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[Usefulness of microdissection of nephron segments and fluorescent indicator for molecular biological studies of nephron functions].

The kidney consists of numerous functional units called nephrons. Thus, the use of individual nephron segments is essential to characterize their functional properties and to clarify the molecular basis of site-specific functions. Nephron segments can be microdissected from collagenase-treated renal slices under a stereomicroscope. A variety of intracellular ionic concentrations or membrane potential can be determined with various fluorescent probes. Fura-2/AM-loaded nephron segments reveal a transient increase of cytosolic free calcium concentrations by agonists such as angiotensin II, vasopressin, kinins, etc. To localize their receptors or to characterize their subtypes, this technique is especially beneficial, because tiny fragments of the nephron are sufficient by combination with a two-wave length microscope fluorometer. As an example, discovery of a novel vasopressin receptor (Vp) is described.

Angiotensin II

Primary culture of mammalian nephron epithelia: requirements for cell outgrowth and proliferation from defined explanted nephron segments.

Nephron segments were dissected from fetal or from early postnatal rabbit kidneys (n = 86) and explanted individually into primary tissue culture. Outgrowth of epithelioid cells and proliferation in monolayers from the collecting tubule and the thick ascending limb of Henle's loop occurred regularly if a special substratum and matrix were used. Media were either supplemented with non-proteins (defined media) or with embryo extract and fetal sera (undefined media). Outgrowth and monolayer spreading were recorded by Differential Interference Contrast Microscopy. Monolayer cells resemble those seen with the same recording technique during tubule in vitro perfusion. Undefined media were essential for growth of convoluted tubule segments from embryonic Nephron Anlagen and outgrowth of cells from proximal straight tubules, in contrast to the distal nephron segments. The specific functional and morphological properties of cells derived from primary culture of nephron segments are under study.

Animals

Effect of diuresis and nephron dimensional heterogeneity on the distribution of nephron filtration rates.

Conflicting data in studies of the effect of natriuresis on intrarenal single nephron glomerular filtration rate (SNGFR) redistribution may arise from the interplay of hitherto largely overlooked factors. In the present work, the effect of diuresis induced by saline, glucose or mannitol, as well as the effect of anatomical nephron heterogeneity, were studied. A highly significant positive correlation was found between the logarithm of the urine flow per gram of kidney weight (log V) and the mean superficial (S) to mean juxtamedullary (JM) SNGFR ratio. The rise in S/JM SNGFR with diuresis was primarily a function of decreased JM SNGFR. Total proximal tubular length (TPL) was used as a measure of nephron size. The distribution of nephron sizes was evaluated as S/JM TPL. The effects of urine flow and anatomical heterogeneity on S/JM SNGFR were spearated by means of multiple regression analysis, which yielded the following equation: S/JM SNGFR = -0.049 + 0.179 log V + 0.818 S/JM TPL. Both slopes were highly significant (P less than 0.001). These findings indicate that S/JM SNGFR increases with urine flow, independently of sodium homeostasis, and that anatomical heterogeneity has a marked effect and must, therefore, be controlled. Conflicting data in the literature are harmonized with the present data when appropriate correction can be made for the dimensional factor. There is no evidence that SNGFR redistributions play a role in sodium homeostasis.

Animals

Developmental changes in nephron number, proximal tubular length and superficial nephron glomerular filtration rate of rats.

1. Post-natal development of single nephron glomerular filtration rate, superficial proximal tubular length, nephron number and kidney weight have been studied in Sprague Dawley and in Wistar rats. 2. Superficial tubular length is a non-linear function of body weight or age. There seems to be a rapid growth until animals weigh about 150 g in Wistar rats. In this strain, growth is slower thereafter. This difference is not as evident in Sprague Dawley rats. 3. Nephron numbers increase over the same period at which rapid tubular growth occurs. 4. Sprague Dawley rats have somewhat fewer, but longer, proximal tubules than do Wistar rats. 5. In all animals weighing more than 100 g, SNGFR is linearly related to weight. For younger, smaller Sprague Dawley rats, the same linearity holds over the age range studied--older than 20 days of age. In Wistar rats, SNGFR relative to weight is less in young animals. 6. By relating SNGFR to total kidney GFR, evidence is obtained that maturation of renal function also involves a greater increase in filtration by superficial than by juxtamedullary nephrons.

