Differential activation of CD95-mediated apoptosis related proteins in proximal and distal tubules during rat renal development.

The CD95-mediated apoptotic pathway is the best characterized of the death receptor-mediated apoptotic pathways. The present study characterized localization and expression of proteins involved in CD95-mediated apoptosis during rat renal development. Kidneys were obtained from embryonic (E) 18 and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups. Immunohistochemical characterization revealed that CD95, FasL and cleaved caspase-3 were strongly expressed in proximal tubules and weakly expressed in distal tubules, but that expression of caspase-8 in distal tubules was stronger than that in proximal tubules. Results from terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that levels of apoptosis in proximal tubules slowly increased after E18, while those of distal tubules slowly decreased after P5. Western blotting demonstrated that expression of CD95, FasL and FADD was very weak during embryonic development, but rapidly increased at P14. Expression of cleaved caspase-3 was maintained at high levels after P1, while caspase-8 expression gradually reached a peak at P7. Results from this study reveal that the CD95-mediated apoptotic pathway is a key driver of apoptosis in proximal tubules during late postnatal kidney development in rats and suggest that apoptosis in distal tubules is mediated by a different apoptotic pathway.

[Morphometric parameters of nutria kidney structures in postnatal ontogeny].

Data on the morphometric parameters of the renal corpuscle, renal tubules, and collecting ducts of male and female nutrias in postnatal ontogenesis were obtained. It was found that the area of the renal corpuscle, glomerulus, the cavity and lumen of the capsule, and the proximal tubule diameter in the right and left kidney of female and male nutrias in the first year of life increase. The distal tubule diameter also increases; however, the dynamics of its changes becomes sinuous after 4.5 months. The collecting duct diameter varies depending on gender, age, and renal topography. The nuclear-cytoplasmic ratio in the cells of proximal and distal tubules and collecting ducts changes in a sinuous manner and depends on the gender and age of nutrias. The minimum mean value of the nuclear-cytoplasmic ratio was found in the proximal tubule cells in the left kidney of 12-month-old female nutrias (0.162 ± 0.002), and the maximum value was found in the distal tubule cells in the left kidney of newborn male nutrias (0.435 ± 0.007).

Expression of Bcl-2 and Bax in mouse renal tubules during kidney development.

Bcl-2 and Bax play an important role in apoptosis regulation, as well as in cell adhesion and migration during kidney morphogenesis, which is structurally and functionally related to mitochondria. In order to elucidate the role of Bcl-2 and Bax during kidney development, it is essential to establish the exact location of their expression in the kidney. The present study localized their expression during kidney development. Kidneys from embryonic (E) 16-, 17-, 18-day-old mouse fetuses, and postnatal (P) 1-, 3-, 5-, 7-, 14-, 21-day-old pups were embedded in Epon. Semi-thin serial sections from two E17 kidneys underwent computer assisted 3D tubule tracing. The tracing was combined with a newly developed immunohistochemical technique, which enables immunohistochemistry on glutaraldehyde fixated plastic embedded sections. Thereby, the microstructure could be described in detail, and the immunochemistry can be performed using exactly the same sections. The study showed that Bcl-2 and Bax were strongly expressed in mature proximal convoluted tubules at all time points, less strongly expressed in proximal straight tubules, and only weakly in immature proximal tubules and distal tubules. No expression was detected in ureteric bud and other earlier developing structures, such as comma bodies, S shaped bodies, glomeruli, etc. Tubules expressing Bcl-2 only were occasionally observed. The present study showed that, during kidney development, Bcl-2 and Bax are expressed differently in the proximal and distal tubules, although these two tubule segments are almost equally equipped with mitochondria. The functional significance of the different expression of Bcl-2 and Bax in proximal and distal tubules is unknown. However, the findings of the present study suggest that the mitochondrial function differs between mature proximal tubules and in the rest of the tubules. The function of Bcl-2 and Bax during tubulogenesis still needs to be investigated.

Ontogeny of renal sodium transport.

One of the main functions of the adult kidney is to maintain a constant extracellular fluid balance. The adult kidney does this, by and large, by filtering a massive quantity of fluid and reabsorbing the solutes needed to maintain volume and electrolyte homeostasis, while leaving the waste products to be excreted in the urine. One of the most precisely regulated functions of the adult kidney is to maintain sodium balance. The challenge of the neonatal kidney is even greater. It must maintain a positive salt balance for growth while the neonate is fed a diet that is very low in sodium. This review focuses on how the neonatal kidney reabsorbs NaCl with a special emphasis on the differences between the neonatal and adult kidney.

