Nestor Schor

Role for intrarenal mechanisms in the impaired salt excretion of experimental nephrotic syndrome.

A unilateral model of puromycin aminonucleoside (PAN)-induced albuminuria was produced in Munich-Wistar rats to examine the mechanisms responsible for renal salt retention. 2 wk after selective perfusion of left kidneys with PAN (n = 8 rats) or isotonic saline (control, n = 7 rats), increases in albumin excretion and decreases in sodium excretion were demonstrated in PAN-perfused but not in nonperfused kidneys of PAN-treated rats although systemic plasma protein concentration remained at control level. Total kidney glomerular filtration rate (GFR) and superficial single nephron (SN) GFR were also reduced selectively in PAN-perfused kidneys, on average by approximately 30%, due primarily to a marked decline in the glomerular capillary ultrafiltration coefficient (Kf), which was also confined to PAN-perfused kidneys. Values for absolute proximal reabsorption (APR) were also selectively depressed in PAN-perfused kidneys, in keeping with a similarly selective decline in peritubular capillary oncotic pressure measured in these kidneys, the latter also a consequence of the fall in Kf. In a separate group of seven PAN-treated rats, however, no differences were detected between PAN-perfused and nonperfused kidneys in the absolute amount of sodium reaching the early (0.77 +/- 0.09 neq/min vs. 0.74 +/- 0.08, P greater than 0.40) and late portions of superficial distal tubules (0.31 +/- 0.02) neq/min vs. 0.32 +/- 0.05, P greater than 0.50), despite the lesser filtered load of sodium in PAN-perfused kidneys. Suppressed sodium reabsorption in both proximal convoluted tubules and short loops of Henle of PAN-perfused kidneys contributed to this equalization of sodium delivery rates to the late distal tubule, as did comparable reabsorption along distal convolutions. In two additional groups of PAN-treated rats, infusion of saralasin (0.3 mg/kg per h, i.v.) led to substantial increases in total kidney GFR and SNGFR in PAN-perfused but not in nonperfused kidneys. Despite these increases in total and SNGFR, urinary sodium excretion by PAN-perfused kidneys remained at a level far below that for nonperfused kidneys, again indicating that the antinatriuresis characterizing the PAN-perfused kidney is due to alterations in sodium handling by the tubules rather than changes in GFR. These results therefore indicate (a) that reductions in Kf and depressed sodium reabsorption by proximal tubules and Henles loop segments in this model are brought about by intrarenal rather than circulating or systemic factors, and (b) assuming that superficial nephrons are representative of the entire nephron population, renal salt retention in this model is due primarily to intrarenal factor(s) acting beyond the distal convolution.

Urolithiasis in childhood : metabolic evaluation

In order to determine metabolic disorders in children with urolithiasis, 50 patients with urinary calculi were studied. Abdominal pain and/or haematuria were the most predominant symptoms. Surgical procedures were required in 22% of these children and urinary tract infection was observed in 34% of this group. Only 2 children had anatomical malformations of the urinary tract. Absorptive hypercalciuria (32%), renal hypercalciuria (34%) and uric acid hyperexcretion (24%) were the most common metabolic abnormalities in these children. We were unable to find an underlying metabolic abnormality in only 14% of the patients. These data suggest that appropriate metabolic study will allow rational management of children with urinary stones.

Purification and characterization of angiotensin I-converting enzymes from mesangial cells in culture