Age Factors

Single nephron glomerular filtration rate ratios of superficial, intercortical and juxtamedullary nephrons in rats during development.

In 20, 40 and 60-day-old rats the filtration rate was studied using Baines modification of Hanssen's Na4Fe (14CN)6 method enabeling the determination of single nephron glomerular filtration rate (SNGFR) ratio between superficial (S), intercortical (I) and juxtamedullary (J) nephrons. The proximal tubule lengths were determined as well. A close correlation was obtained between age and S/I, I/J and S/J of proximal tubule lengths and between age and I/J, S/J 14C-activities of ferro-cyanide. Presented findings confirm the data on the increasing role of superficial nephrons in the course of postnatal development of rat.

Age Factors

Deep nephron function after release of acute unilateral ureteral obstruction in the young rat.

The effects of acute unilateral ureteral obstruction (UUO) of 18 h duration on deep nephron function was evaluated in 14 weanling rats with the technique of micropuncture. After release of UUO, 3.4 +/- 0.66% (SE) of the filtered water remained at the tip of the collecting duct nearly fivefold greater than in controls (0.75 +/- 0.10%). Similar differences were seen in fractional sodium that remained at this site. The ratio of tubular fluid osmolality to that of plasma was also reduced in the UUO group (1.53 +/- 0.06 vs. 4.60 +/- 0.26 in controls, P less than 0.001). Single nephron glomerular filtration rate of cortical and deep nephrons was significantly less (P less than 0.001) after release of UUO. Although the percentage of filtering nephrons was significantly reduced in both nephron populations, the decline in glomerular filtration rate was greater in cortical than in juxtamedullary nephrons (cortical:juxtamedullary nephrons = 27.6 +/- 4.5% vs. 53.3 +/- 5.2% in controls, P less than 0.005) which suggests that single nephron glomerular filtration rate is redistributed to deep nephrons after release of UUO. In contrast to cortical nephrons, the amount of tubular fluid which remains near the bend of the loop of Henle of deep nephrons was greater after release of UUO. This appeared to be the result of a decrease in the reabsorption of both water (tubular fluid:plasma inulin = 2.41 +/- 0.16 vs. 7.94 +/- 0.69 in controls, P less than 0.001) and sodium (52.3 +/- 4% vs. 40.7 +/- 2.9% of the filtered sodium in controls, P less than 0.02). It is suggested that this altered reabsorption occurs along both the proximal tubule and descending limb of the loop of Henle of juxtamedullary nephrons. Inner medullary plasma flow (IMPF), as measured with the [125I]albumin-accumulation technique, was significantly depressed before release of UUO, but exceeded control values 90 min postrelease. Such changes imply that the filtration fraction of deep nephrons is decreased and that physical factors in the proximal tubular reabsorption of sodium have been altered. When papillary solute content was measured before release of UUO it was low (428 +/- 23 vs. 1,205 +/- 106 mosmol/kg in controls, P less than 0.001) which indicates that the decline in papillary osmolality is not a consequence of the increased IMPF seen after ureteral release, but rather precedes it. In fact, the decline in papillary osmolality may contribute to the increase in IMPF after release of UUO and to the decreased reabsorption of fluid along the descending limb of the loop of Henle.

Animals

Tubule-tubule and tubule-arteriole contacts in rat kidney distal nephrons. A morphologic study based on computer-assisted three-dimensional reconstructions.

