Biomedical subjects

John F Bertram

Publications and source records attributed to John F Bertram.

36 records · Page 2Linked to original sources

PubMed2005

Exogenous BMP-4 amplifies asymmetric ureteric branching in the developing mouse kidney in vitro.

BACKGROUND: Exogenous bone morphogenetic protein 4 (BMP-4) has been reported to inhibit ureteric branching morphogenesis and regulate the anterior-posterior axis of the developing kidney in vitro. We examined the role of BMP-4 on ureteric branching in vitro using three-dimensional image analysis software and statistical models. Additionally, in vivo ureteric branching was analyzed and the effect of reduced levels of BMP-4 in vivo on nephron number was examined. METHODS: Embryonic day 12.5 (E12.5) Balb/c mouse metanephroi cultured for 48 hours with or without 260 ng/mL recombinant human BMP-4 (rhBMP-4) were immunostained to identify the ureteric epithelium which was quantified in three dimensions. In vivo ureteric branching morphogenesis in Hoxb7/GFP mice was also analyzed. The effect of reduced in vivo levels of BMP-4 on nephron number was examined in BMP-4(+/-) and wild-type mice using an unbiased stereologic method. RESULTS: Qualitative and quantitative studies identified a decrease in total ureteric length and branch number in wild-type mouse metanephroi cultured in the presence of BMP-4. A marked anterior-posterior asymmetry in both ureteric length and branch number was observed in BMP-4-treated metanephroi. A similar asymmetry was revealed in control metanephroi, both in vitro and in vivo. This asymmetry is the result of reduced ureteric branching morphogenesis in the posterior region of the kidney and appears to be due to slower growth rather than the adoption of an alternate branching pattern. Reduction of endogenous BMP-4 in BMP-4(+/-) mice resulted in no change in total nephron number in macroscopically normal kidneys. CONCLUSION: These results suggest that BMP-4 plays an important role in the regulation of ureteric branching morphogenesis, and that excess BMP-4 in vitro can amplify the existing asymmetry of the normal mouse kidney.

Animals↗

PubMed2005

The where, what and why of the developing renal stroma.

In recent years, a great deal has been learnt about the molecular regulation of kidney development. While most research has focused on the molecular regulation of ureteric branching morphogenesis and nephron formation, significant insights into the definition and functions of the renal stroma have emerged. Many molecules expressed in the developing renal stroma are now known to play significant regulatory roles in kidney development. However, the term 'renal stroma' continues to have different meanings to different researchers. This review clarifies this situation and defines the derivation, location and functions of the stroma in the developing metanephros.

Animals↗

PubMed2004

Does a nephron deficit in rats predispose to salt-sensitive hypertension?

AIM: This study tested the hypothesis that a nephron deficit predisposes rats to salt-sensitive hypertension in adulthood. METHODS: Female Wistar-Kyoto rats were fed a low (9%) or a normal (20%) protein diet during pregnancy and lactation. Male, birth-weight-matched offspring were paired. One rat from each pair was perfusion fixed at 4 weeks of age and the other rat at 40 weeks of age. Kidneys were removed and nephron number and total renal filtration surface area (FSA) determined using unbiased stereological techniques. The rats that were allowed to grow to adulthood had tail-cuff systolic blood pressure and body weight determined twice weekly. Between 30 and 40 weeks of age, a normal or a high-salt diet was fed to the rats. RESULTS: The offspring of rats fed the low-protein diet were significantly smaller at birth, and at 4 weeks of age they had a significant reduction in kidney volume, nephron number, and total renal FSA when compared to controls. Tail-cuff systolic blood pressure in the offspring from 4 to 29 weeks of age did not significantly differ between the two groups. Administration of a high-salt diet from 30 to 40 weeks of age led to a significant increase in blood pressure in both dietary treatment groups; however, it was not exacerbated in the rats exposed to the low-protein diet in utero. CONCLUSIONS: Maternal protein restriction in rats did not lead to salt-sensitive hypertension. Nephron endowment and FSA did not correlate with blood pressure in adulthood.

