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Nephrology in Rome began in the 1960s with the arrival of Ernico Fiaschi in the wake of Cataldo Cassano at the Institute of Medical Pathology (later on Clinica Medica II). A group of doctors interested in nephrology was set up, with among them Giulio A. Cinotti, who was to become full professor of nephrology at the University of Rome ''La Sapienza'' in 1980. By the end of the 1960s, the renal transplant activity had become an important asset at the Institute of Surgical Pathology (later on Clinica Chirurgica II) thanks to Paride Stefanini. A chair of surgical nephrology was instituted at the Urology Clinics of Ulrico Bracci; the chair was first held by Nicola Cerulli, who developed an intensive hemodialysis program. Around the same time, the Center for the Research and Treatment of Arterial Hypertension and Kidney Diseases became operational at the hospitals of Rome (under the responsibility of Vito Cagli at the Policlinico Umberto I), while a nephrology and dialysis unit, directed by Giancarlo Ruggieri, was set up at the San Giacomo Hospital. Many nephrology-related ''cultural'' activities started to be undertaken thanks to the ''Gruppo Laziale di Nefrologia Medica e Chirurgica'' founded by Drs Cagli, Cerulli, and Cinotti. Two national congresses were organized by Giulio Cinotti in 1979 (Fiuggi) and 1992 (Rome).

An 11-year old boy with acute lymphoid leukemia underwent umbilical cord stem cell infusion. This was followed at day 15 by the onset of asymptomatic hypotonic isovolemic hyponatremia. The disorder could be attributed to a syndrome of inappropriate ADH secretion (SIADH), most probably related to the massive i.v. induction treatment with cyclophosphamide. The major causes and clinical variants of SIADH are reviewed, with particular emphasis on the complications of chemotherapy in hematological diseases. Worsening of hyponatremia during routine parenteral feeding, as opposed to normalization of plasma Na+ by infusion of hypertonic saline, emphasize the importance of early accurate diagnosis and careful follow-up of these iatrogenic sequelae of stem cell allograft.

Background: Adhesion of monocytes triggers apoptosis, cytotoxicity, cytokine release, and later proliferation of cultured human mesangial cells (HMC). In the search for transmembrane signals transducing the interaction of HMC adhesion molecules with leukocyte counterreceptors, we measured variations of cytosolic Ca(2+) ([Ca(2+)](i)) in HMC and monocytes of the U937 cell line during 6-h co-cultures., Methods: Monolayer cultures of HMC and suspensions of U937 cells were loaded with the fluoroprobe fura 2-AM and subsequently co-cultured for 6 h while separately monitoring by microfluorometry the Ca(2+)-dependent 500 nm fluorescent emission of each cell line at fixed intervals upon excitation at 340/380 nm., Results: U937 and peripheral blood monocyte adhesion was followed in HMC by a slow, progressive rise of [Ca(2+)](i) from basal levels of 96+/-9 nM to 339+/-54 at 60 min and 439+/-44 nM at 3 h. The [Ca(2+)](i) elevation reached a steady state thereafter, while parallel monolayers incubated with control media maintained resting levels throughout the co-culture with stable fluoroprobe retention. Receptor sensitivity to vasoconstrictor agents, including compounds not released by monocytes, such as angiotensin II, was rapidly downregulated in HMC co-cultured with U937 cells. No [Ca(2+)](i) changes could be elicited by the octapeptide or by the TxA(2) analogue, U-46619, as early as 30 min after exposure to U937 cells. No [Ca(2+)](i) changes were observed in U937 cells throughout the co-culture. Conditioned media from monocytes and from co-cultured HMC+U937 cells had no effect on [Ca(2+)](i) of HMC. Ca(2+) entry leading to fura 2 saturation was still inducible by Ca(2+) ionophores, such as ionomycin and 4-Br-A23187, which also inhibited the responses to vasoconstrictors. Ca(2+)-free solutions prevented the [Ca(2+)](i) rise as well as subsequent receptor inactivation, implicating Ca(2+) influx through store-operated Ca(2+) channels (SOC), a major pathway for Ca(2+) entry in these cultured cells. Ca(2+) influx was confirmed by Mn(2+)-quenching of fura 2., Conclusions: In HMC, early changes in [Ca(2+)](i) signal for monocyte adhesion in a co-culture model of glomerular inflammation. This signalling mechanism may mediate the functional responses elicited in glomerular cells by leukocytes, including downregulation of receptors for vasoactive agents.

Background: Several risk factors of IgA nephropathy (IgAN) have been identified, but their importance in predicting outcome is still controversial., Methods: We conducted a retrospective study on 119 patients (pts) with IgAN. All had a follow-up of over five years (mean 134+/-56 months). For each patient we recorded age, 24h proteinuria, hematuria, renal function (RF), arterial hypertension (AH) and histological features. Multivariate analysis was done for predictive purposes (segmentation, using Chi-squared Automatic Interaction Detection-CHAID)., Results: AH at the time of renal biopsy was the principal and independent predicting factor: 30/50 (60%) hypertensive pts had serum creatinine > or =1.5 mg/dL at the end of follow-up compared to 9/69 (13%) pts with normal blood pressure. Age was a further predictive parameter: 21/28 (75%) pts with AH and age over 39 years had reduced RF at the last examination. In this subgroup, 18/19 (95%) with evidence of tubulo-interstitial lesions showed a decline of RF., Conclusions: AH and age alone are significant prognostic factors; tubulo-interstitial lesions are an additional pointer to poor outcome in these pts. The algorithm obtained with segmentation analysis may be a guideline for prognosis in single patients with IgA nephropathy.