BACKGROUND: Functional investigations of the tubulo-glomerular feedback mechanism have indicated the existence of a contact between the distal nephron and the macula densa region. The structural justification of such a contact is investigated. EXPERIMENTAL DESIGN: Tubule-tubule and tubule-arteriole contacts were investigated in distal nephrons from normal rat kidneys. Computer-assisted three-dimensional reconstructions of distal nephrons were made from serial sections of renal cortical tissue and selected sections were examined by electron microscopy. RESULTS: In 14 of 15 reconstructed nephrons, the distal convoluted tubule or the connecting tubule approached the macula densa region. A wall-to-wall contact between two tubules corresponding to a three-dimensional distance below 28 microns between the axes of the two tubules was found in only five of the reconstructed tubules. The distal nephron contacts to afferent and efferent arterioles of the same nephron were also examined. The efferent arteriole revealed no consistent contacts but the afferent arteriole contacted the distal convoluted tubule/connecting tubules consistently in all 10 of the superficial nephrons and in 3 of 5 midcortical nephrons. Electron microscopy confirmed a close contact between the distal tubule and the afferent arteriole in superficial nephrons and small nerves were often found at or near the site of contact, but the morphology at the site of contact was not unique. The arteriole contacts were made with late distal convoluted tubules, connecting tubules, or cortical collecting ducts. CONCLUSIONS: In conclusion, the present study shows that tubule-tubule contacts are inconsistent between the macula densa region and the distal nephron but that the tubule-afferent arteriole contact is consistent and close in superficial nephrons. This morphology is compatible with the existence of a feedback mechanism between the superficial distal nephron and the afferent arteriole, apart from the one located at the juxtaglomerular apparatus.

Animals

A basic study of the Hanssen technique for evaluation of single nephron glomerular filtration rate.

The purpose of this study was mainly to examine some basic problems of the Hanssen technique for the evaluation of single nephron glomerular filtration rate (SNGFR). Firstly, experiments were performed using Wistar rats to determine the amount of C14-ferrocyanide necessary to give sufficient C14 radioactivity in dissected nephrons. Using 200 muCi of C14-ferrocyanide, the samples of dissected nephrons provided only twice as much as background. Hence, we chose the amount of 600 to 700 muCi of C14-ferrocyanide for each rat in the later experiments, which yielded the radioactivity 5 to 6 times as much as background. Secondly, the homogeneity of intrarenal C14-ferrocyanide distribution was tested. Five superficial (SUP) and juxtamedullary (JM) proximal tubules four different regions were dissected and the radioactivity of those nephrons was counted. The distribution of C14-ferrocyanide in each piece was representative of the overal distribution. The summary of morphologic and clearance studies were as follows. The mean lengths of proximal tubules of 40 SUP nephrons, 5.3 +/- 0.2 mm, and 39 JM nephrons, 6.3 +/- 0.1 mm, were significantly different (P less than 0.01). The mean glomerular diameter of the JM nephrons, 110 +/- 2.7 mu, was significantly greater than that of the SUP nephrons, 95 +/- 3.1 mu (P less than 0.01). The mean SNGFR of 129 SUP nephrons, 23.4 +/- 4.2 nl/min/100g Body Weight (BW), and that of 130 JM nephrons, 34.5 +/- 5.1 nl/min/100g BW, were significantly different (P less than 0.01). The ratio of superficial/juxtamedullary nephron glomerular filtration rate (S/JM Ratio) was 0.85 +/- 0.02. We conclude that C14-ferrocyanide is regularly distributed to the kidney and that it is necessary to infuse sufficient amount of C14-ferrocyanide in this technique.

Animals

Nephron functional heterogeneity in the postobstructive kidney.

