Formation Of New Nephrons Research Articles

LIM kinase function and renal growth: Potential role for LIM kinases in fetal programming of kidney development

AimsMaternal dietary restriction during pregnancy impairs nephron development and results in offspring with fewer nephrons. Cell turnover in the early developing kidney is altered by exposure to maternal dietary restriction and may be regulated by the LIM-kinase family of enzymes. We set out to establish whether disturbance of LIM-kinase activity might play a role in the impairment of nephron formation. Main methodsE12.5 metanephric kidneys and HK2 cells were grown in culture with the pharmacological LIM-kinase inhibitor BMS5. Organs were injected with DiI, imaged and cell numbers measured over 48h to assess growth. Cells undergoing mitosis were visualised by pH3 labelling. Key findingsGrowth of cultured kidneys reduced to 83% of controls after exposure to BMS5 and final cell number to 25% of control levels after 48h. Whilst control and BMS5 treated organs showed cells undergoing mitosis (100±11 cells/field vs 113±18 cells/field respectively) the proportion in anaphase was considerably diminished with BMS5 treatment (7.8±0.8% vs 0.8±0.6% respectively; P<0.01). This was consistent with effects on HK2 cells highlighting a severe impact of BMS5 on formation of the mitotic spindle and centriole positioning. DiI labelled cells migrated in 100% of control cultures vs 0% BMS5 treated organs. The number of nephrogenic precursor cells appeared depleted in whole organs and formation of new nephrons was blocked by exposure to BMS5. SignificancePharmacological blockade of LIM-kinase function in the early developing kidney results in failure of renal development. This is likely due to prevention of dividing cells from completion of mitosis with their resultant loss.

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury.

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Epigenetic States of nephron progenitors and epithelial differentiation.

In mammals, formation of new nephrons ends perinatally due to consumption of mesenchymal progenitor cells. Premature depletion of progenitors due to prematurity or postnatal loss of nephrons due to injury causes chronic kidney disease and hypertension. Intensive efforts are currently invested in designing regenerative strategies to form new nephron progenitors from pluripotent cells, which upon further differentiation provide a potential source of new nephrons. To know if reprogramed renal cells can maintain their identity and fate requires knowledge of the epigenetic states of native nephron progenitors and their progeny. In this article, we summarize current knowledge and gaps in the epigenomic landscape of the developing kidney. We now know that Pax2/PTIP/H3K4 methyltransferase activity provides the initial epigenetic specification signal to the metanephric mesenchyme. During nephrogenesis, the cap mesenchyme housing nephron progenitors is enriched in bivalent chromatin marks; as tubulogenesis proceeds, the tubular epithelium acquires H3K79me2. The latter mark is uniquely induced during epithelial differentiation. Analysis of histone landscapes in clonal metanephric mesenchyme cell lines and in Wilms tumor and normal fetal kidney has revealed that promoters of poised nephrogenesis genes carry bivalent histone signatures in progenitors. Differentiation or stimulation of Wnt signaling promotes resolution of bivalency; this does not occur in Wilms tumor cells consistent with their developmental arrest. The use of small cell number ChIP-Seq should facilitate the characterization of the chromatin landscape of the metanephric mesenchyme and various nephron compartments during nephrogenesis. Only then we will know if stem and somatic cell reprogramming into kidney progenitors recapitulates normal development.

Compensatory renal growth after unilateral or subtotal nephrectomy in the ovine fetus

Clinical and experimental studies show that unilateral (1/2Nx) and subtotal nephrectomy (5/6Nx) in adults result in compensatory renal growth without formation of new nephrons. During nephrogenesis, the response to renal mass reduction has not been fully investigated. Ovine fetuses underwent 1/2Nx, 5/6Nx, or sham surgery (sham) at 70 d of gestation (term: 150 d), when nephrogenesis is active. At 134 d, renal function was determined, fetuses were killed, and kidneys were further analyzed at the cellular and molecular levels. Additional fetuses subjected to 5/6Nx were killed at 80 and 90 d of gestation to investigate the kinetics of the renal compensatory process. At 134 d, in 1/2Nx, a significant increase in kidney weight and estimated glomerular number was observed. In 5/6Nx, the early and marked catch-up in kidney weight and estimated glomerular number was associated with a striking butterfly-like remodeling of the kidney that developed within the first 10 d following nephrectomy. In all groups, in utero glomerular filtration rates were similar. Compensatory renal growth was observed after parenchymal reduction in both models; however, the resulting compensatory growth was strikingly different. After 5/6Nx, the remnant kidney displayed a butterfly-like remodeling, and glomerular number was restored.