Background: The aim of the study was to determine whether Lisinopril, an ACE-inhibitor (ACEi), was more effective than other antihypertensive agents in slowing the progression of non-diabetic chronic renal diseases in patients with baseline proteinuria < or =1.0 g/day., Methods: In an open, multicentre study all eligible patients entered a 3 months run-in phase during which antihypertensive therapy (with exclusion of ACEi) was adjusted in order to obtain a supine diastolic blood pressure < or =90 mmHg and urinary protein excretion and renal function stability were verified. One hundred and thirty-one patients with chronic renal insufficiency (Clcr between 20-50 ml/min) because of primary renoparenchymal diseases and proteinuria < or =1.0 g/day, were randomized to Lisinopril (L=66) or alternative antihypertensive therapy (C=65). Changes in renal function were assessed by inulin (Clin) clearance., Results: During the follow-up period of 22.5+/-5.6 months, Clin did not change significantly in group L (-1.31+/-0.6 ml/min/1.73 m(2)) differing significantly from group C in which it declined markedly (-6.71+/-3.6 ml/min/1.73 m(2)) (P<0.04). Seven patients experienced adverse events that prompted discontinuation of treatment: four in group L and three in group C; in addition seven patients showed severe deterioration in renal function requiring dialysis: two in group L and five in group C. The overall risk of the combined end-points: need for dialysis or halving of GFR was significantly higher in group C versus group L. During the study the mean value for systolic blood pressure was 137.8+/-14.6 SD mmHg in group L and 140.8+/-14.1 SD mmHg in group C; the mean difference between groups, during and at the end of the study, was 2 mmHg (NS). The mean diastolic blood pressure during the study was 83.8+/-8.6 SD mmHg in group L and 84.3+/-7.56 SD mmHg in group C; during and at the end of the study the mean diastolic difference between groups was 1 mmHG:, Conclusion: This study, employing a sensitive measurement of renal function and with similar blood pressure in both groups, provides support to the hypothesis that ACEi have a specific renoprotective effect, in addition to blood pressure control, also in patients with mild proteinuria.

Background: Monocytes bind to human mesangial cells (HMC) in a co-culture model of leukocyte/ glomerular cell interactions. Since monocytic infiltration has been demonstrated in the early stages of diabetic glomerulopathy, we examined whether co-culture with myelomonocytes of the U937 cell line in media mimicking the diabetic microenvironment modulated phenotype, growth, and extracellular matrix production patterns of HMC., Methods: HMC monolayers grown for 5 days in 5.5 mmol/l (NG) or 30 mmol/l (HG) glucose media were examined 3, 24 and 48 h after addition of U937 cells by computer-assisted image analysis/fluorescence microscopy following fixation, staining for cell adhesion, and TUNEL/propidium iodide labelling for apoptosis. As matrix components may be relevant to both phenotype of cultured HMC and monocyte adhesion, reverse transcription-polymerase chain reaction, zymography, and ELISA were used to detect urokinase-plasminogen activator (uPa), collagen type IV (COL IV), transforming growth factor beta1 (TGF-beta1), matrix metalloproteinases (MMP), and relative inhibitors (tissue inhibitor of MMP (TIMP)) expression in co-cultures in NG/HG., Results: U937 adhesion at 1-3 h was increased in HG (from 54.9+/-6.6 to 87.1+/-5.8% U937/HMC). Control HMC proliferating in NG supplemented with 10% fetal bovine serum had an average cross-sectional area of 9993+/-505 micro(2) with 1.2+/-0.1 hillocks/high-power field, which increased to 13 651+/- 1114 micro(2) with 0.5+/-0.2 hillocks/high-power field in HG (P<0.05). TUNEL+HMC were nearly identical (4.9+/-1.7 vs 4.2+/-0.4% in HG, P=NS). Enhanced transcription and secretion of urokinase (uPA, +656%), COL IV (+137%), TGF-beta1 (+590%) were observed in co-cultures in HG. COL IV and TGF-beta1, but not uPA, were also increased in HMC alone, exposed to HG for 5 days. MMP-2/TIMP-2 ratio was decreased while MMP-1/TIMP-1 was increased in HG co-cultures. In both NG and HG, U937 adhesion reduced HMC number and hillocks at 24 h, with constant apoptosis. The effects of U937 were no longer detectable at 48 h, when apoptosis was 2.1+/-0.6 vs 4.0+/-0.4% in HG, and cell counts returned above basal, possibly due to a delayed proliferative response., Conclusions: High glucose medium increases U937 cell adhesion to HMC. In turn, monocytes modulate number and spatial distribution of HMC, which are also markedly affected by ambient glucose levels. These interactions may be relevant to leukocyte infiltration, mesangial expansion, and glomerulosclerosis in diabetes.

Background: Available data on changes in serum levels of bone markers after parathyroidectomy (PTx) in dialysis patients are not uniform. Changes are thought to be due to either a reduction in PTH activity per se or to a direct effect of vitamin D therapy on bone cells. We aimed to verify whether treatment with vitamin D modifies serum levels of markers of bone synthesis (alkaline phosphatase (AP), osteocalcin (BGP), procollagen type I C-terminal peptide (PICP)) and resorption (collagen type I C-terminal peptide (ICTP)) within a period of 15 days in haemodialysis patients with severe secondary hyperparathyroidism following PTx., Methods: We randomized two groups (A, treatment and B, placebo, 10 patients each) with comparable basal PTH values and measured bone markers 3, 7 and 15 days after surgery. All patients were treated with calcium supplements (i.v. and p.o.), and group A also received calcitriol (2.4+/-1.0 microg/day, p.o.)., Results: In both groups, PTx induced significant changes in all the markers evaluated, except for BGP in group B. Compared to basal values, ICTP decreased from 481+/-152 ng/ml in group A and 277+/-126 ng/ml in group B to 267+/-94 and 185+/-71 ng/ml (M+/-SD) respectively, and PICP increased from 307+/-139 ng/ml in group A and 309+/-200 ng/ml in group B to 1129+/-725 and 1231+/-1267 ng/ml (M+/-SD) respectively, within 3 days of surgery. AP values increased after 15 days from 1115+/-734 mU/ml in group A and 1419+/-1225 mU/ml in group B to 1917+/-1225 and 1867+/-1295 mU/ml (M+/-SD) respectively. On the contrary, mean values of BGP were never different from basal levels after PTx in either group. In the two groups, the pattern of changes of all the bone markers after PTx was almost identical. Group A patients predictably required lower doses of oral calcium supplements to correct hypocalcaemia (16. 9+/-5.7 vs 22.1+/-5.0 g/10 days; M+/-SD, P<0.04)., Conclusions: The opposite behaviour of serum PICP and ICTP after PTx, in both the treated and untreated groups suggests that quantitative uncoupling between bone synthesis and resorption is responsible for hypocalcaemia. This phenomenon, as reflected by the evaluated bone markers, is unaffected by calcitriol. Based on our data we conclude that immediately after parathyroid surgery, vitamin D therapy does not influence bone cell activity, but improves hypocalcaemia mainly through its known effect on intestinal calcium absorption.