The purpose of this study was to determine the distribution of nephron filtration rates between superficial and juxtamedullary nephrons (S/J ratio) in kidneys studied immediately after relief of 24 hr total obstruction (acute) and after relief of prolonged partial obstruction (chronic). Injection of 14C-ferrocyanide and microdissection (modified Hanssen's technique) was used to provide an index of superficial and deep nephron glomerular filtration rate (GFR), and standard clearance determinations were done. In normal kidneys S/J ratio of 14C content (nephron GFR) was 0.73 plus or minus 0.03, a value similar to those obtained by other workers. After relief of acute obstruction, nephron GFR was too low for accurate measurement in 22% of superficial and 13% of deep nephrons. The mean S/J ratio of 14C content was similar to control, being 0.67 plus or minus 0.07, with only three of seven kidney showing loss of the normal S/J ratio. Since redistribution of nephron GFR was an inconsistent finding, while marked diuresis and natriuresis occurred in all rats, it appears that redistribution of nephron GFR is not an important factor in the phenomenon of postobstructive diuresis. After relief of chronic obstruction, diuresis and natriuresis were less marked but the mean S/J ratio of 14C content was 0.95 plus or minus 0.11, and in the majority of kidneys (six of eight), there was loss of distinction between superficial and deep nephron GFR. This redistribution of nephron function after relief of chronic, rather than acute, obstruction may be due to the more severe structural damage to the renal medulla of such kidneys and, although not responsible for postobstructive diuresis, it may be important in the diminished capacity of the chronically hydronephrotic kidney to conserve salt and water.

Acute Disease

Renal potassium transport: contributions of individual nephron segments and populations.

General features of the processes that contribute to renal potassium excretion are understood from clearance, stop-flow, micropuncture, and in vitro microperfusion experiments. However, the complex architecture of the kidney has made it difficult to examine individual nephron segments in all parts of the kidney. Accordingly, the extent to which distinguishable nephron populations, such as superficial and deep, may differ in their contributions to overall potassium excretion are not known. Also, the nature of transport processes across the successive segments of the nephrons (including not only the underlying cellular mechanisms, but even the direction of transport) is not known for all segments in any one nephron population. Excreted potassium is derived both from filtered potassium that escapes reabsorption and from secreted potassium. The filtered portion is large in amphibians and may be larger than generally recognized in mammals. The remainder is secreted primarily by distal nephron segments (distal tubule and cortical collecting duct). Potassium is also secreted into descending limbs of Henle loops; apparently this fraction is recycled from collecting ducts, and so does not represent an additional quantity of potassium transferred from blood to tubule fluid. Systemic factors that affect potassium excretion (potassium intake, sodium chloride intake, mineralocorticoid hormone levels, acid-base balance, and diuretic treatments) do so by modifying the net uptake of potassium from blood to cell and by altering the rate of fluid flow through the distal nephron. Under most circumstances, the distal nephron in the cortex appears to secrete potassium and the medullary collecting duct reabsorbs potassium. Although it is clear that successive nephron segments transport potassium in different ways, evidence to date does not indicate that potassium is handled differently by superficial nephrons compared to nephrons whose glomeruli lie in the deeper levels of the cortex.

Acid-Base Equilibrium

Immunomorphometric studies of proteinuria in individual nephrons of rats.

BACKGROUND: Heterogeneity of proteinuria among nephrons has been directly shown by micropuncture analysis of experimental models. Since the number of nephrons per kidney that are accessible for micropuncture is very small, we have developed a new immunomorphometric method for directly studying proteinuria in a much larger number of individual nephrons. This new method is based on the luminal surfaces of thick ascending limb of the loop of Henle (TAL) cells collectively functioning as an immunoabsorbent column for anti-Tamm-Horsfall protein antibodies that enter the urinary filtrate of that nephron. EXPERIMENTAL DESIGN: The distribution of luminal IgG deposits formed in TALs after injection of anti-Tamm-Horsfall protein antibodies was studied in three models of experimental proteinuria in rats to define the relationship between the formation of luminal deposits and the overall level of albuminuria and to test the utility of this method in analyzing the heterogeneity of luminal deposits among nephrons in other models. RESULTS: A close relationship between the magnitude of albuminuria and the distances that luminal IgG deposits extended along the straight course of TALs in individual nephrons of rats injected with rabbit anti-Tamm-Horsfall protein antibodies was established in a model of proteinuria with a uniform pattern of glomerular hemodynamics, heterologous immune complex nephropathy in rats. In models with more heterogeneous glomerular hemodynamics, autologous immune complex nephropathy and aminonucleoside nephrosis, greater heterogeneity in luminal IgG deposit formation among nephrons was demonstrated. The distances that luminal IgG deposits extended along TALs was more variable in these models than in heterologous immune complex nephropathy rats with comparable levels of albuminuria. CONCLUSIONS: Variability in the distances that luminal IgG deposits extend along TALs reflects heterogeneity among nephrons. The luminal deposit technique provides a new means for direct analysis of variability of dysfunction in many individual nephrons not accessible to micropuncture.