Objective Previous analysis of the angiotensin I-converting enzyme (ACE) gene in this laboratory showed that primary mesangial cells in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on glomerular microcirculation. The aim of this study was to purify and characterize the secreted and intracellular ACE forms from mesangial cells in culture. Methods and results Medium from Wistar rats mesangial cells was collected (third passage), incubated for 20 h with RPMI without fetal bovine serum and concentrated 29 times in an Amicon concentrator. The concentrated medium was submitted to gel filtration on an AcA-34 column and two peaks (ACE1, mol. wt 130 000 and ACE2, 60 000) with ACE on activity Hippuryl-His-Leu and Z-Phe-His-Leu were separated. The mesangial cells were collected and ACE enzyme was extracted using Triton X-114, followed by centrifugation and concentration. The supernatant was submitted to the same chromatography as described above and two peaks with ACE activity (ACEInt1, mol. wt 130 000 and ACEInt2, 68 000) were separated. The purified ACE were inhibited by enalaprilat and captopril, two potent competitive inhibitors of ACE and by EDTA, using Hippuryl-His-Leu as a substrate. The Km values were 2 mM for ACE1 and ACE2 and 3 mM for ACEInt1 and ACEInt2. The enzymes ACE1 and ACE2 presented an optimum pH of 8.0 and ACEInt1 and ACEInt2 an optimum pH of 7.5. Conclusion The activities of full-length wild-type and N-domain ACE were characterized by the ratio of the hydrolysis of Z-Phe-His-Leu/Hippuryl-His-Leu, which was 1 and 4, respectively. The ratios found for ACE1, ACE2, ACEInt1 and ACEInt2 in the present study were similar to those described above, suggesting that mesangial cells, besides showing the presence of intracellular ACE, are able to secret both full-length wild-type ACE and N-domain ACE. Thus, they may potentially have an effect, not only on bradykinin and angiotensin I (ACE wild-type), but also on substance P, luteinizing hormone-releasing hormone and Met-enkephalin to interfere with glomerular haemodynamics and with the renal microcirculation.

Effects of long-term training on the progression of chronic renal failure in rats

To evaluate the effects of long-term exercise on the progression of chronic renal failure (CRF), adult Munich-Wistar rats with 5/6 renal mass ablation were submitted to treadmill exercise for 30 min 5 times/wk for 60 d. Whole kidney function and glomerular hemodynamics, proteinuria, and glomerular sclerosis were evaluated in 4 groups: Control, Sham trained (Sham + Ex), rats submitted to 5/6 nephrectomy (CRF) and maintained sedentary, and rats with 5/6 nephrectomy and trained (CRF + Ex). The groups with chronic renal failure (sedentary and trained) presented a reduction in total glomerular filtration rate (GFR) and in renal plasma flow (RPF), accompanied by an increase in single nephron GFR (SNGFR) and glomerular plasma flow (QA). However, the CRF + EX group did not show the glomerular hypertension observed in the CRF group. Despite the normalization of glomerular hypertension, proteinuria and sclerosis index were not different from the CRF sedentary group. Physical training provoked a vasodilatation of efferent arterioles, which induced the normalization of glomerular hypertension. These results suggest that the reduction alone of glomerular hypertension induced by exercise does not prevent the progression of renal disease, indicating the participation of other associated factors in this experimental model.

Role of angiotensin II in the physiologic regulation of glomerular filtration

Abstract Angiotensin II and arginine vasopressin are capable of triggering glomerular mesangial cell contraction in vitro. A similar mechanism acting in vivo to reduce glomerular capillary surface area could account for the decrease in the ultrafiltration coefficient which occurs in single glomeruli in response to infusion of these substances. Less clear is the mechanism whereby similar decreases in the ultrafiltration coefficient are induced with infusions of dibutyryl cyclic adenosine monophosphate (cAMP), parathyroid hormone and prostaglandins since the former two, at least, are incapable of eliciting mesangial cell contraction in vitro. To further explore the factors which regulate the ultrafiltration coefficient in vivo, micropuncture experiments were performed in 47 euvolemic Munich Wistar rats. Infusions of dibutyryl cAMP, parathyroid hormone, and prostaglandins I 2 and E 2 led to lower mean values for plasma flow rate and the ultrafiltration coefficient in superficial glomeruli than were found in control animals given vehicle alone, whereas average values for glomerular transcapillary hydraulic pressure difference and total renal arteriolar resistance tended to be higher. These increases in glomerular transcapillary hydraulic pressure difference and total renal arteriolar resistance and decreases in plasma flow rate and the ultrafiltration coefficient with dibutyryl cAMP, parathyroid hormone, prostaglandin I 2 and prostaglandin E 2 are typical of changes induced by angiotensin II. Indeed, when saralasin, a competitive angiotensin II antagonist, was infused together with these various vasoactive substances, the effects on glomerular transcapillary hydraulic pressure difference, plasma flow rate, total renal arteriolar resistance and the ultrafiltration coefficient were largely abolished. Therefore, the actions of dibutyryl cAMP, parathyroid hormone, prostaglandin I 2 and prostaglandin E 2 on the glomerular microcirculation appear to depend on an intermediate action of angiotensin II. By contrast, although Pitressin infusion also led to a significant decrease in the ultrafiltration coefficient, saralasin administration did not reverse this change, suggesting that the action of antidiuretic hormone on the glomerular microcirculation is independent of a pathway involving angiotensin II. Based on these studies, it seems reasonable to propose that angiotensin II and antidiuretic hormone are both potentially important regulators of mesangial cell contraction and thereby of glomerular capillary filtering surface area and the ultrafiltration coefficient.