Low dialysate to blood flow rate ratios are a unique characteristic of continuous arteriovenous hemodiafiltration (CAVHDF) that should allow complete saturation of dialysis fluid with small-molecular-weight blood solutes. The aim of the investigation was to evaluate the performance of different hemofilters in CAVHDF. In 10 critically ill patients with acute renal failure, the efficiency of four hollow-fiber hemofilters, polyamide 0.6 m(2), polyacrylonitrile (PAN) 0.3 and 0.6 m(2), acrylonitrile sodium methallylsulfonate (AN69HF) 0.6 m(2), has been evaluated. For comparison, dialysate flow rates (Q(di)) were standardized to 16.6 and 25 ml/min. Samples for urea nitrogen were obtained from the arterial blood line (C(bi)) and from the dialysate exit port (C(do)) within 24-hour running time. Outflowing dialysate (Q(do)) was also measured at the same time. Blood flow (Q(b)) was calculated by the bubble transit time technique. Diffusive and total urea clearances were determined. AN69HF and PAN hemofilters provided higher clearances than the polyamide hemofilter. Despite the smaller surface area, PAN 0.3 m(2) had a total urea clearance comparable to that of PAN 0.6 m(2) and AN69HF at Q(di) = 16.6 ml/min. While at Q(di) = 16.6 ml/min equilibrium between blood and dialysate (C(do)/C(bi) congruent with 1) occurred with the AN69HF and PAN hemofilters, at Q(di) = 25 ml/min the equilibrium was obtained only with the AN69HF hemofilter. In conclusion, almost complete urea saturation of dialysis fluid has not been obtained with all hemofilters tested here. In our experience, membrane characteristics play an important role in determining diffusive efficiency in CAVHDF., (Copyright 2000 S. Karger AG, Basel)

Advanced glycation end product (AGE) accumulation in a high glucose (HG) environment is thought to mediate some of the vascular complications of diabetes. Transmembrane signaling of contractile cells is generally inhibited by HG, with implications for systemic and target organ hemodynamics. In the kidney, glomerular mesangial cells grown in HG media are hyporesponsive to the effects of vasoconstrictor agents, possibly explaining the hyperfiltration and increased capillary pressure that eventually lead to diabetic glomerulopathy. To verify whether AGE binding to specific mesangial receptors could mediate these effects of HG, cultured human mesangial cells (HMC) were exposed to in vitro glycated bovine serum albumin (BSA) for 60 min at 37 degrees C before measurement of cytosolic Ca2+ ([Ca2+]i) by microfluorometric techniques in monolayers or single cells. AGE-BSA (2 mg/ml) reduced Ca2+ release from intracellular stores by 1 microM angiotensin II from peak [Ca2+]i levels of 843+/-117 to 390+/-50 nM in monolayers and from 689+/-68 to 291+/-36 nM in individual cells (P < 0.05). Nonglycated BSA and BSA exposed to 250 mM glucose-6-phosphate for 30 d in the presence of 250 mM aminoguanidine (AMGD), an inhibitor of nonenzymatic glycation, had no effect on the angiotensin II-induced [Ca2+]i spike (peak 766+/-104 and 647+/-87 nM, monolayers/ single cells, respectively, P = NS). AGE also inhibited store-operated Ca2+ influx through plasma membrane channels, assessed by addition of 1 to 10 mM extracellular Ca2+ to cells previously held in Ca2(+)-free media (control 339+/- 46/593 +/- 51, +AGE-BSA 236 +/- 25/390 +/- 56, +AMGD 483+/-55/ 374+/-64 nM [Ca2+]i, monolayers/single cells at 10 mM Ca2+, respectively; +AGE-BSA, P < 0.05 versus control). Contrary to HG, AGE-BSA did not translocate protein kinase C isoforms alpha, zeta, and delta to the plasma membrane. Culture of HMC in HG supplemented with 1 mM AMGD prevented downregulation of [Ca2+]i signaling. These data suggest that glycated macromolecules or matrix components may inhibit transmembrane Ca2+ signaling of glomerular cells through binding to a specific AGE receptor, thus mediating some of the known functional effects of HG on the kidney.

Background: A recent retrospective study has clearly demonstrated a reduction of cases with positive bone aluminium (Al) staining in the Italian dialysis population, which in general has had a low prevalence of bone Al toxicity. In the present study we tried to better address the relative role played, in our study population, by enteral and parenteral exposure to Al in reducing bone accumulation., Methods: We retrospectively examined the data of 105 DFO tests and bone Al determinations performed in dialysis patients from 1984 to 1995. Enternal exposure was analysed by accurate anamnestic records, while parenteral exposure was evaluated by the determination of Al content in dialysis fluids. Bone Al content was assayed chemically and histochemically, while serum Al was assayed spectrophotometrically. Data pertinent to the patients were allotted into three period groups: 1984-1987; 1988-1991; 1992-1995. As for Al concentrations in dialysis fluids, the interval 1980-1983 (immediately before the start of our study), which could clearly have influenced bone Al content, was also considered., Results: Basal serum Al showed some fluctuations (42.7 +/- 34.1; 24.8 +/- 21.9 and 38.9 +/- 34.9 micrograms/l respectively in the three groups, ANOVA P < 0.01) but only values of the period 1988-1991 were significantly lower than those of the period 1984-1987 (P < 0.05). Increments after DFO did not differ in the three periods (136.5 +/- 105.7; vs 98.7 +/- 91.7 and 106.1 +/- 96.2 micrograms/l respectively, P = n.s.). Enteral exposure to drugs containing Al was comparable (4.1 +/- 2.9 vs 4.0 +/- 4.6 and 5.8 +/- 7.9 total kg ingested respectively; P = n.s.), but bone Al was dramatically reduced (from 60.7 +/- 43.0 to 29.0 +/- 24.4 and 31.9 +/- 29.9 mg/kg/dw respectively; P < 0.0001), along with the definite disappearance of Aluminon-positive cases and Al-related bone disease (ARBD) after 1991. Parenteral exposure through the dialysate dropped from a mean of 26 +/- 14 micrograms/l in the 4-year period prior the start of the study (1980-1983) to 9 +/- 6 micrograms/l in the period 1984-1987 and to 4.9 +/- 2.1 micrograms/l and 5.0 +/- 2.0 micrograms/l respectively thereafter (P < 0.0001)., Conclusions: Despite the persistence of oral exposure to Al, responsible for the observed stability of serum Al levels, a definite reduction of bone Al content has been recorded in our dialysis population, and ARBD has disappeared. This result has to be referred essentially to the optimal control of Al content in dialysis fluids, which is confirmed as a major factor for Al intoxication.

