Cryopreservation of immature bovine oocytes to reconstruct artificial gametes by germinal vesicle transplantation.

Joining immature gamete cryopreservation and germinal vesicle transplantation (GVT) technique could greatly improve assisted reproductive technologies in animal breeding and human medicine. The present work was aimed to assess the most suitable cryopreservation protocol between slow freezing and vitrification for immature denuded bovine oocytes, able to preserve both nuclear and cytoplasmic competence after thawing. In addition, the outcome of germinal vesicle transfer procedure and gamete reconstruction was tested on the most effective cryopreservation system. Oocytes, isolated from slaughterhouse ovaries, were stored after cumulus cells removal either by slow freezing or by vitrification in open pulled straws. After thawing, oocytes were matured for 24 h in co-culture with an equal number of just isolated intact cumulus enclosed oocytes, and fixed in order to evaluate the stage of meiotic progression and cytoskeleton organization. Our results showed that after warming, vitrified oocytes reached metaphase II (MII) in a percentage significantly higher than oocytes cryopreserved by slow freezing (76.2% and 36.5% respectively, p < 0.05). Moreover, vitrification process preserved the organization of cytoskeleton elements in a higher proportion of oocytes than slow freezing procedure. Therefore vitrification has been identified as the elective method for denuded immature oocytes banking and it has been applied in the second part of the study. Our results showed that 38.3% of oocytes reconstructed from vitrified gametes reached the MII of meiotic division, with efficiency not different from oocytes reconstructed with fresh gametes. We conclude that vitrification represents a suitable method of GV stage denuded oocyte banking since both nuclear and cytoplasmic components derived from cryopreserved immature oocytes can be utilized for GVT.

Cellular and molecular mechanisms mediating the effects of polychlorinated biphenyls on oocyte developmental competence in cattle.

Polychlorinated biphenyls (PCBs) can interfere with normal reproductive functions acting as endocrine disruptors. Aroclor-1254 (A-1254), is a pool of more than 60 congeners used for in vitro studies because its composition is representative of PCBs environmental pollution. We previously demonstrated that the exposure of bovine oocytes to A-1254 during in vitro maturation (IVM) was detrimental not only to the maturation process but also induced a significant increase of polyspermy and a reduction of developmental competence. Therefore, we investigated whether A-1254 acts on two processes that occur during IVM and may be related with its negative effects: maternal mRNA polyadenylation and cortical granules (CGs) migration and exocytosis. Bovine cumulus-oocyte complexes (COCs) were exposed to 0.1 microg/ml of A-1254 during IVM, a level of exposure known to affect oocyte maturation, fertilization, and developmental competence. Oocyte exposure to A-1254 altered the poly(A) tail length of 5 out of 10 genes examined. PCBs effect on mRNA polyadenylation was different depending on the gene considered and resulted either in a shorter or in a longer poly(A) tail. At the end of maturation, Aroclor treated oocytes presented clustered CG in a significantly higher percentage than the control group. In addition, CG exocytosis after 8 hr of fertilization occurred at significantly lower extent in zygotes derived from the exposed group compared to control. Our results indicated that the lower developmental competence of oocytes exposed to PCBs during IVM can be related to the interaction of these contaminants with mechanisms regulating maternal mRNA storage in the ooplasm and normal CGs function.

Human parvovirus B19 infection mimicking systemic lupus erythematosus in an adult patient.

We report a case of widespread immune activation with moderate cytopenia during acute infection with human parvovirus B19 in an adult female patient, in whom five criteria for the diagnosis of systemic lupus erythematosus were present at disease onset. Our case is unusual due to the presence of a cutaneous rash mimicking leukocytoclastic vasculitis at presentation, moderate leukopenia with thrombocytopenia and the presence of a broad array of autoantibodies. Diagnosis was established on the grounds of serological tests confirming recent infection with human parvovirus B19; spontaneous regression of clinical and laboratory abnormalities was observed within 16 weeks, ruling out classic systemic lupus erythematosus. We conclude by proposing that human parvovirus B19 infection should be included in the differential diagnosis of lupus-like syndromes in adult patients.

-344C/T polymorphism of CYP11B2 gene in Italian patients with idiopathic low renin hypertension.