Animals

Phosphate transport in superficial and deep nephrons in phosphate-loaded rats.

We tested the hypothesis that greater phosphate delivery from deep nephrons than from superficial nephrons contributes to the addition of phosphate to the collecting system during phosphate loading. In the first group of eight anesthetized Munich-Wistar rats infused with phosphate and parathyroid hormone (PTH), fractional delivery of phosphate (FDP%) from superficial distal tubules was 56 +/- 6%, significantly less than the amount appearing in the urine, 67 +/- 6% (P less than 0.01). In the second group of six rats, we determined whether this addition of phosphate could be accounted for by a higher FDP% from the deep nephrons. Free-flow micropuncture collections were taken from deep nephrons (ascending limb of the loop of Henle in the papilla), superficial nephrons (distal tubules in the cortex), and urine (duct of Bellini). The FDP% to the ascending limb of the loop of Henle in deep nephrons was 78 +/- 10%, significantly greater than to the distal convoluted tubules in superficial nephrons, 51 +/- 6% (P less than 0.005), and the fractional excretion of phosphate in urine, 72 +/- 10% (P less than 0.05). Although a difference between FDP% in superficial and deep nephrons due to reabsorption in the ascending limb of the loop of Henle cannot be ruled out from the present data, other studies indicate that this interpretation is unlikely. We conclude that greater phosphate delivery by deep nephrons contributes to the addition of phosphate to the collecting system of phosphate-loaded rats.

Animals

The development of various generations of nephrons during postnatal ontogenesis in the rat.

The structural development of individual nephrons has been investigated in the kidneys of young rats by microdissection. Superficial, intercortical and juxtamedullary nephrons in Wistar-Konarovice rats aged 2 to 60 days have been followed. At two and five days of age the superficial nephrons were in an early stage of anatomical development. At this time intercortical and juxtamedullary nephrons were more developed, and basically resembled similar structures in adult animals with the exception that the loops of Henle did not have a thin ascending limb. At ten days of age all three types of nephrons were differentiated with the exception that the intercortical nephrons did not possess a thin ascending limb of Henle's loop. At selected stages of development there appears to be a direct relation between the length of the superficial nephrons and the weight of the kidney. Since individual segments of these nephrons do not grow at the same proportional rate, this relationship does not hold when compared to kidney weight.

Age Factors

Development of the metanephros in the chick: maturation of glomerular size and nephron length.

In the avian kidney three nephron types may be identified: mammalian-type nephrons with long (MTN-I) and short (MTN-II) loops of Henlé, and reptilian-type nephrons (RTN). By the method of microdissection the maturation of the nephrons of domestic fowl kidney has been studied. From the 14th day of incubation it is possible to isolate the MTN-I which appear first; all three nephron types may be isolated after 18 days of incubation. The thin limb of Henlé's loop in MTN-I appears after hatching, and the length of this segment in 1-day-old chicks is 0.1-0.13 mm. In 60-day-old chicks its length has approximately doubled. The transition of the thin segment to the thick segment is situated in the descending part of Henlé's loop. In the course of development, the relative length of all nephron segments in MTN-I increases uniformly, while in MTN-II and in RTN the relative length of the proximal and distal convoluted tubules increases. At all stages of development, MTN-I are the longest among the three groups of nephrons and have the largest glomeruli. The comparison between developing avian and mammalian kidneys shows great similarities in the process of maturation in analogous nephron types in these two classes of vertebrates.