Ontogeny of NO synthase and renin in juxtaglomerular apparatus of rat kidneys.

The presence of NO synthase (NOS) in cells of the macula densa (MD) suggests a role for arginine-derived NO in tubulovascular information transfer. To investigate the postnatal development of the neuronal isoform of NOS and of renin in the kidney, the cellular distribution of these enzymes was examined in perfusion-fixed kidneys of 2-, 6-, and 15-day-old rats at both the protein and mRNA level (n = 4 rats/group). NOS and renin and their mRNAs were localized by immunohistochemical and in situ hybridization methods. In addition, NOS levels were assessed by using NADPH diaphorase (NADPH-d) histochemistry. For quantification, the fraction of NOS- and renin-positive glomeruli as well as the number of NOS-positive MD cells was evaluated at all stages. Presence of NOS in single cells of the developing distal tubule was encountered already in the S-shaped body. Full expression of a NOS signal in MD cells was seen as soon as a glomerular urinary space was developed. Double labeling with NADPH-d and antibody to Tamm-Horsfall protein (THP) indicated mutual exclusiveness of NADPH-d-positive MD cells and neighboring THP-positive distal tubule cells at all levels of development. The relative intensity of renin status was 2 day > 6 day > 15 day, whereas NOS expression was maximal on postnatal day 6. Our data are consistent with an involvement of MD NO synthesis in the early organization of the juxtaglomerular apparatus during nephrogenesis and suggest an interdependent relation with renin-producing cells.

Epidermal growth factor in the neonatal mouse salivary gland and kidney.

In the adult mouse, epidermal growth factor (EGF) is synthesized in granular convoluted tubule (intralobular) duct cells of the submandibular gland and in distal tubule cells of the kidney. The presence of EGF in developing tissues and maternal milk and the localization of EGF receptors in developing tissues suggest a role for EGF in developmental processes. The primary aims of the present study were to: (1) localize EGF and EGF-binding sites in the kidney and submandibular gland during neonatal development and (2) to determine the effect of exogenously administered EGF on cell proliferation in these two developing organs. In the present study, EGF was localized by immunocytochemistry in granular convoluted tubule cells of the submandibular gland initially on day 21 after birth and in distal tubule cells of the kidney on postnatal day 6. EGF binding in the kidney decreased after birth with some localization to the glomerulus. In submandibular glands of newborn and 10-day-old mice, EGF-binding sites were associated with both acinar and duct cells with peak binding at 10 days postnatally. Submandibular glands from 20-day-old mice demonstrated primarily ductal EGF-binding sites. Exogenously administered EGF induced a mitogenic response in acinar and interlobular duct cells of submandibular glands during the first week after birth. EGF treatment during this period had an inhibitory effect on 3H-thymidine incorporation into cellular compartments in the developing kidney. The identification of EGF-binding sites in the kidney and submandibular gland before the presence of EGF suggests that an EGF-like molecule such as transforming growth factor alpha (TGF-alpha) may be present as a potential ligand in these organs. In order to assess this possibility, developing kidneys and submandibular glands were stained with anti-TGF-alpha. These immunocytochemical studies localized TGF-alpha to the proximal tubule of the kidney and immature acinar cells of the newborn mouse. Our data strongly support an autocrine, juxtacrine or paracrine role for EGF and/or TGF-alpha in the regulation of cell proliferation and cytodifferentiation in the kidney and submandibular gland.

Development of the urinary system of the marsupial native cat Dasyurus hallucatus.

The development of the mesonephros and metanephros was examined in the marsupial Northern native cat, Dasyurus hallucatus, from birth through to the end of lactation. The mesonephros was present at birth, reached a maximum volume 11 days after birth and had regressed completely by day 30. The metanephros was present on day 2; glomeruli were first seen on day 8, and nephrogenesis continued until day 89 post partum. The newborn native cat is at a very primitive stage of development compared to other marsupials. However, at weaning, like other marsupial species, the native cat has developed a fully morphological and functional urinary system.

Renal reabsorption of phosphate during development: tubular events.