Most patients with low renin essential hypertension are not qualitatively different from patients with idiopathic hyperaldosteronism, as in both conditions aldosterone secretion is not appropriately reduced. The aim of the study was to investigate allele and genotype frequencies of the -344C/T polymorphism, located in the promoter region of the aldosterone synthase gene, in 83 patients with idiopathic low renin hypertension characterized by an increased aldosterone to renin ratio, including both patients with low renin essential hypertension (n=53) and subjects with idiopathic hyperaldosteronism (n=30), compared with 78 patients with normal to high renin essential hypertension and 126 normotensive control subjects. The relationship of -344C/T genotypes to basal and postcaptopril plasma aldosterone/plasma renin activity ratio was also examined in the entire hypertensive population. An increased frequency of the T allele and a relative excess of TT homozygosity over CC homozygosity were found in patients with idiopathic low renin hypertension in comparison with both normal to high renin hypertensives and normotensive controls. A higher post-captopril aldosterone to renin ratio was found in the hypertensives with TT genotype than in those with CC genotype, and TT+TC genotypes were associated with a smaller decrease in the aldosterone-to-renin ratio elicited by captopril administration. The present study suggests that the -344C/T polymorphism, or a functional variant in linkage disequilibrium with it, may play a role in the abnormal regulation of aldosterone secretion in idiopathic low renin hypertension.

In vitro reproductive toxicity of polychlorinated biphenyls: effects on oocyte maturation and developmental competence in cattle.

Polychlorinated biphenyls (PCBs) are one of the most persistent and widespread group of endocrine disrupting compounds in the ecosystem. High concentrations of these substances are known to be present in sewage sludge from industrial, agricultural, and domestic origin that is spread in increasing amounts on arable land and pasture as fertilizer and is found in water, representing an increasing risk for the reproductive health of farm animals. Objective of this study was to determine the impact of PCBs on maturation and developmental competence of cattle oocytes. Since PCBs are a family of 209 molecules present in the environment as a mixture, Aroclor-1254, a pool of more than 60 congeners, was used in these experiments as its composition is considered to be environmentally relevant. Cumulus-oocytes complexes were exposed during IVM to serial concentrations of Aroclor-1254 (between 1 microg/ml and 0.0001 microg/ml) and compared with control groups. Aroclor decreased the percentage of oocytes that reached metaphase II stage after 24 hr, at doses as low as 0.01 microg/ml. Groups treated with 0.001 microg/ml or above, showed an impaired fertilization rate and a dramatic increase of polyspermy. Moreover, exposure during maturation resulted in a reduced proportion of oocytes that cleaved and developed until blastocyst stage although no differences in embryo cell numbers were observed. The present study indicates that very low PCBs concentrations are sufficient to disrupt bovine oocyte maturation, its fertilization, and developmental competence. These results also provide a set of reference data for the assessment of the risk posed by these substances to animal reproductive health, though further work will be necessary to equate in vitro doses to in vivo exposures.

Heart rate variability in patients with Sjögren's syndrome.

Heart rate variability (HRV) gives information about sympathetic parasympathetic autonomic balance. Our purpose was to determine whether HRV is abnormal in patients with Sjögren's syndrome. In 16 patients with Sjögren's syndrome and 30 matched controls, a short time analysis of HRV was performed for both the frequency and the time domain. In the time domain, patients tended to display a slower heart rate, greater R-R variability and higher standard deviation of the mean (SDNN) than did healthy subjects, but the differences were not statistically significant. In the frequency domain the spectral measures of HRV showed a slight reduction of LF and an increase of HF; as a result, the ratio between high and low frequencies, representative of sympathovagal modulation, was significantly reduced. Our data suggest an increase in the parasympathetic control of heart rate in patients with Sjögren's syndrome. This predominance in vagal tone could exert a protective and antiarrhythmic role in patients with primary Sjögren's syndrome, and may be relevant with reference to the lower incidence of sudden death in this disorder compared to other major autoimmune diseases.

Liquorice-induced sodium retention. Merely an acquired condition of apparent mineralocorticoid excess? A case report.