In cultured mesangial cells (MC), capacitative Ca2+ influx via store-operated channels (SOC) is potentiated by agents that release Ca2+ from intracellular stores, and inhibited by protein kinase C (PKC). Cells grown under high glucose conditions, as a model of the diabetic microenvironment, display reduced Ca2+ signalling in response to vasoconstrictors, probably due to downregulation by elevated PKC activity. Since SOC might be relevant to this phenomenon, we assessed Ca2+ influx by microfluorometry of fura-2-loaded rat MC cultured for 5 days in normal (5.5 mmol/l, NG) or high glucose (30 mmol/l, HG). The addition of 1-10 mmol/l Ca2+ to NG cells equilibrated in Ca(2+)-free media induced an immediate Ca2+ influx with a free cytosolic Ca2+ ([Ca2+]i) plateau of 155 +/- 50 and 318 +/- 114 nmol/l, respectively. Basal influx was reduced to 88 +/- 8 and 145 +/- 17 nmol/l [Ca2+]i (1-10 mmol/l Ca2+, p < 0.01) by 30 mmol/l D-glucose. This effect of HG was confirmed by Mn2+ quenching of fura-2, indicating reduced entry of divalent cations via the capacitative pathway. Equimolar L-glucose had no effect on Ca2+ influx, consistent with a non-osmotic mechanism. Arginine vasopressin (10 mumol/l) elicited weaker release of stored Ca2+ and subsequent influx in HG cells (191 +/- 33 vs 153 +/- 24 nmol/l, 400 +/- 76 vs 260 +/- 33 nmol/l, 1-10 mmol/l Ca2+, NG/HG, p < 0.05). To examine the involvement of PKC in the effect of HG on capacitative Ca2+ influx, the enzyme was activated or downregulated by treatment with 0.1 mumol/l phorbol myristate acetate (PMA) for 3 min or 24 h, respectively. PMA acutely inhibited Ca2+ influx in NG cells, while PKC downregulation restored it in HG cells. Similarly, the PKC inhibitors staurosporin or H-7 normalized SOC activity in HG cells. In summary, impairment of Ca2+ influx via SOC by HG is one mechanism of the reduced MC [Ca2+]i responsiveness to vasoconstrictors. This event is mediated by PKC and may contribute to the glomerular haemodynamic changes in the initial stages of diabetes mellitus.

The effect of a regular haemodialysis session on the plasma concentrations of beta-endorphin, ACTH and cortisol was investigated in 14 patients with end-stage renal disease and 20 healthy controls. Blood for analysis of beta-endorphin, ACTH and cortisol was sampled before and immediately after haemodialysis. In four patients the dialysate was studied for presence of these hormones, but showed no specific activity. The predialysis beta-endorphin, ACTH and cortisol levels did not differ significantly from the control values. The postdialysis levels were significantly higher than the predialysis. Significant linear correlation was found between plasma ACTH and beta-endorphin values in the postdialysis samples. The similarity of plasma beta-endorphin, ACTH and cortisol levels in patients with end-stage renal disease before dialysis and in normal controls indicated integrity of the hypothalamic pituitary-adrenal axis. The significantly increased levels after the dialysis session and the significant correlation between postdialysis plasma beta-endorphin and ACTH suggest that the haemodialysis session was a stressful event.

In an in vitro model of monocyte adhesion to glomerular cells, U-937 myelomonocytic leukemia cells irreversibly bind to human mesangial cell monolayers. Adhesion is enhanced in mesangial cells proliferating in response to fetal bovine serum, and in the presence of several cytokines and vasoactive agents. In the present study, co-culture with U-937 followed by removal of non-adherent cells time-dependently decreased viability of mesangial cells, measured either by fluorometry after dual labeling with calcein acetoxymethylester and ethidium homodimer, or by the release of lactate dehydrogenase. The cytotoxic effects of co-culture with U-937 cells were significantly reduced by a combination of free radical scavengers, indicating involvement of reactive oxygen species. U-937 cells also stimulated subsequent proliferation of mesangial cells, assessed by [3H]-TdR incorporation and direct cell counts 24 hours later (from 1,034 +/- 83 to 14,611 +/- 959 and from 2,931 +/- 201 to 19,400 +/- 2,124 cpm/well, quiescent/cycling mesangial cells, respectively, P < 0.01). Controls to rule out TdR incorporation by adherent U-937 cells included selective [3H]-TdR labeling and demecolcine pretreatment. Cell counts at 24 hours confirmed U-937-induced proliferation of quiescent HMC, from 50,575 +/- 3,596 to 143,012 +/- 10,039 cells/cm2 (P < 0.01). Agents that promote U-937 cell adhesion, such as the TxA2 mimetic, U-46619, or angiotensin II, enhanced cytotoxicity while inhibiting the proliferation of both quiescent and cycling mesangial cells, when added during co-culture and the subsequent 24 hours (+1 microM U-46619, 1,875 +/- 131 and 2,546 +/- 125 cpm/well, respectively, 79,793 +/- 5,744 cells/cm2, P < 0.01 vs. U-937 only; +1 microM Ang II, 5066 +/- 560 and 5,784 +/- 306 cpm/well, respectively, 81,068 +/- 4,671 cells/cm2, P < 0.05). Blocking antibodies against the adhesion molecule ICAM-1 and leukocyte counterreceptors (LFA-1, VLA-4) prevented the proliferative response, which could not be duplicated with the conditioned media of U-937 alone or co-cultured with mesangial cells. These findings may reflect the interactions occurring in vivo between infiltrating leukocytes and resident cells during glomerular inflammation.