Studies performed in our laboratory on the isolated perfused kidney of the guinea pig have demonstrated that the rate of Pi reabsorption is substantially greater in the newborn than in the adult, when appropriate corrections are being made either for differences in glomerular filtration rate (GFR) or in renal tubular mass. In order to determine the location of this enhanced reabsorption along the nephron, micropuncture experiments were performed on euvolemic, non-fasted guinea pigs 5-14 and 42-49 days of age, maintained on standard guinea-pig chow diet (0.76% Pi). Concomitant measurements of overall kidney function were also obtained. The results confirmed that fractional reabsorption of Pi (TRPi%) across the entire kidney was significantly higher (P less than 0.01) in the newborn (89.93 +/- 2.55%) than in the adult (78.25 +/- 2.89%) animals. The difference was also significant (P less than 0.05) when TRPi was expressed in mol/ml GFR (1.87 +/- 0.14 vs 1.53 +/- 0.12, respectively). At comparable locations along the proximal tubule (TF/Pin of 1.90 +/- 0.16 in the newborn, and 1.79 +/- 0.15 in the adult, P greater than 0.70), the fraction of the filtered load of Pi reabsorbed was significantly higher (P less than 0.001) in the immature (76.66 +/- 2.74%) than in the mature (67.21 +/- 2.74%) guinea pigs. Estimates based on the differences between proximal Pi reabsorption and the urinary excretion of Pi indicate that the reabsorption of Pi in tubular segments located beyond the proximal tubule is also enhanced in the newborn when compared with the adult (15.62 +/- 2.11% vs 10.51 +/- 1.83%, respectively, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological study of cell organelles during development. I - The nuclear sac and the endoplasmic reticulum on the rat nephron.

The maturation of the endoplasmic reticulum (ER) of rat kidney tubule cells was studied with an osmium impregnation technique. Thick sections (0.3-0.6 micron) of kidney tissue were made after a five-day impregnation with osmium tetroxide and examined by standard transmission electron microscopy at 80-100 kV. Studies were performed on rat foetuses from 18-21 days of gestation, on newborns, and on 2-20 day old animals. At the undifferentiated stage, only a small percentage of the tubule cells were impregnated; in these, the perinuclear sac was stained and a few nuclear pores were already seen. Rudimentary, but thick canalicular projections seemed to originate from the perinuclear sac and become more extensive with maturity. Flattened saccules appeared later and fenestrations were seen in proximal tubule cells only when they seemed to have reached their functional specialization. In some cells, only the Golgi apparatus was stained. In the distal tubule cells, there was also progressive formation of a network consisting first of canaliculi and later of saccules which were rarely fenestrated. The osmium impregnation technique appears to be useful as an index of the ER organization development.

The differentiation of proximal and distal tubules in the male rat kidney: the appearance of aldolase isozymes, aminopeptidase and alkaline phosphatase during ontogeny.

1. The specific activities of aminopeptidase, alkaline phosphatase and aldolase isozymes were measured in homogenates of kidneys taken at different stages of ontogeny. The cellular localization of these enzymes was studied in cryostat tissue sections using substrate linked assays for aminopeptidase and alkaline phosphatase and the mixed aggregation immuno-cytochemical technique for aldolase isozymes; local enzyme concentrations were estimated photometrically. 2. The presence of both aldolase-A and aldolase-B was demonstrated in all metanephrogenic cells (and at still higher concentrations in collecting tubule cells) of the rat fetus 16 days after conception and in the undifferentiated cells of the neogenetic zone of kidney up to 8 days after birth; no aminopeptidase or alkaline phosphatase could be found in these cells. 3. Measurements made on stained tissue sections show that the shift towards aldolase-B, seen in homogenate analyses, is due to a change in the relative amounts of proximal tubules. No evidence was seen for repression in the synthesis of aldolase-A or aldolase-B monomers in the different kidney cells during ontogeny. 4. Two transitions in the mode of nephron differentiation were observed: one was shortly after birth, the other followed weaning. Before the first transition the concentrations of the enzymes increased to different degrees, such that the enzymes reached concentrations comparable with those as in the cells of adult rats by 2 to 4 days post partum. After the second transition proximal tubule size and specific activity of brush border membrane enzymes increased 3 fold. In contrast, the distal tubules did not increase significantly in size, but their aldolase-A concentration increased 3 fold. 5. Evidence based on enzyme quantification and morphometry in kidney sections is presented to demonstrate that the proximal tubule cells show functional adaptation by two independent mechanisms: specific amplification of gene expression and hypertrophy. In contrast, the distal tubule shows functional adaptation only by specific amplification of gene expression.

A micropuncture study of the development of renal function in the young rat.

Total kidney function and function of individual surface nephrons were measured under hypotonic saline load conditions in young rats 22-, 30- and 42-days old. The highest values of urine flow rate and GFR were found in 30-day-old rats. The water reabsorption was higher in 42-day-old rats than in two younger groups. This decreased fractional reabsorption of water in younger animals was detectable already in the late proximal tubule and in the early distal tubule. Fractional reabsorption of sodium was significantly lower at the age of 22 and 30 days than at the age of 42 days in both late proximal tubule and in the early distal one.