Excessive ingestion of liquorice may result in sodium retention, hypertension, hypokalemia, and suppression of renin and aldosterone. Similarities between liquorice-induced effects and congenital apparent mineralocorticoid excess have recently been emphasized, as in both conditions, reduced activity of the enzyme 11 beta-hydroxysteroid dehydrogenase type 2 allows cortisol to act as a potent mineralocorticoid. We report a case of generalized edema without any increase in blood pressure, with biochemical and hormonal features of apparent mineralocorticoid excess, in a young woman who had been ingesting substantial amounts of liquorice for several years. Liquorice-induced wide-spread edema without hypertension in our patient, as well as in a few other cases previously reported, and the more common occurrence of edema associated with hypertension challenge the current explanation of liquorice syndrome as a purely acquired apparent mineralocorticoid excess. Indeed, in both congenital apparent and true mineralocorticoid excess, edema is typically absent, as a result of the sodium escape phenomenon. As pressure-natriuresis may be an essential mechanism accounting for the sodium escape phenomenon, some component of liquorice could partially or completely oppose the circulatory response that converts liquorice-induced sodium retention into blood pressure elevation. In patients with unexplained generalized edema and hypokalemia without hypertension, liquorice ingestion should be carefully investigated and the renin-aldosterone system should be assayed.

Necrotizing Lymphadenitis in Systemic Lupus Erythematosus Kikuchi's Disease or a Kikuchi's-Like Disease?

Kikuchi's disease or histiocytic necrotizing lymphadenitis is a recognized cause of benign lymphadenopathy, although the current clinical literature also supports a link with systemic lupus erythematosus (SLE). Eight patients represent all the cases of Kikuchi's disease admitted to our hospital from 1990 to 1997. They were all young women of median age of 31 years (range 22-43 years). All patients showed cervical lymphadenopathy, and in two cases splenomegaly was present. In one patient, the diagnosis of lupus preceded the onset of Kikuchi's disease by 5 months. In 4 other patients, antinuclear antibodies (ANA) were positive at titers ranging between 1:80 and 1:2560 and were associated with leukopenia ranging from 1190 to 2800 white blood cells/mm3 when the diagnosis of Kikuchi's disease was made. Among these, 3 patients developed SLE after periods ranging from 6 to 25 months. The other ANA positive patient and the 2 remaining ANA negative patients did not develop any autoimmune disease after 2-A years of follow-up.The differential diagnosis between Kikuchi's disease and lupus lymphadenitis cannot always be clarified histologically. We recommend that patients with necrotizing lymphadenitis, leukopenia, and ANA be closely followed. This should ensure an early diagnosis of development of an autoimmune disorder that will require treatment, rather than always considering these patients to be affected by a benign self-limited disorder like Kikuchi's disease.

Increased plasma levels of platelet-derived growth factor (PDGF-BB + PDGF-AB) in patients with never-treated mild essential hypertension.

Platelet-derived growth factor (PDGF) could play a role in both vascular hypertrophy and atherosclerotic disease associated with hypertension. To assess whether plasma PDGF level is increased in mild essential hypertension, we measured plasma PDGF concentration in 25 never-treated patients with uncomplicated mild essential hypertension and in 22 normotensive healthy subjects. To evaluate the contribution of platelets to plasma PDGF in the two groups, we also measured plasma beta-thromboglobulin (BTG). Measurement of PDGF was carried out through an enzyme-linked immunoadsorbent assay, which detects two PDGF dimers, namely PDGF-BB and PDGF-AB. Both plasma PDGF and BTG were higher in the hypertensive than in the normotensive subjects. The ratio of PDGF to BTG was similar in the two groups. Plasma PDGF was weakly correlated with plasma BTG in the normotensive subjects, whereas this relationship was lost in the hypertensive patients. Our results suggest that the increase in plasma PDGF (PDGF-AB + PDGF-BB) in never-treated essential hypertension is mainly due to platelet activation. The increased circulating level of PDGF could play a role in the vascular structural changes associated with hypertension.

Acute effects of intravenous sodium chloride load on calcium metabolism and on parathyroid function in patients with primary aldosteronism compared with subjects with essential hypertension.

To elucidate the mechanisms involved in increased parathyroid function in primary aldosteronism (PA), we evaluated the effects of an intravenous NaCl load on Ca metabolism and plasma level of intact parathyroid hormone (PTH) in patients with PA compared with that in patients with essential hypertension (EH). Sixteen PA patients and 16 EH patients who were well matched for age, gender, body mass index, renal function, and systolic (SBP) and diastolic blood pressure (DBP) were examined. In each subject, after 6 days of a controlled intake of Na, K, and Ca, isotonic saline was infused at a rate of 500 mL/h for 4 h. At baseline, in spite of similar BP values and urinary Na excretion (U[Na]V), urinary excretion of Ca (U[Ca]V) and PTH were higher in the PA group than in the EH group. In both groups, the NaCl load caused a decrease of serum ionized Ca (Ca2+) and an increase in PTH, U(Na)V, and U(Ca)V. However, these changes were significantly greater in the PA group. The increased baseline U(Ca)V in PA could be due to reduced reabsorption of sodium in aldosterone insensitive tubular sites, as a result of the "escape phenomenon." The increased U(Ca)V may explain the higher basal PTH in PA patients, which is needed for maintaining a normal Ca2+. The greater changes in the Ca2+/PTH profile elicited by the saline load in PA patients are apparently due to a higher calciuretic response following a more exaggerated natriuresis in PA.