Morphology, development and comparative anatomical evaluation of the testicular excretory pathway in Acipenser

In the differentiated state, the testicular excurrent duct system of the sturgeon begins as a longitudinal marginal network of the testis, extending along the entire length of the male gonad. From here, mesorchial transversal ducts travel to the ventral aspect of the pars sexualis of the opisthonephros where they merge behind the dorsal coelomic wall to form the longitudinal marginal network of the kidney. Then, the seminal pathways enter the confines of the pars sexualis of the opisthonephros and divide into a complicated, multipartite system consisting of (1) centropapillary ducts, situated in the center of a group of urinary collecting ducts, (2) lacunary basal sinuses, located on the bases of opisthonephric columns and (3) intracolumnar ducts running inside the renal columns, the latter representing typical functional units of the adult sturgeon kidney. The contacts between intracolumnar ducts and the vascular poles of corresponding renal corpuscles represent the urogenital junction in the sturgeon. The nephrons of the pars sexualis involved in sperm transport do not lose their urinary functions, but are histologically identical to those of the pars excretoria which are solely urinary. The opisthonephros of sturgeons grows continuously by the formation of new nephrons from an opisthonephric blastema located on the base of each renal column. A close topographical association between this blastema tissue and the lacunary basal sinuses of the testicular excurrent duct system guarantees that new renal corpuscles in the pars sexualis are included in the seminal passage from their beginning. From the urogenital junctions, on their way to the exterior, the spermatozoa have to travel through Bowman's capsules and tubules of the nephrons involved, then through the urinary collecting ducts, the wolffian duct and finally the sinus urogenitalis. The development of the testicular excurrent duct system begins in 8-month-old animals in the pregonadal area of the gonadal fold. Here, a primary gonoductal blastema proliferates to form a longitudinal network of anastomosing strands, situated in the dorsal mesogonadal attachment. From this primary longitudinal network, small tubules grow into the direction of the opisthonephros and into the direction of the testis. In the period from 8 to 18 months, the testicular excurent duct system reaches the adult state. In conclusion, the testicular excurrent ducts of sturgeons initially develop similar to those of Polypterus and in modern teleosts from a primary longitudinal system, beginning in the pregonadal area, localized in the mesogonadal attachment and extending caudally. Then, in a second step of development, the phylogenetically older situation, using parts of the kidney as passage, already seen in Chondrichthyes, but preserved also in higher vertebrates, is achieved in Acipenser. For this, seminal ducts grow into the opisthonephros and establish here the urogenital junctions with corresponding renal corpuscles. Furthermore, the initially longitudinally oriented ducts in the mesogonadal attachment partly lose their continuity and become integrated into the course of the transversal mesorchial ducts, represented by their portions with the widest lumina and the thickest walls.

Compensatory renal growth after unilateral nephrectomy in the ovine fetus.

Unilateral nephrectomy of the adult animal results in compensatory renal growth but does not involve formation of new nephrons. It is not clear whether compensatory growth can occur during the period of active nephrogenesis in utero and if so, whether more nephrons can be formed. Male ovine fetuses (n = 20) underwent unilateral nephrectomy (n = 10) or sham nephrectomy (n = 10) at 100 d of gestation (term, 150 d). After 27 to 34 d, ewes and fetuses were killed and the right kidney of each fetus was removed and weighed. The wet weight of the right kidney was greater in the unilaterally nephrectomized fetuses (16.3 +/- 1.3 g compared with 12.2 +/- 0.7 g; mean +/- SEM, P < 0.05) as was the kidney to body weight ratio (5.2 +/- 0.3 g/kg compared with 3.8 +/- 0.2 g/kg; P < 0.001). Nephron number in the right kidney was estimated by an unbiased stereologic technique. There was a 45% increase in the number of nephrons in the kidneys from unilaterally nephrectomized animals compared with the kidneys from sham-operated animals (530,763 +/- 37,136 nephrons in the unilaterally nephrectomized group compared with 365,672 +/- 36,016 nephrons in the sham-operated group; P < 0.01). Mean glomerular volume was lower in the unilaterally nephrectomized group; however, total glomerular volume per kidney was not different between groups. This study demonstrates that there is a significant amount of compensatory growth and nephron endowment in a remaining kidney after unilateral nephrectomy during the period of active nephrogenesis in the sheep. This is the first time such events have been shown to occur in utero.

