazem. Plasma aldosterone concentrations remain constant or are slightly decreased.In contrast to classic vasodilators, the long-term administration of calcium antagonists usually does not result in tachycardia (nifedipine), but slight sinus bradycardia (verapamil, diltiazem). Peripheral edema may occasionally occur after nifedipine. A tolerance has been observed during long-term treatment of hypertension.Combining these drugs (verapamil, diltiazem) with betablockers is not recommended due to the negative inotropic and bathmotropic effects. Simultaneous administration of nifedipine and beta-blockers enhances the hypotensive action, but favours the development of peripheral edema and in rare cases (especially in severe coronary heart disease) results in a dramatic drop in blood pressure and/or congestive heart failure.Further clinical evaluation and long-term trials of calcium antagonists as antihypertensive agents will be needed before definite conclusions can be drawn.

Role of urinary alpha1-antitrypsin in padutin® (kallikrein) inactivation

SummaryAn inverse relationship between proteolytic activity in the presence of kallikrein 0.4 IU and urinary alpha1-antitrypsin concentration has been demonstrated. This protease inhibitor can directly inactivate kallikrein activity. The inhibition was abolished by removal of urinary alpha1-antitrypsin by trypsinsepharose treatment. Inhibition could be reversed by addition of purified alpha1-antitrypsin. These effects could not be demonstrated with inter-alpha-trypsin inhibitor or alpha2-macroglobulin. The inhibitory effect of alpha1-antitrypsin on kallikrein activity should be taken into account in studies in which kallikrein activity is estimated.

The Gluconeogenetic Ability of Hepatocytes in Various Types of Acute Uraemia

Liver cells were prepared from untreated controls, rats with various models of acute uraemia (uranyl nitrate-treated, bilaterally nephrectomised and ureter-ligated rats, rats with acute ischaemic renal failure) and sham-operated animals. Hepatocyte glucose output, pyruvate utilisation and lactate production were determined in the presence of Krebs-Ringer bicarbonate buffer with different pH values (7.1, 7.4, 7.6) using pyruvate, dihydroxyacetone, serine and fructose as substrates. In the presence of pyruvate and dihydroxyacetone a significant increase of glucose production in hepatocytes from bilaterally nephrectomised and ureter-ligated rats was observed. However, pyruvate-generated glucose production in the hepatocytes of uranyl nitrate-treated animals was unchanged, while a diminished glucose output was seen in the presence of dihydroxyacetone. A marked increase in glucose and lactate production in the presence of serine was observed in the hepatocytes of uranyl nitrate-treated, ureter-ligated and binephrectomised rats. However, lactate production from dihydroxyacetone in the liver cells of uranyl nitrate-treated animals was inhibited. In contrast to other types of uraemia, in acute ischaemic renal failure there is significantly lower hepatocyte glucose production using pyruvate as a substrate, but unchanged glucose generation from dihydroxyacetone or serine.