Sustained Ca2+ influx follows discharge of intracellularly stored Ca2+ in a variety of cell types previously equilibrated in Ca(2+)-free media, including cultured human mesangial cells. This Ca2+ influx pathway has been referred to as capacitative Ca2+ entry or Ca2+ release-activated Ca2+ influx (iCRAC). This study investigated two cellular mechanisms potentially controlling iCRAC in human mesangial cells, protein kinase C (PKC), a key signalling kinase activated by vasoconstrictors that release Ca2+ from internal stores, and calmodulin, a Ca(2+)-binding protein that may couple Ca2+ release to the putative channel(s). The PKC activator phorbol myristate acetate (PMA) dose-dependently inhibited both Ca2+ influx in resting cells and iCRAC, assessed by microfluorometry in fura-2-loaded monolayers, when added before or after 1 uM angiotensin II (AngII) (Ca2+ influx at 1 mM (Ca2+)e +278 +/- 56%/+80 +/- 8%, at 10 mM + 473 +/- 59%/+250 +/- 24% (Ca2+)e, -/+ PMA, respectively, P < 0.05). PMA did not affect 5 uM ionomycin-induced iCRAC, possibly because it downregulated Ca2+ release by AngII but not by ionomycin, suggesting a key role of released Ca2+ in triggering subsequent Ca2+ influx. This was confirmed by buffering the (Ca2+)i elevation induced by AngII with intracellularly trapped 1,2-bis-(0-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), which abolished any subsequent iCRAC. Moreover, the calmodulin inhibitors calmidazolium (10 uM), trifluoperazine (0.1 mM), or W-7 (0.1 mM) significantly inhibited AngII- or ionomycin-activated iCRAC (+106 +/- 38/229 +/- 53, +58 +/- 9/195 +/- 29, +161 +/- 38/180 +/- 40% at 1/10 mM (Ca2+)e, all P < 0.05), but did not affect basal Ca2+ entry, consistent with a direct role of cytoplasmic Ca2+ in the regulation of ion gating. These results indicate that iCRAC is under the control of both PKC and calmodulin, and that the site of regulation is distal to the emptying of Ca2+ stores. iCRAC may represent a key mechanism for the control of Ca(2+)-regulated mesangial functions.

Leukocyte adhesion to kidney cells is an early event in renal inflammation, such as glomerulonephritis. We developed an experimental model of monocyte adhesion to cultured human mesangial cells. U-937 myelomonocytic leukaemia cells, similar to peripheral blood human monocytes, irreversibly bound to mesangial cell monolayers upon 30-180 min coincubations (to a max. of 13,600 +/- 1100/cm2 monolayer), as assessed by cell counting, U-937 labelling with 3H-thymidine, and colorimetry of nuclear staining with crystal violet. Adhesion was enhanced in mesangial cells proliferating in response to 17% fetal bovine serum, indicating expression of a proinflammatory phenotype. E. coli lipopolysaccharide (LPS), tumour necrosis factor-alpha (TNF-alpha) and protein kinase C activation with phorbol myristate acetate (PMA) potentiated monocyte binding during either coincubation or 24-h pretreatment (0.1 microM PMA, +200 +/- 21%). Binding was also promoted by pretreatment with vasoconstrictors, such as the thromboxane A2 mimetic, U-46619 (10 nM-1 microM, max. +35 +/- 3%), or 1 microM angiotensin II (+64 +/- 4%). To elucidate the mechanisms of monocyte adhesion, we analysed the adhesion molecules expressed by human mesangial cells, employing reverse transcription/polymerase chain reaction to detect ICAM-1, VCAM-1 and E-selectin gene expression. Proliferating cells express VCAM-1 and ICAM-1, confirmed by immunocytochemical staining and 79 +/- 3% inhibition of stimulated adhesion by pretreatment of mesangial cells with an anti-ICAM-1 monoclonal Ab. E-selectin transcription was not detectable.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to examine serum angiotensin converting enzyme (SACE) activity and the renin-angiotensin-aldosterone system in patients on chronic haemodialysis during one routine dialysis session. Fourteen patients (8 men and 6 women; mean age 51.9 +/- 17 years) with end stage renal disease, receiving regular haemodialysis treatment for an average of 6 months, were studied. The patients were dialysed for 4 hours three times a week using cellulose membranes (cuprophan). After an overnight fast blood samples were taken from the patients before and after the haemodialysis session. Serum and plasma were separated and stored at -20 degrees C until assayed for SACE, plasma renin activity (PRA) and plasma aldosterone (PA). For comparison, SACE, PRA and PA were also measured in 8 patients after renal allotransplantation and on treatment with cyclosporin A (5 men, 3 women; mean age 38.9 +/- 12.3 years) and in 19 healthy subjects (13 men, 6 women; mean age 38.9 +/- 12.3 years). SACE levels in patients with chronic renal failure and on haemodialysis (17.55 +/- 9.03 nmol/ml/min) and in patients with renal transplantation (18.12 +/- 3.92) were significantly higher than those of the healthy subjects (9.27 +/- 1.67) (p < 0.0001, respectively). At the end of the dialysis session SACE levels in patients with chronic renal failure (14.9 +/- 7.19) did not increase in respect to pre-dialysis levels (17.55 +/- 9.03; p = 0.132). PRA and PA values increased after the dialysis session (p < 0.026 and p < 0.044, respectively). Correlation of SACE with PRA and PA was not demonstrated before or after the dialysis session. In patients with chronic renal failure and on haemodialysis our findings suggest that a disarrangement exists between the circulatory components of the reninangiotensin-aldosterone system before and after the dialysis session.

Ca2+ influx is a major component of the response of cultured human mesangial cells (HMC) to vasoconstrictors. Activators of phospholipase C such as angiotensin II (Ang II) release Ca2+ from intracellular stores and enhance Ca2+ influx, which in turn is modulated by Na+/Ca2+ exchange. By microfluorometry we studied the mechanisms of Ca2+ entry in resting and stimulated fura-2-loaded monolayers or single HMC. Addition of 1 to 10 mM extracellular Ca2+ to cells equilibrated in Ca(2+)-free media resulted in a rapid, persistent elevation of free cytosolic Ca2+ ([Ca2+]i), from 52 +/- 5 to 113 +/- 18 and 226 +/- 37 nM, respectively. Ca2+ influx was blocked by lanthanum or chelation with EGTA, while it was only partially inhibited by voltage-operated Ca2+ channel (VOC) blockers, such as nifedipine or verapamil. The rise of [Ca2+]i at high external [Ca2+] was not due to a Ca(2+)-sensing mechanism with release of intracellular stored Ca2+, since it was prolonged, and it was not seen in cells maintained in normal 1.25 mM [Ca2+] media. Moreover, it was not abolished by prior depletion of Ca2+ stores with 0.5 microM thapsigargin or 5 microM ionomycin in Ca(2+)-free media, which transiently increased [Ca2+]i (to 281 +/- 39 and 380 +/- 51 nM, respectively). On the contrary, both agents markedly potentiated Ca2+ influx upon addition of 1 to 10 mM [Ca2+]e, (to a maximum of 686 +/- 111 and 633 +/- 150 nM, P < 0.05 vs. control).(ABSTRACT TRUNCATED AT 250 WORDS)