Diagnosis of the human fetal age based on the development of the normal kidney

Background and aims: The diagnosis of human fetal age is usually estimated based on the measurement of crown-rump length or crown-heel length and the weight of the fetus. However, this estimate is not totally accurate and sometimes is necessary to combine other data to determine the fetal age. An analysis of the normal embryological development of the kidney may assist in this determination. The histology of this process, although well described, lacks photographic documentation. We intend to fill this gap by providing histologists and pathologists, especially inexperienced ones, with information about the staging of the renal development through microphotography. The objective of the present study was to achieve greater accuracy for the diagnosis of human fetal age through the proposed classification and the photographic documentation presented. Material and methods: Normal embryological development of the human kidney was studied by light microscopy. The fetal period from 6 to 40 weeks of gestation was observed according the stage of maturity of glomeruli and tubules; localization of glomeruli, occurrence of nephrogenic tissue and cortico-medullary differentiation. At least 5 different exams were observed from each week of development. Two hundred four exams were analyzed in the whole study. The histological characteristics were quantified and the process was documented by microphotography. Results and final considerations: The fetal development of the kidney was divided into 8 stages, which was documented through microphotography. Nephron structural formation occurred until the 34th week of prenatal development. From the 35th week on, tubules and glomeruli continued to mature without the formation of new nephrons. The proposed classification intends to improve the accuracy of the fetal age diagnosis.

Communications: Gentamicin Concentrations in Toadfish and Goldfish Serum

Abstract Previous work in our laboratory has demonstrated that goldfish Carassius auratus produce new nephrons in response to gentamicin-induced nephrotoxicity. The oyster toadfish Opsanus tau, however, does not undergo nephron neogenesis. To explore possible reasons for the differences in the formation of new nephrons, we evaluated the elimination of gentamicin from the serum of both species. Fish (20 toadfish and 20 goldfish) were given intramuscular injections of 3.5 mg gentamicin/kg of body weight or an equal volume of sterile water. Blood samples were taken by venipuncture from goldfish at days 1, 2, 3, and 4 postinjection and from toadfish at days 1, 2, 7, 14, 28, and 49 postinjection. Gentamicin levels were measured in serum via a competitive binding immunoassay. Gentamicin levels in serum declined rapidly in goldfish, reaching near baseline levels 4 d after the injection, with a half-life of 49.6 h for the β component. Toadfish had a much longer gentamicin retention time with a half-life of 602 h ...

Morphological and functional comparisons of normal and hypertrophied kidneys of adult domestic fowl (Gallus gallus)