Screening for primary aldosteronism with a logistic multivariate discriminant analysis.

OBJECTIVE: Primary aldosteronism (PA) is the most common endocrine cause of curable hypertension, but no single test unequivocally identifies it. Accordingly, we investigated the usefulness of a logistic multivariate discriminant analysis (MDA) approach for PA screening. DESIGN: Generation of a logistic MDA function based on retrospective analysis of biochemical tests in a large cohort of referred patients with/without confirmed Conn's adenoma (CA), followed by prospective validation of the model. PATIENTS: We investigated 574 selected hypertensives: 206 (32 with and 174 without CA) retrospectively, 48 (with a 13% prevalence of CA) prospectively for the validation of the model, and 320 referred hypertensives (with a 3.4% prevalence of CA) similarly evaluated. Patients were referred to a specialised centre for hypertension (4th Clinica Medica--University of Padua) and to a department of Internal Medicine of a regional hospital (Reggio Emilia). MEASUREMENTS: In all patients we measured several demographic and biochemical variables and performed a captopril test. A stepwise analysis of variance, based on a model fitted with several different variables, identified baseline (sALDO) and captopril-suppressed plasma aldosterone (cALDO), supine plasma renin activity (sPRA) and K+ as the most informative. Therefore, two models of logistic MDA with sPRA, K+, and either sALDO (model A) or cALDO (model B) were developed and used. ROC analysis was also performed to assess the optimal cut-off values. RESULTS: The model B of MDA provided the best performance and identified CA with 100% sensitivity and 81% accuracy. When used prospectively it showed 100% sensitivity, both in the Padua (88% accuracy) and in the Reggio Emilia series (90% accuracy). However, at both institutions most patients with idiopathic hyperaldosteronism (IHA) were also detected. CONCLUSIONS: Thus, although developed from patients with confirmed Conn's adenoma, a strategy based on multivariate discriminant analysis can be used prospectively for accurate screening for primary aldosteronism. Furthermore, it was proven to be accurate and applicable to patients tested with similar modalities at a different institution. Although this approach did not provide a clear-cut discrimination of Conn's adenoma from idiopathic hyperaldosteronism, it may avoid unnecessary and costly further testing in patients with a low probability of primary aldosteronism.

Alterations of calcium metabolism and of parathyroid function in primary aldosteronism, and their reversal by spironolactone or by surgical removal of aldosterone-producing adenomas.

In order to investigate the possible existence of abnormal calcium metabolism and parathyroid function in primary aldosteronism (PA), we have compared the calcium/parathyroid hormone (PTH) profile of patients with PA with the profile of healthy normotensive subjects and of patients with essential hypertension (EH). Furthermore, we have evaluated the effects of spironolactone and the surgical removal of aldosterone-producing adenomas on the calcium/PTH profile in the PA patients. Four groups of 10 subjects each participated in the study: 1) hypertensive patients with PA, 2) patients with low-renin EH (LREH), 3) patients with normal-renin EH (NREH), 4) normotensive healthy subjects (NS). The four groups were well-matched for age, sex, body mass index, and renal function. The three hypertensive groups were also matched closely for blood pressure values and for duration of hypertension. In all subjects, after 1 week of a controlled intake of Na and K, the following parameters were measured: urine excretion of Na, K, Ca, Mg, and P, plasma levels of K, Mg, inorganic P, total calcium and ionized calcium, and plasma renin activity, aldosterone concentration, and intact PTH. Blood pressure and laboratory parameters were determined again in all the PA patients after 1 month of 100 mg daily spironolactone administration, and in four out of the 10 PA patients 2 months after surgical removal of aldosterone-producing adenomas. All of these subjects had undergone the same controlled intake of Na and K indicated above. Serum intact PTH was higher in PA patients than in the other three groups (P < .01), and serum ionized calcium was significantly higher in normotensive subjects than in the three hypertensive groups (v PA P < .01, v LREH and v NREH P < .05). An increase in serum ionized calcium and a decrease in PTH level were associated with both spironolactone administration (P < .001) and surgical treatment (P < .05). These results suggest the presence of calcium metabolism alterations in both PA and EH patients, but that these alterations are more exaggerated in PA, so that higher PTH levels are needed for maintaining low-normal levels of serum ionized calcium.