Focal segmental glomerulosclerosis evolves toward chronic renal failure (CRF) with a highly variable rate; in particular, a group of patients with a much more rapid decline of renal function has been described. The purpose of this study was to evaluate the usefulness of morphometry in identifying those cases with a faster decline in renal function. Two groups of patients have been studied: six with rapid evolution toward CRF (group 1) and six without reduction in renal function during a follow-up of up to 10 years (group 2). The results of the morphometric analysis of glomeruli were as follows: mean glomerular area: 30,550 +/- 5,259 microns2 (group 1) versus 22,667 +/- 5,078 microns2 (group 2) (P = 0.01); maximum glomerular area: 40,827 +/- 9,508 microns2 (group 1) versus 30,445 +/- 7,224 microns2 (group 2) (P = 0.02); mean glomerular diameter: 193.9 +/- 15.8 microns (group 1) versus 161.8 +/- 16.8 microns (group 2) (P = 0.003); and caliper diameter: 328.6 +/- 20.6 microns (group 1) versus 260.6 +/- 36 microns (group 2) (P = 0.001). Values of body surface area were not different between the two groups (1.85 +/- 0.34 m2 v 1.6 +/- 0.13 m2) (P = NS). Our results suggest that glomerular hypertrophy in the course of focal segmental glomerulosclerosis is correlated with a faster decline toward CRF. It might represent a compensatory hypertrophy that would immediately precede a rapid decline in renal function or it might be the expression of the preexisting condition (meganephronia), which predisposes to the development of CRF.

Na+/Ca2+ exchange contributes to the control of cytosolic free Ca2+ levels ([Ca2+]i) in resting and activated cultured human mesangial cells. We have previously shown that activation of phospholipase C by vasoconstrictors enhances Ca2+ influx upon extracellular Na+ withdrawal. This effect is not mediated by concurrent activation of protein kinase (PK) C, since it occurs even after PKC inhibition, and phorbol esters actually blunt both basal and stimulated Na+/Ca2+ exchange. We now studied the effects of PKA and PKG activation by adenylate/guanylate cyclase stimuli or by permeant analogues of cyclic nucleotides in monolayer cultures loaded with the fluorescent Ca(2+)-sensitive probe, fura-2. The exchanger was inhibited by the stable prostaglandin I2 analogue, iloprost, which is transduced by cAMP (peak [Ca2+]i inhibition by 1 microM iloprost 35 +/- 3%). Similarly, non-receptor activation of adenylate cyclase by 10 microM forskolin inhibited basal and agonist-stimulated Na+/Ca2+ exchange by 52 +/- 4 and 66 +/- 4%, respectively. Dibutyryl-cAMP (0.1 mM) also inhibited stimulated Na(+)-dependent Ca2+ influx by 72 +/- 2%. The particulate guanylate cyclase agonist, atriopeptin III, and the soluble guanylate cyclase activator, glyceryltrinitrate, also inhibited both basal and angiotensin II-stimulated Na+Ca2+ exchange (to a maximum of 53 +/- 5 and 62 +/- 3%, respectively). Dibutyryl-cGMP (1 mM) mimicked the effects of cGMP stimuli, reducing stimulated Na+/Ca2+ exchange by 79 +/- 2%. Therefore, similar to PKC, cyclic nucleotide activation of PKA and PKG regulates Na+/Ca2+ exchange, providing a functional link between transmembrane signalling systems for vasoactive agents in cultured human mesangial cells.

Clinical and experimental studies have markedly expanded our understanding of the causes of renal disease. Although they have not resulted in comparable therapeutic advances, new lines of treatment have emerged which cannot be underestimated. The prognosis of lipoid nephrosis has been substantially modified. The treatment of membranous glomerulonephropathy with steroids and immunosuppressive drugs may modify the course of the disease, at least in a subset of patients with decreased and deteriorating renal function. Encouraging results have been reported after the use of inhibitors of platelet aggregation in mesangiocapillary glomerulonephritis. We have demonstrated that a receptor antagonist of thromboxane can significantly influence the renal functional parameters in patients with lupus nephritis. ACE-inhibitors, cyclosporine and NSAID's have proved useful in reducing nephrotic proteinuria. Lipid lowering agents are able to ameliorate the glomerular lesions in experimental models of renal injuries. Since systemic hypertension may initiate the development of renal disease or accelerate loss of function in the kidney in which parenchymal disease is already established, controlling hypertension by any effective means helps to slow the progression of renal failure. Preliminary reports have suggested that there may be advantages to using ACE-inhibitors and/or calcium entry blockers.

Glomerular vasodilatation in the early stages of type I diabetes mellitus apparently results from arteriolar insensitivity to vasoconstrictors. Since cytosolic free calcium ([Ca2+]i) is a major signaling mechanism for smooth muscle contraction, we studied whether growth of smooth muscle-like rat glomerular mesangial cells in media with high glucose concentration affects [Ca2+]i responses to vasoconstrictors. In cells grown for five days in 22 mM glucose, we observed blunted responsiveness to three structurally unrelated vasoconstrictors that elevate [Ca2+]i via a phospholipase C-dependent mechanism, angiotensin II, prostaglandin F2 alpha, and arginine vasopressin. Inhibition of [Ca2+]i responses was not due to an osmotic effect of high glucose, since it was not mimicked by hypertonic mannitol. While the size of intracellular Ca2+ pools was unaffected by elevated glucose, Na+/Ca2+ exchange was markedly inhibited, thus ruling out both impaired filling of Ca2+ stores and enhanced counter-regulatory mechanisms. Impaired myoinositol transport or intracellular sorbitol accumulation were not responsible for the effects of high glucose, since supplementation of media with myo-inositol or with the aldose reductase inhibitor. Alcon 1576, failed to reverse insensitivity to vasoconstrictors. On the other hand, down-regulation or pharmacological inhibition of protein kinase C completely reversed the effects of high glucose, thus indicating involvement of this signal transduction pathway. These data suggest a possible intracellular mechanism for the impaired vascular sensitivity underlying early renal hemodynamic changes in diabetes mellitus.