Electroacupuncture and Moxibustion Decrease Renal Sympathetic Nerve Activity and Retard Progression of Renal Disease in Rats

Background/Aim: Chronic kidney disease (CKD) is an increasing major public health problem worldwide. The sympathetic nervous system and nitric oxide play an important role in the pathogenesis of CKD. Traditional Chinese medicine has accumulated thousands of years of therapeutic experiences. Electroacupuncture (EA) and moxibustion (MO) are two such therapeutic strategies. The aim of this study was to investigate the renal and hemodynamic effects of EA-MO in an experimental model of a CKD. Methods: Male Wistar rats submitted to 5/6th nephrectomy (5/6 NX) were studied for 8 weeks. There were four groups: (1) control, normal rats; (2) NX, 5/6 NX only; (3) NX-AS, 5/6 NX and EA-MO session using sham points, and (4) NX-AM, 5/6 NX and EA-MO session using real acupoints. Biochemical and blood pressure studies, renal sympathetic nerve activity measurements, nitric oxide levels and the histopathological indices were assessed. Results: The EA- and MO-treated group presented significant improvement in all measured functional and histopathological parameters. Conclusion: These findings suggest that EA-MO had beneficial effects on CKD. This effect was probably achieved by the modulation of the renal sympathetic nerve activity and nitric oxide levels, leading to decreased blood pressure, which is associated with less proteinuria.

EFFECT OF CYCLOSPORIN A ON NITRIC OXIDE PRODUCTION IN CULTURED LLC-PK1 CELLS

The effect of Cyclosporin A on nitric oxide production was studied in cultured LLC- PK1 cells. For this purpose the cells were incubated with vehicle (olive oil, 10 μg/ml in DMSO), Cyclosporin A (CsA, 10 μg/ml), tumor necrosis factor (TNF-α, 150 U/ml) + interferon (IFN-γ, 500 U/ml) to upregulate NOS synthesis, and therefore NO production (used as a positive control), or CsA + TNF-α + IFN-γ. After 72 hours the culture medium was collected and nitrite was determined by the Griess method. The results were normalized to the protein harvested from these cells as measured by the Lowry method. Viability was determined by the exclusion of the fluorescent dyes (acridine orange and ethidium bromide). Intracellular calcium was measured spectrophotometrically using the fluorescent calcium indicator fura-2 AM. In CsA treated cells, the nitrite (pmoles/mg of protein) was decreased when compared to control (12.8 ± 0.5 vs. 18.3 ± 0.6; p < 0.05; both n = 8). TNF-α + IFN-γ increased the nitrite synthesis (52.0 ± 0.2; p < 0.05 vs. control; n = 6). This effect was decreased significantly by the simultaneous treatment with CsA (38.8 ± 0.3; p <0.05; n = 6). Cell viability in CsA group was decreased when compared to the control (84.7 ± 0.2% vs. 93.6 ± 0.1%; p < 0.05; both n = 10). TNF-α + IFN-γ had no effect on viability (93.0 ± 0.3%; n = 10). However, when combined with CsA, viability was decreased relative to the control (85.0 ± 0.2%; p < 0.05; n = 10). Acute (1 h) or chronic (72 h) treatment of LLC- PK1 cells with CsA had no effect on basal calcium levels. Our results demonstrate a reduced level of nitric oxide production in LLC- PK1 cells treated with CsA. There was no effect of the drug on intracellular calcium levels, however CsA treatment did reduce cellular viability. We suggest that, in part, the decreased levels of NO production are a secondary consequence of direct cell damage. However, CsA may also be exerting direct effects on NO synthesis through its interactions with both iNOS and cNOS. These results also provide a dual mechanism of action for CsA induced nephrotoxicity, that is, direct cell damage and interference with the NO system within the nephron.