Renal effects of nifedipine and captopril in patients with essential hypertension and reduced renal reserve.

In this study we investigated the short-term effects of calcium channel blockers and angiotensin-converting enzyme inhibitors on renal hemodynamics and the urinary excretion of proteins with different relative mass in subjects with mild to moderate essential hypertension and apparently normal glomerular filtration rate but reduced renal functional reserve. Sixteen subjects underwent the following four treatments: (1) low-protein meal (0.2 g protein/kg body wt), (2) high-protein meal (1.3 g protein/kg body wt), (3) high-protein meal plus oral nifedipine (20 mg), and (4) high-protein meal plus oral captopril (50 mg). Two urine samples were obtained after meals. Blood samples were drawn at the midpoint of each 120-minute urine collection period. Urine and serum were tested for total protein, immunoglobulin G, albumin, alpha 1-microglobulin, retinol binding protein, and beta 2-microglobulin. Glomerular filtration rate and renal plasma flow were assessed by iothalamate and p-aminohippuric clearance, respectively. Compared with the high-protein meal alone, nifedipine elicited a clear-cut increase in the urinary excretion of total protein (+60%, P < .01), immunoglobulin G (+58%, P < .01), albumin (+25%, P < .05), retinol binding protein (+47%, P < .05), and beta 2-microglobulin (+52%, P < .05); captopril decreased the urinary excretion rate of immunoglobulin G (-26%, P < .05), albumin (-22%, P < .05), and beta 2-microglobulin (-34%, P < .05). The ratio between the clearances of immunoglobulin G and albumin was higher after nifedipine (+21%, P < .01) and unchanged after captopril (-9%, P = NS) compared with the high-protein meal alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Significance of albuminuria in the follow-up of acute poststreptococcal glomerulonephritis.

The present study was aimed at assessing the diagnostic value of urinary albumin (uA) excretion rate in the long-term follow-up of patients suffering from acute post-streptococcal glomerulonephritis (APSGN). 26 patients, who had presented primarily with nephritic syndrome and showing increased uA without a concomitant rise in total proteinuria (uTP) were followed-up for 131 months on average (range 36-288). At the last check, 14 patients did not show urinary abnormalities, 9 had a persistent increase in uA, 1 increased uTP and 2 renal insufficiency. Urinary and clinical signs of the disease were not seen during observation periods prolonged for 79 months on average (range 20-156) after normalization of uA. No pathological findings were remarked in biopsy specimens obtained in 3 patients when uA was normalized; in contrast, when both uTP and uA (12 cases) or when isolated uA (14 cases) were increased a pattern of diffuse mesangial proliferative glomerulonephritis was constantly observed. These results indicate that the abnormal uA excretion rate during long-term follow-up of APSGN allows to identify a subset of patients with persistent renal disease; conversely, the occurrence of normal uA seems to point to a good diagnostic and prognostic significance.

[Lymphocyte subpopulations in workers exposed occupationally to styrene]. / Sottopopolazioni linfocitarie in lavoratori professionalmente esposti a stirene.

In a group of 32 workers occupationally exposed to styrene, the distribution of lymphocyte subsets was investigated by automated flow cytometry. The group under study consisted of 22 male and 10 female workers aged 39.7, D.S. = 10 years and employed for 6.8 years in factories manufacturing glass fibre-reinforced plastics. A control group (12 males and 7 females) recruited according to the same selection criteria was simultaneously examined. Environmental and biological monitoring was used to characterize styrene exposure. In styrene-exposed workers, phenotypic analysis of peripheral blood revealed a reduced proportion of T helper lymphocytes and a relative increase in the T suppressor subset, leading to a significant inversion of the helper/suppressor ratio (0.92) among heavily exposed workers (greater than 50 ppm, 8h-TWA) as compared to less exposed workers and controls (1.37 and 1.43 respectively). The proportion of natural killer (NK) T lymphocytes was significantly increased among styrene workers. The proportion of B lymphocytes was unchanged as compared to the control group. Dose-response relationships were clearly apparent for the observed increases in the prevalence of abnormalities: none of control subjects showed more than 2 (out of 8) abnormal values as compared to 20 and 40% of styrene workers belonging respectively to the low and high exposure group (p less than 0.007). Only a minority of controls exhibited abnormally high levels of both NK and suppressor T lymphocytes (0-5%), the corresponding figures for styrene workers being 7-10% (low exposure) and 45% (high exposure). As a whole, the findings support the hypothesis of the immunotoxicity of styrene, which could be due either to direct effects on lymphocytes or to indirect mechanisms possibly mediated by neuroendocrine changes.