Chronic renal parenchymal disease is the most common cause of secondary hypertension: By the time end-stage renal disease develops, its prevalence approaches 75-85%. It was previously believed that hypertension caused arteriolar nephrosclerosis and ischemic renal injury superimposed on primary renal parenchymal disease, contributing to progressive renal insufficiency. Recently, the importance of an increased intraglomerular hydraulic pressure due to loss of renal autoregulation has been emphasized. There are several potential explanations concerning clinical studies which have not uniformly demonstrated slowing of progressive renal damage with antihypertensive therapy: Random BP measurements may not adequately reflect average BP levels, or, alternatively, the widely accepted target level of BP control may be inadequate. In a retrospective study, we found that hypertensive nephropathics had higher serum creatinine levels than normotensives; in another prospective trial we have demonstrated that enalapril is an effective antihypertensive agent in patients with IgA nephropathy, and it also ameliorates the evolution of the disease.

Cultured human glomerular mesangial cells express functional receptors for vasoconstrictor metabolites of arachidonate, such as thromboxane A2. Binding of thromboxane A2 analogs triggers phosphatidylinositol breakdown and a rise of intracellular free Ca2+, followed by complex effects on cell growth. We have evaluated the effects of the thromboxane A2 mimetic, U-46619, on the biosynthesis of human mesangial cell proteins. After 4 to 24 hours of incubation with U-46619, cells were metabolically labeled with tritiated leucine or sulfur 35-methionine and analyzed by one-dimensional and two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis and fluorography. U-46619 modestly stimulated total protein synthesis in both quiescent and cycling cells to a maximum of 12% and 23% above control at 1 mumol/L after 24 hours, respectively. The eicosanoid selectively enhanced the labeling of crude membrane and cytosolic proteins of molecular weights of approximately 38 to 53 kd and 125 to 200 kd in the presence or absence of serum, respectively. Cellular tubulin and collagen type IV, but not actin, were markedly stimulated, as determined by immunoblotting of cell-associated proteins. On the contrary, U-46619 potently inhibited labeling of soluble proteins that were released into the media, to a maximum of 49% after 24 hours. Inhibition was confirmed by immunoblotting of collagen type IV. Intraglomerular accumulation of thromboxane A2 during chronic inflammation may modify the functional characteristics of the mesangium by regulation of the biosynthesis of structural proteins.

We evaluated the importance of the net charge of polyionic macromolecules in the regulation of cultured human mesangial cell growth. Structurally unrelated polyanionic compounds, i.e., heparin, suramin, poly-L-aspartic acid, and poly-L-glutamic acid, strongly inhibited 10% fetal bovine serum-stimulated cell proliferation. On the other hand, two polycations, protamin sulfate and poly-L-lysine, were equally effective in inhibiting cell growth. The antiproliferative activity of each compound was neutralized by molecules with opposite net charge. These data indicate that both anionic and cationic macromolecules exert an antimitogenic effect on cultured human mesangial cells. This inhibitory effect is dependent upon charge density rather than on the net electric charge of each compound.

Phenotype, growth, and functional characteristics of glomerular mesangial "myofibroblasts" are under the control of multiple hormones, vasoactive agents, autacoids, and cytokines. Several parallel signal transduction pathways couple receptor occupancy with functional changes, including phospholipases C, A2, and D breakdown of membrane phospholipids, and adenylate/guanylate cyclase activation. Changes of cytosolic ion concentrations, cyclic nucleotide accumulation, and eicosanoid biosynthesis are currently interpreted as intracellular signals for protein kinase activation. Phosphorylation of multiple substrates by serine/threonine kinases C, A, and G or by tyrosine kinases directly coupled to receptors, is a final step in cell activation. Cross-talk between signal transduction pathways, along with the release of eicosanoids and cytokines acting as intercellular mediators, provides the potential for interactive regulation of glomerular cell functions.

Na(+)-Ca2+ exchange contributes to regulation of cytosolic free Ca2+ levels ([Ca2+]i) of cultured human mesangial cells following phospholipase C stimulation, as shown by larger responses to vasoconstrictors such as angiotensin II (ANG II) or endothelin 1 in Na(+)-free media. In turn, previous activation of phospholipase C by vasoconstrictors significantly enhances the amplitude of the [Ca2+]i elevation induced by Na+ withdrawal. We studied the mechanisms of upregulation in monolayer cultures loaded with the fluorescent Ca(2+)-sensitive probe fura-2. The exchanger was stimulated by insulin and inhibited by chronic exposure to serum. A rise of [Ca2+]i was not sufficient per se to enhance exchange activity, as prior elevation of [Ca2+]i with the ionophores ionomycin or 8-bromo-A23187 failed to augment the response to Na+ withdrawal. Protein kinase C (PKC) activation by phorbol 12-myristate-13-acetate (PMA), alone or in combination with a rise of [Ca2+]i, potently inhibited basal and vasoconstrictor-enhanced Na(+)-Ca2+ exchange. Suppression of the effects of ANG II was not due to frustrated phospholipase C activation by PMA, because addition of PMA after ANG II also inhibited Na(+)-Ca2+ exchange. PKC downregulation by 24-h pretreatment with PMA or inhibition with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine or staurosporine did not prevent activation by ANG II. The exchanger was markedly potentiated by Na+ loading the cells with gramicidin D or reducing extracellular K+. ANG II failed to stimulate Na(+)-Ca2+ exchange when added in the absence of extracellular Na+. Therefore vasoconstrictors promote Na(+)-Ca2+ exchange by a mechanism independent of [Ca2+]i and PKC while presumably linked to Na+ influx.

