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Mechanisms and precise targets of nephrotoxicants have been evidenced in experimental animals, a very important contribution for decades. But, structural and functional heterogeneity in renal tissue makes often difficult the exact determination of the molecular targets at renal cell level. Indeed, some new in vitro models have been gradually reported. This non exhaustive review purposes to present the different recent models, ever more reductionist, as isolated kidneys, kidney slices, isolated renal cells or renal cultured cells to assess the nephrotoxicity of new molecules. For every model are presented a brief history, the principle of the technique, viability criteria and its relative interest for renal toxicology. In conclusion, a general discussion points out the main advantages and limits of each one.

We have prepared in rabbits antibodies against rat GBM, with GBM prepared from isolated glomeruli issued from rat renal cortex. It is shown with immunodiffusion technique the excretion of basement membrane-like fragments into the normal rat urine. This result confirms the earlier works studying the human and rabbit urines. The origin and the nature of this patterns, the signification of this presence into the mammalian urines are discussed.

The present study originates in two experimental data: circadian variations evidence of water, electrolytes and solutes urinary excretion and theophylline diuretic and salidiuretic effects knowledge; we purpose to evidence theophylline-induced water and sodium renal excretion in rats as modified by the time of drug administration. Theophylline single dose is injected in 100 animals (20 lots of 5 rats) at 8 h, 14 h, 20 h or 2 h and urines are collected during a consecutive to injection hours long period: 8 h-14 h (I), 14 h-20 h (II), 20 h-2 h (III) or 2 h-8 h (IV). Diuresis increases in + 40,4 p. cent (I), in + 123,7 p. cent (II), in + 123,3 p. cent (III) in + 65,4 p. cent (IV). So, natriuresis increases in 39,6 p. cent (I), in 223,2 p. cent (II), in 114,3 p cent (III) and in 109,6 p. cent (IV). These results evidence that theophylline diuretic and natriuretic effects change strongly with injection time, being largest if it is injected at 14 h and slightest if injected at 8 h. Such observations prompt to study if the other pharmacological properties of theophylline, especially at pulmonary level, response also with a time-dependant intensity.

The present microscopic study evaluates the quantitative variations of ferritin particules density within the glomerular ultrastructures after theophyllin perfusion in the Rat. The ferritin particules density increases 3,65 times in the glomerular basement membrane and decreases 3,28 times in the glomerular capillary lumen. So, theophyllin increases considerably the glomerular pathway of the protein macromolecules. This results confirm the increased volumic flow (Jv) and macro-solutes flow (Js') noted with clearance methods and urinary excretion studies.

The purpose of the study was to determine a direct vascular effect of beta-blockers on isolated glomeruli of rat kidney. The glomeruli were isolated by sieving and their diameters measured with a micrometer before and after addition of each of three beta-blockers. The diameter of the glomeruli increases (6,7%) with verapamil, a calcium inhibitor, but remains unchanged in the same experimental conditions as well with propranolol, drug which decreases renal plasma flow, than with nadolol and tertatolol, drug increasing renal haemodynamics. The location and the mechanism of the glomerular vasoactivity of these different beta-blockers are discussed.

In 3 lots of 15 rats, urinary proteins excretion (U.P.E.) during 6 hours periods is compared : 1) daily and nightly in physiological conditions, where U.P.E. increases nightly (+ 97,47%) : 2) daily, before and after theophylline injection, where U.P.E. increases after theophylline injection (+ 78,62%) ; 3) nightly, before and after theophylline injection, where U.P.E. increase is only tiny (+ 3,53%) after theophylline injection. So, in nightly physiological and in daily experimental conditions the important U.P.E. increases can be in part explained by those of renal functions. This result can explain after nightly theophylline injection (where renal functions are already very higher than in daily period) the so small nightly proteinuria increase with the xanthic diuretic ; it cannot be compared to the daily value, because the renal functions cannot increase beyond some limits. Lastly, the present paper exhibits how a pharmacomolecule (as theophylline here) can act differently daily and nightly and open a new investigation field in chronopharmacology of diuretics.

The present study defines morphological and enzymatic characteristics of human healthy renal tubular cells in primary culture, and during three passages. The results confirm the ability to culture human renal tubular cells and to quantitatively evaluate the activity of specific tubular enzymes in the cells and in the culture medium. Such determinations can be compared to similar enzyme activity research in renal biopsies or in urines, to investigate human renal cell functions, in clinical, pharmacological or toxicological approaches.

We have studied in rats the influence of renal ischemia on urinary excretion of three brush border membrane enzymes (gamma glutamyl transferase, alkaline phosphatase and leucine aminopeptidase) and of a lysosomal one (N-acetyl-B-D-glucosaminidase). Urines were collected over 24 hours periods during three days before and after a 45 minutes renal artery clamping. Urinary GGT, PAL and LAP excretion were significantly increased on the first day after renal ischemia, but returns to normal values on the second day. Urinary NAG activity increases on the first day, but contrary to the latter enzymes, reached to normal values only on the third day. Enzymuria seems to be a useful marker of tubular injury occurring after a temporary renal ischemia in the rat.

This paper presents the repartition of 989 glomeruli in 8 parallel concentric renal cortex zones, by projecting on a calibrated screen rat kidney slices photographies. The glomerular density increases regularly from the outside to the middle cortical zones and then decreases to the deepest juxtamedullary zones.

Agar-agar and indian ink mixture intrarenal injections enable the precise glomerular afferent arteriole diameter and the evaluation of its variations after theophyllin perfusion. The increased diameter (+ 35, 78 %) confirms the peripheral vasodilating theophyllin effect, never to our knowledge proven in situ in the kidney.