Glomerular hyperfiltration in essential hypertension: hormonal aspects.

Glomerular hyperfiltration is thought to play a pivotal role in causing renal damage in essential hypertension. An increase in glomerular filtration rate can be experimentally induced by an acute oral protein load through still unclarified mechanisms, although hormonal factors have been postulated; in already hyperfiltering nephrons, the capacity to further increase glomerular filtration rate upon stimulation with an acute protein load (i.e. renal functional reserve) would conceivably be reduced, even in the presence of apparently normal renal function. The present study aimed at assessing whether renal functional reserve is preserved and/or is affected by different antihypertensive drugs in essential hypertensive patients without signs of renal function impairment; moreover, we tried to highlight changes in the plasma levels of natriuretic and antinatriuretic hormones potentially involved in the modulation of renal hemodynamics under the chosen experimental conditions. Renal hemodynamic parameters, plasma renin activity, aldosterone and atrial natriuretic factor were measured in fourteen essential hypertensives submitted to an acute oral protein load, alone or with a concomitant administration of either nifedipine or enalapril, as compared with a control carbohydrate meal. Glomerula filtration rate and renal plasma flow rose slightly but not significantly following an acute oral protein load as compared with a carbohydrate meal; no changes were noted in plasma atrial natriuretic factor levels, whereas plasma renin activity decreased. When nifedipine was administered together with the protein meal, both glomerular filtration rate and renal plasma flow increased significantly; there were also, parallel increases in plasma renin activity and atrial natriuretic factor. Administration of enalapril was associated with a decrease in both glomerular filtration rate and renal plasma flow; plasma renin activity showed an expected marked rise, whereas the plasma levels of atrial natriuretic factor were only slightly but not significantly reduced and plasma aldosterone fell. In conclusion, our data suggest that in our patients renal functional reserve was blunted. Clear-cut hyperfiltration was brought about by administration of nifedipine together with a protein meal, whereas enalapril completely abolished even the small increase in glomerular filtration rate seen after protein meal alone. The concomitant alterations in plasma renin activity, aldosterone and atrial natriuretic factor seemed to play no major role in the determinism of the observed renal hemodynamic changes.

Protein-induced changes in kidney function depend on the time of administration but not on the dietary source.

Two separate experiments were carried out to study the effects of the same acute protein load given at different hours of the day and to assess the ability of proteins from different sources to induce hyperfiltration. In the first experiment, 9 healthy volunteers were kept at strict bedrest for 48 h, during which both a meat high-protein meal (protein load, PL) and a vegetable low-protein meal (control load, CL) were given either at lunch or at suppertime. As compared to a CL, PL determined a significant increase in GFR, total proteinuria (uTP), albuminuria (uA), and urinary retinol-binding protein (uRBP). These effects were much more significant after lunch PL than after supper PL, thus indicating an interaction between the PL and the time of the day. The existence of a circadian rhythm for GFR, uTP, uA, and uRBP was corroborated by spontaneous changes over baseline levels, which also were prominent after lunch CL as compared to those following supper CL. In the second experiment, 7 healthy volunteers ingested at lunch three protein-rich meals at 1-week intervals. The three protein loads consisted of about 80 g protein in the form of cooked red meat, cheese, and soya, respectively. The only significant differences between groups were urea appearance and urea clearance, lower and higher, respectively after soya load. These findings suggest that when evaluating the renal functional reserve after acute protein load both the spontaneous changes and the time-dependent sensitivity of kidney functions to acute challenges should be considered. Finally, the amount rather than quality of dietary proteins seems to be the determinant factor for protein-induced glomerular hyperfiltration.