Animals

Nephron obstruction in nordihydroguaiaretic acid-induced renal cystic disease.

Studies were performed to characterize conditions in rat kidneys whose nephrons were made cystic by feeding 2% nordihydroguaiaretic acid (NDGA) to the animals. Using two micropipettes, we monitored intratubular hydrostatic pressures while perfusing single surface nephrons in NDGA-exposed (5 to 7 weeks) and normal rat kidneys. The introduction of 50 nl of Ringers solution labeled with 3H-inulin at a flow rate of 25 nl/min was associated with a significant mean (+/- SEM) increase (167 +/- 61%; P less than 0.02) in pressure in cystic but not in nondilated (-0.5 +/- 27.2%) or normal (31 +/- 23%) nephrons, respectively. The relative amount of 3H-inulin excreted in 40 min from cystic (4.0 +/- 2.0%) was less than that excreted from either nondilated (19 +/- 7%; P less than 0.05) or normal (105 +/- 26%; P less than 0.01) nephrons. Intralumenal pressures in nondilated but not other nephron groups correlated with urinary flow rates (r = 0.51; P less than 0.02). Single nephron filtration rates and tubular-fluid-to-plasma 3H-inulin rations in additional rats were similar among all groups of tubules. Concluding that these data reflected increased resistance to outflow from cystic nephrons, we examined these and additional NDGA-exposed (1 to 24 weeks) kidneys. 3H-thymidine radioautography demonstrated maximum collecting tubular cell hyperplasia (13% labeling) at 2 to 3 weeks of NDGA-exposure. Microscopy and microdissection demonstrated tiny mural polyps along outer medullary segments of collecting tubules. Thirteen tubules were traced to their outlets; polyps impinging on outflow lumens were found in all 13 instances. We conclude that partial nephron obstruction exists in NDGA-exposed kidneys and that obstruction is a likely contributor to cyst formation in this model.

Animals

The effect of intrauterine growth retardation on the development of renal nephrons.

OBJECTIVE: To investigate the effect of Type II (asymmetrical) intrauterine growth retardation (IUGR) on renal development. DESIGN: A prospective descriptive study. SETTING: Department of Fetal and Infant Pathology, Liverpool Children's Hospital. SUBJECTS: Six (severely) affected IUGR stillbirths of known gestational age with a control group of stillbirths with birthweight greater than 10th centile, and eight liveborn IUGR infants who died within a year of birth with a control group of appropriately grown infants who died within a year of birth (postnatal groups). TECHNIQUES: The kidneys from all the groups studied were analysed using unbiased, reproducible and objective design-based stereological techniques. MAIN OUTCOME MEASURES: Total renal nephron (glomerular) numbers and average volumes of total nephron and cortical and medullary nephron segments. RESULTS: Nephron number estimates lay below the control group's 5% prediction limit in five out of the six growth-retarded stillbirths, and were significantly (P less than 0.005, IUGR at 65% of the control mean) reduced in the postnatal group. Estimates of nephron (segment) volume did not differ between control and IUGR groups. CONCLUSIONS: Type II intrauterine growth retardation may exert a profound effect on renal development. The reduced nephron number at birth, together with the lack of any early postnatal compensation in either nephron number or nephron size, emphasizes the need for vigorous antenatal surveillance for IUGR and consideration of elective preterm delivery of affected fetuses. A systematic review of other organs, which develop in a similarly rapid fashion during the late intrauterine period, is indicated by this work. With one exception, all birthweights in the growth-retarded groups were below the third centile, thus the precise quantitative relation between progressive IUGR and renal function requires further assessment.

Birth Weight