Similar to mammals, kidneys of domestic fowl undergo compensatory hypertrophy after loss of functional renal mass. Because this species continues to develop new nephrons for up to 12-wk posthatch, renal hyperplasia might play a significant role in compensatory growth. Either transient or permanent loss of approximately 60% of the right kidney was produced in 2- to 3-wk-old roosters by simple ureteral transection or by removing a 1-mm segment of ureter at the level of the ischiadic artery, respectively. In the latter (experimental) group, right anterior and medial divisions atrophied leaving only the posterior division intact. Spontaneous reanastomosis occurred in the former (reconnected) group, and all three divisions were present at death. Control birds were untouched as were the left kidneys of experimental and reconnected birds. At 40-50 wk, renal function was measured separately in right and left kidneys of all groups during five different infusion protocols. Compared with control kidneys, experimental kidneys had a 50-60% weight gain, and their glomerular size distribution profile was shifted to the right (larger glomeruli). Reconnected kidneys were not hypertrophied, and their profile was shifted to the left (smaller glomeruli). Neither group had significant formation of new nephrons. Once variations in kidney weight were taken into account, there were no differences between hypertrophied (experimental) and control kidneys in urine flow rate (UFR), glomerular filtration rate (GFR), paraaminohippuric acid (PAH) clearance, UFR/GFR, urine osmolality, urine/plasma osmolality, osmolal clearance, free water clearance, Na and K load, absolute Na and K excretion, and fractional Na and K excretion except as follows: 1) during infusion of isotonic mannitol-dextrose at 0.1 ml.min-1.kg body wt-1 experimental kidneys had a lower fractional excretion of K than control kidneys, and 2) during brisk osmotic diuresis (isotonic mannitol-dextrose at 0.4 ml.min-1.kg body wt-1) experimental kidneys had higher UFR and free water clearance and lower urine osmolality and urine/plasma osmolality than control kidneys. Reconnected kidneys differed from control kidneys in only 1 of 210 comparisons. Permanent loss of functional renal mass in young birds produces significant compensatory renal hypertrophy that is due to enlargement of existing nephrons rather than formation of new nephrons. Hypertrophied kidneys function like normal kidneys except under conditions of brisk osmotic diuresis.

Maturation of glomerular size distribution profiles in domestic fowl (Gallus gallus)

Previous histological evaluations of chick kidneys indicated nephrons continue to develop from embryonic foci for up to 6 weeks after hatching. The present study was conducted using an in vivo alcian blue staining technique to quantify posthatch changes in glomerular numbers and sizes in female domestic fowl at 1, 3, 5, 9, 12, 21, and 30 weeks of age. Changes in glomerular size distributions reflect changes in the heterogeneous nephron populations of avian kidneys. Foci of embryonic tissue were observed at the periphery of renal lobules up to 12 weeks of age. Glomerular numbers increased from 69,800/kidney at 1 week to 586,000/kidney at 12 weeks, with no further significant increase up to 30 weeks (599,000/kidney). The increase in glomerular number per gram kidney weight remained constant as kidney mass increased up to 12 weeks of age, after which the number of glomeruli per gram kidney weight declined significantly as kidney size increased without further addition of new nephrons. Glomerular size distribution profiles were constructed using eleven circumference categories. The peak number of glomeruli fell within the 0.11-0.14 mm category at 1 and 3 weeks; within the 0.15-0.18 mm category at 5, 9, and 12 weeks; and within the 0.19-0.22 mm category at 21 and 30 weeks. One and 3-week-old chicks had no glomeruli within the largest (greater than or equal to 0.35 mm circumference) size categories, and 9-12-week-old birds had significantly fewer glomeruli in these categories than 21-30-week-old birds. These results demonstrate that posthatch renal maturation in domestic fowl involves the ongoing formation of new nephrons up to 12 weeks of age, with subsequent kidney growth (12-30 weeks of age) accomplished by enlargement of existing nephrons (nephron hypertrophy). The cumulative evidence indicates that nephrons destined to develop loops of Henle (mammalian-type) develop first, with shorter (reptilian-type) nephrons developing later as the kidneys enlarge.

Renin immunocytochemistry of the differentiating juxtaglomerular apparatus.

The differentiation of the juxtaglomerular apparatus in fetuses and newborn mice was investigated by renin immunocytochemistry and electron microscopy. Three to four days before delivery and prior to other organs renin was found in the fetal kidney. At this early time immunoreactivity was preferentially located in cells of the media of interlobular arteries. In newborn mice the formation of new nephrons and maturation of their glomeruli was accompanied by a shift in renin localization from the interlobular arteries to the afferent arterioles. At the same time, kidney renin content and concentration increased rapidly. Synchronously with renin immunoreactivity, during the capillary loop stage of glomerular development, granulated epitheloid cells became visible in the afferent arteriole.