Urinary inhibitors of crystallization in hypercalciuric children with hematuria and nephrolithiasis.

Abstract. Urinary inhibitors are suggested to play a significant role in reducing crystallization in calcium (Ca) stone former and idiopathic hypercalciuria (IH). Urinary inhibitors such as magnesium (Mg), citrate, and glycosaminoglycans (GAGs) were evaluated, as well as urinary Ca and creatinine (Cr), in IH children with nephrolithiasis (LIT) or with hematuria plus IH (HEM) and were compared with a control group. The mean 24-h urinary excretion of Mg was similar in all groups. However, the urine Ca/Mg ratio was significantly increased (P <0.005) in LIT and HEM groups. A higher mean value for GAGs and citrate was found in the HEM group, but a very low level of GAGs (less than 60% of the normal value) and citrate (less than 30% of the normal value) was found in the LIT group. These data suggest that, despite a high urinary Ca excretion (3.6±0.1 mg/kg per day) in the HEM group, elevated urinary GAGs (32.0±1.0 mg/g Cr) and a normal urinary citrate (428.7±62.3 mg/24 h) excretion may prevent Ca crystallization and thus renal stones. In contrast, in the LIT group low urinary GAG (10.3±0.9 mg/g Cr) and citrate (235.2±52.3 mg/24 h) excretion may precipitate stone formation in the presence of a high urinary Ca excretion. Thus, it is reasonable to suggest that patients with hematuria and IH may not develop overt renal stone due to the presence of normal levels of renal stone inhibitors.

Nephrotoxicity of cyclosporine: The role of platelet-activating factor and thromboxane

Cyclosporine (CsA), an immunosuppressive agent, is potentially nephrotoxic. We had previously observed that acute administration of CsA to Munich-Wistar rats induced a decrease in single nephron glomerular filtration rate, due to a decline in glomerular plasma flow, and in the glomerular ultrafiltration coefficient. Moreover, these alterations were prevented when an antagonist of platelet-activating factor (PAF) was administered. In the present study we examined whether the protective effect of the PAF blocker in CsA nephrotoxicity could have been mediated by thromboxane (TxA2). Our data show that the PAF effects were not mediated by TxA2, since administration of dazmegrel, a thromboxane synthetase inhibitor, did not ameliorate the acute renal failure caused by CsA. Thus, PAF appears to be a direct mediator of acute CsA nephrotoxicity, while TxA2 is not significantly involved in this process.

Electroacupuncture and Moxibustion Attenuate the Progression of Renal Disease in 5/6 Nephrectomized Rats

Background/Aim: Chronic kidney disease is a worldwide public health problem and the prevention of its progression is still a major challenge in nephrology. Specific therapies that inhibit or attenuate this process are neither available nor satisfactory. Traditional Chinese medicine has been increasingly recognized as an effective therapeutic approach in several fields of medicine. The aim of this study was to investigate the effects of electroacupuncture (EA) and moxibustion (MO) in an experimental model of progressive renal disease in rats. Methods: Twenty-one male Wistar rats were submitted to 5/6th nephrectomy (NX) and assessed 8 weeks later and were divided into three groups: NX = only 5/6 NX, NX-AS = 5/6 NX and a 20-min EA-MO session in sham points, and NX-AM = 5/6 NX and a 20-min EA-MO session in three real acupuncture points. The treatment consisted of 16 sessions twice a week. Renal function, urine volume, serum creatinine, 24-hour proteinuria, direct and indirect blood pressure, glomerulosclerosis and tubulointerstitial fibrosis indices were assessed. Results: The NX-AM group showed a significant decrease in all investigated parameters when compared to the control groups. Conclusion: Our results suggest that EA and MO attenuated the progression of renal disease in the experimental model of 5/6 NX.