Serotonin, a release product of activated platelets, stimulates proliferation and prostaglandin synthesis in cultured smooth muscle-like glomerular rat mesangial cells by activation of phospholipase and protein kinase C. To further characterize the signaling mechanisms used by serotonin, we monitored its effects on intracellular free Ca2+, pH, and membrane potential of cultured rat mesangial cells with sensitive fluorometric techniques. Activation of a 5-HT2 receptor, blocked by the specific receptor antagonists ketanserin and ritanserin, triggered immediate discharge of intracellular Ca2+ stores. The resulting rise of cytosolic free Ca2+ was accompanied by simultaneous membrane depolarization and followed within 30-60 seconds by prolonged cytosolic alkalinization. Depolarization and cytosolic free Ca2+ elevation were persistent in the continued presence of serotonin and were rapidly reversed by competitive receptor displacement with ketanserin or ritanserin. Depolarization is secondary to enhanced Cl- conductance, whereas it is relatively independent of Na+, K+, and Ca2+ fluxes. The putative Cl- channel is regulated by Ca2+ since ionomycin and other stimuli of cytosolic free Ca2+ mimic the effects of serotonin on membrane potential, whereas serotonin-induced depolarization is blunted in cells pretreated with the intracellular Ca2+ chelator BAPTA. Cytosolic alkalinization occurs in HCO3(-)-free solutions resulting from enhanced activity of a Na(+)-H+ exchanger and blocked by extracellular Na+ removal or amiloride. In the presence of HCO3-, serotonin elicits a persistent acidification, revealing simultaneous enhancement of a Na(+)-independent Cl(-)-HCO3- countertransport. These findings indicate multiple pathways for contraction and long-term functional changes induced by serotonin in mesangial cells, with potential relevance to glomerular and systemic hypertension.

Excitable cells express Na+/Ca2+ exchange activity among other mechanisms modulating rapid fluctuations of cytosolic free Ca2+ ([Ca2+]i). We studied functions and regulation of a Na+/Ca2+ exchanger in cultured human glomerular mesangial cells. Fura-2-loaded confluent monolayers reacted to removal of ambient Na+ with an immediate, transient elevation of [Ca2+]i, assessed by single/dual wavelength fluorometry. Peak [Ca2+]i was inversely correlated with the extracellular Na+ concentration. Ca2+ influx was the sole mechanism implicated, as the [Ca2+]i rise was prevented by EGTA. The process was inhibited by 1 mM amiloride, but not by blockers of voltage-operated Ca2+ channels. Re-addition of Na+ resulted in a rapid decrease of [Ca2+]i, indicating bimodal operation of the exchanger. Na(+)-loading the cells with monensin and ouabain enhanced Ca2+ uptake. Prior stimulation of [Ca2+]i with the thromboxane A2 mimetic, U-46619, or angiotensin II also increased Ca2+ uptake upon subsequent Na+ removal, suggesting induction of the exchanger by vasoconstrictors. Moreover, the magnitude of agonist-induced [Ca2+]i transients was amplified by Na+ removal, indicating that the exchanger modulates the effects of vasoconstrictors. These results demonstrate that an inducible Na+/Ca2+ antiporter is operative in resting and stimulated human mesangial cells, further confirming their smooth muscle origin and potential regulatory role on glomerular hemodynamics.

The glomerular polyanion comprises all anionic sites of glomerular cell surfaces, basement membranes and extracellular matrix. Charged structures may play a critical pathophysiologic role within the glomerular microcirculation, as loss of charges results in altered permselectivity of the filtration barrier and proteinuria. Neutralization of cell surface negative charges of cultured glomerular epithelial and mesangial cells by the polycation poly-L-lysine (PL) is accompanied by increased prostanoid synthesis. Recent studies have shown that PL enhances mesangial cell proliferation in culture. Conversely, the polyanion heparin prevents the effect of PL, and inhibits the growth-stimulatory effect of serum. Thus, our data suggest that the glomerular polyanion, in addition to maintaining the integrity of the filtration barrier, regulates key cell functions such as eicosanoid biosynthesis and proliferation.

Mesangial cells are contractile pericytes of the kidney glomerulus. Mesangial contraction/relaxation contributes to the regulation of glomerular hemodynamics. Additionally, mesangial cells process filtered macromolecules, synthesize extracellular matrix, respond to and release a number of cytokines and vasoactive mediators. Cultured mesangial cells express receptors for circulating and local agents that affect glomerular function. These receptors are coupled to distinct signaling pathways, namely phospholipase C and A2, transducing vasoconstrictor stimuli, and adenylate/guanylate cyclase, transducing vasodilators. Early intracellular signals include changes of cytosolic ions and cyclic nucleotides. They translate into short-term responses, such as cell depolarization and contraction, and later events, such as prostanoid synthesis and cell proliferation. Studies of mesangial cell behavior in culture may largely enhance our current understanding of glomerular pathophysiology.

The present study deals with the appropriate statistical approach to the histomorphometric technique carried out on bone tissue with the Merz and Schenk integrating eyepiece. Statistical analysis of the results obtained from ten bone biopsies of renal osteodystrophic patients enabled to calculate the values of standard errors for each histomorphometric parameter as a function of measured histological fields. The examination of 42 fields chosen at random on histological sections from different sites of the bone biopsy guarantees sufficiently low standard error values. This technique is of invaluable help in the study of metabolic bone diseases, and extremely useful in the evaluation of treatment trials in renal osteodystrophy.

We investigated whether the glomerular synthesis of prostacyclin modulates the renal blood flow and glomerular filtration rate in chronic glomerular disease. The urinary excretion of 6-keto-prostaglandin F1 alpha, a stable breakdown product of prostacyclin, was significantly (P less than 0.01) reduced in 20 women with chronic glomerular disease, as compared with 19 controls, whereas excretion of urinary prostaglandin E2 was unchanged. In 10 patients randomly assigned to one week of treatment with ibuprofen, excretion of urinary 6-keto-prostaglandin F1 alpha and prostaglandin E2 was reduced by 80 per cent, the level of serum creatinine was increased by 40 per cent, and creatinine and para-aminohippurate clearances were reduced by 28 and 35 per cent, respectively. The reduction of both clearances was inversely related (P less than 0.01) to the basal urinary excretion of 6-keto-prostaglandin F1 alpha but not of prostaglandin E2. No functional changes were detected in five healthy women, despite a similar suppression of renal prostacyclin synthesis by ibuprofen. In contrast, one week of treatment with sulindac did not affect renal prostacyclin synthesis or renal function in the other 10 patients, despite a marked inhibition of extrarenal cyclooxygenase activity. We conclude that in patients with mild impairment of renal function, the renal blood flow and glomerular filtration rate are critically dependent on prostacyclin production. In such patients sulindac may be a safe substitute for other nonsteroidal antiinflammatory drugs.