The present microscopic study evaluates precisely the density of ferritin-particules in several substructures of the glomerular barrier, particularly the endothelial barrier, and the glomerular basement membrane, 15 and 60 minutes after the injection of the tracer. This quantitative method opens the way to numerous studies on the pathway of this protein-tracer through the glomerular substructures in the normal and pathologic rat.

The present study evidences blood and urine urea, creatinine and total proteins circadian variations in 50 male rats. Venous blood is sampled at behind socket sinus once a week at different hours (8, 11, 14, 17, 20, 23, 2 and 5) and urines are collected during 4 consecutive six hours periods in animals living in metabolism cages (8-14, 14-20, 20-2, 2-8). Blood three nitrogen substances circadian variations bring out the decrease at 17 h and increase at 23 or 2 h. Urinary excretion variations curves shows, in all cases, an increase more than 40% during the nightly periods. The influence of feeding rhythms on the blood and urine three derivates circadian rhythms is discussed. Moreover, night diuresis increase and urea and creatinine urinary remarkable constancy suggest water, solutes and macrosolutes transglomerular pathway nightly increase existence. Urea (+ 40%) and creatinine (+ 30%) clearance nightly significative increase confirms glomerular filtration circadian variations and its nightly increase.

The purpose of the study was to establish an in vitro model for the quantification of the melanin produced in human melanocytes in culture. Melanin content in these cultured human melanocytes is determined by spectrofluorimetric assay. Fluorescence intensity is quantitatively related to the melanin content present in cultured melanocytes.

After confirmation of the intense and very fast myorelaxant effect of theophylline on isolated glomeruli, xanthic reagent myotoxic effects are shown during more or less long contacts with these glomerular suspensions. We studied isolated glomeruli diameter evolution according to reagent dose (0,25 to 10 mg/ml) and its contact length (1, 5, 10 minutes). If the diameter remains practically constant during all the period for the lowest doses, it begins to decrease after the 5th minute for higher (1 and 2,5 mg/ml) and cracks up in the first minutes for the biggest ones (5 and 10 mg/ml). Pharmacologic and toxic actions are discussed ; hypothesis of a toxic effect at smooth muscular cells level is proposed.

The present study considers the quantitative variations of the physiologic proteinuria with theophyllin in the rat. The urins are collected, during 8 hours, every Rat being its own reference. The single theophyllin injection (20 mg/kg) induces an increased diuresis (+ 42,1 %) and proteinuria (+ 73,7 %). The repetitive theophyllin injections (during 5 days) confirms this constation. The hypothesis of a modification of the permeability of the glomerular barrier to proteic macromolecules is proposed to agree this result.

Water, sodium and proteins renal excretion in with collodion Page's method hypertensive rats is compared with that of same Wistar strained control rats. Blood pressure of treated animals (n = 16) is 193.1 Hg mm (138 in control rats). Urinary flow increases from 19.5 to 33.7 ml/24 h (+ 72.8%), sodium excretion from 29.6 to 37.5 mg/24 h (+ 26.7%) and total proteins excretion from 23.5 to 63 mg/24 h (+ 169%. This data are correlated with the renal cortex morphological changes with photon and electron microscope. Severe damages are seen in Bowman's capsule and in glomerular copruscules, especially at the epithelial layer level. Important proteic pools occur within tubular lumen. Proximal tubular epithelium seems normal. On the other hand, distal tubular epithelium seriously scales. So, important water, sodium and proteins excretion increase in our experimental hypertension model can be explained. It is also discussed about functional and structural modifications analogy in our model's rats and in spontaneously hypertensive rats (SHB).

Amelioration of sieving technics for glomeruli isolation aims at obtention of purer and more homogeneous preparations, presenting a high preservation degree for glomerular functional studies. We have recently demonstrated the necessity to use sieves adapted to the glomeruli size of different considered animals as well as to use kidneys having exactly the same weight in order to obtain very homogeneous glomeruli suspensions. This study presents a new amelioration in the homogeneity of the glomeruli diameter through a preliminary dissection of the renal cortex in order to isolate glomeruli situated in the same particular cortical zone. Pig renal cortex, because of its size, agrees well with a fine dissection in order to dissociate the superficial and the juxta-medullary zones. Glomeruli are isolated with 160/120 micro mesh sieves. Diameter mean value is 218.8 +/- 30.1 micro in superficial glomeruli and 270.4 +/- 30.1 micro in juxta-medullary ones, highly significant difference, (p less than 0.001). Moreover, repartition frequency histograms of the juxta-medullary glomerular populations diameter presents a large shifting to higher values. This renal dissection preceding the glomeruli isolation sieving technique contributes to better the homogeneity of the isolated glomeruli suspensions and opens the way to the original study of the comparative in vitro vasoreactivity of these two different glomerular populations after incubation with physiological or pharmacological reagents.

Circadian rhythms in urinary water, sodium, potassium and proteins excretion are studied in 45 rats living alone in metabolism cages. Urines are collected during 4 consecutive 6 hours long periods during 2 consecutive days. Large circadian variations of these parameters (especially water and proteins excretion and urinary protein concentration) are described. The influence of feeding rhythms on the circadian urinary excretion rhythms is discussed. It is proposed that nightly renal hemodynamic changes (during meal digestion or with high renin plasma levels) can induce modifications in glomerular filtration rate and electrolytes and macromolecules transglomerular flow.