THE EFFECT OF NORMAL DEVELOPMENT ON COMPENSATORY RENAL GROWTH

Rats were nephrectomized (Nx) or sham-operated (S) at the age of 5, 12 and 40 days. One group of rats was Nx in utero 3-4 days before delivery. Light microscopy studies of renal structural development were carried out in the postnatal Nx and S rats at the age of 6-26 days. Renal structural development followed the same pattern in Nx as in S rats. The formation of new nephrons was completed at the age of 6-7 days. There was no structural evidence of formation of new nephrons. Furthermore, glomerular counting showed the same number of glomeruli in Nx and S rats at the age of 60 days. The number of glomeruli in fetal nephrectomized rats was the same as in control animals from the same litter. GFR, SNGFR and kidney weight were estimated at 60 days of age in Nx and S rats. The compensatory increase in renal weight and GFR was most pronounced in rats Nx at the age of 5 days, i.e. just before the formation of nephrons was completed.The quotient between the recorded SNGFR and GFR was the same in all groups studied, indicating a homogenous increase in SNGFR of the nephrons at all cortical levels.

Effect of normal development on compensatory renal growth

Sprague-Dawley rats were nephrectomized unilaterally, or sham-operated at 5, 12, and 40 days of age. The kidney weight, number of glomeruli, GFR, and single nephron GFR (SNGFR) were determined in about 60 days of age. In both sham-operated and nephrectomized rats operated on at 5 and 12 days of age, the kidney was studied with light microscope for 1 to 14 days. Unilateral nephrectomy performed just before or just after completion of nephrogenesis or in adulthood did not result in formation of new nephrons. Renal structural development followed the same pattern in nephrectomized and sham-operated rats. A compensatory increase in renal size and GRF was inversely related to the age at which the nephrectomy was carried out but was more pronounced if nephrectomy had been performed just before rather than just after completion of nephrogenesis. In all nephrectomized rats, the compensatory increase in filtration rate appears to be the same in nephrons at all cortical levels judging by comparison of SNGRR and GFR. Conclusion. Compensatory renal growth does not involve the formation of new nephrons in the postnatal kidney of the rat. The potentiation of compensatory changes after nephrectomy at an early age is largely dependent on the development stage of the kidney at the time of nephrectomy.

Age-dependent characteristics of compensatory renal growth

The age-dependent characteristics of compensatory renal growth were studied in neonatal, weanling and young adult rats. Compensatory growth was demonstrated to be greater in young, sexually immature animals than in adults following unilateral nephrectomy. In the four-week interval following surgery in 21-day-old weanling rats, renal mass increased 240% in control animals (group A) and 407% after unilateral nephrectomy (group B). In similarly prepared adult animals, renal weight increased 31 and 81% in the same groups, respectively. The remarkable response in compensatory growth achieved by the young animal was further demonstrated by a greater absolute increase in renal mass in the experimental kidney of young rats compared to adults. Further studies were performed to determine whether the mechanism of compensatory growth in young rats involved formation of new nephrons. The total glomerular count in the remaining kidney of rats after removal of one kidney at 5 (neonatal) and 21 (weanling) days of life did not differ from the value obtained in control neonatal, weanling or adult kidneys. These data indicate that compensatory renal hypertrophy is age-dependent and is greater in young animals. The mechanism for enhanced compensatory renal growth in young animals does not involve glomerular neogenesis since there was no evidence for formation of additional nephrons following nephrectomy either in weanling or neonatal animals.

Influence of Increased Postnatal Nutrient Intake on Kidney Cellular Development in Progeny of Protein-deficient Rats

Kidney development was studied in the progeny of both control and protein-deficient rats from birth to 90 days after birth. Retarded development in the kidneys of deficient progeny was indicated by reduced weight, cell number and nephron number. Increased postnatal nutrient intake, achieved by raising pups in litters of four animals, resulted in an increase in kidney cell number which was temporary in the progeny of deficient dams. The increase in cell number occurred in existing nephrons and did not appear to involve the formation of new nephrons. An increase in proximal convoluted tubule length occurred during the suckling period in kidneys of deficient progeny raised in small litters compared to those raised in normal-sized litters. The data suggest that the progeny of protein-deficient rats show a permanent retardation in kidney development which is not reversed by increased postnatal nutrient intake. A deficit of nephron number is specifically involved.