Animals↗

PubMed2004

Nephron endowment and blood pressure: what do we really know?

It has been hypothesized that a reduced number of nephrons at birth contributes to the development of essential hypertension. Nephron number in normal human kidneys has been shown to vary up to eightfold. Therefore, a significant proportion of the population appears to be at risk for developing hypertension. Furthermore, nephron deficits might explain why some racial groups have a higher incidence of hypertension and end-stage renal disease than others. Animal studies have demonstrated that maternal limitations in nutrient supply, both gross and nutrient-specific; exposure to elevated levels of hormones or toxins; and genetic factors can lead to permanent deficits in nephron number and, when examined, elevated blood pressure. In this review, maternal and genetic factors influencing nephron endowment and the implications of nephron deficit for hypertension and renal disease in humans are discussed.

Animals↗

PubMed2004

Cardiovascular hypertrophy in one-kidney, one-clip renal hypertension is resistant to heparin.

OBJECTIVE: Heparin inhibits vascular hypertrophy in angiotensin-induced hypertension, in addition to its well-known role in inhibiting injury-induced vascular smooth muscle proliferation. We tested whether hypertension and vascular hypertrophy could be reduced by heparin independently from the renin-angiotensin system. METHODS: Rats were made hypertensive with a one-kidney, one-clip (1K1C) procedure and received heparin from osmotic minipumps (0.3 mg/h per kg i.v.) or saline vehicle for 2 weeks. Blood pressure was measured by the tail-cuff method and vessel cross-sectional area was measured by morphometry in the aorta and mesenteric arteries. Proliferation was assessed with bromodeoxyuridine labelling. RESULTS: Blood pressure elevation and cardiovascular hypertrophy were evident in 1K1C rats. The media of mesenteric arteries was increased by 25%, and the media : lumen ratio by 35%, in hypertensive rats. DNA synthesis by smooth muscle cells in the mesenteric arteries was increased sevenfold in renal hypertension. Heparin treatment did not influence either the increase in blood pressure, the cardiovascular hypertrophy response or hypertension-mediated proliferation of arterial smooth muscle cells. CONCLUSIONS: These data suggest that the vascular hypertrophy mechanisms operating in 1K1C renal hypertension are not inhibited by heparin and thus are different from those in angiotensin-mediated hypertension. Identifying such mechanisms in the future will be important for devising appropriate intervention strategies in angiotensin-independent forms of vascular hypertrophy.

Angiotensin II↗

PubMed2004

Is there an association between level of adult blood pressure and nephron number or renal filtration surface area?

BACKGROUND: Reductions in renal filtration surface area (FSA) have been linked to development of hypertension. This study investigated whether there are direct relationships, in the adult rat, between levels of blood pressure and nephron number or total renal FSA. METHODS: F1 and F2 offspring were generated from a spontaneously hypertensive rat (SHR)/Wistar Kyoto (WKY) rat cross. Tail-cuff systolic blood pressure was measured twice weekly from 5 to 15 weeks of age and mean arterial blood pressure determined prior to sacrifice. At 15 weeks of age, the rats were perfusion-fixed and glomerular (and thereby nephron) number, glomerular size, total length, and surface area of glomerular capillaries and total renal FSA were determined using unbiased stereologic techniques. RESULTS: In F1 offspring, blood pressure levels were midway between the SHR and WKY rats. Nephron number was significantly higher in the WKY rats compared to the SHR and F1 offspring. However, there was no difference in nephron number between the F1 rats and SHR and no difference in renal FSA between the three groups. In the F2 generation, where there is random segregation of the SHR and WKY genes, there was no significant correlation between either nephron number and adult blood pressure (r2= 0.16, P= 0.11) or total renal FSA and adult blood pressure (r2= 0.02, P= 0.58). There was a significant inverse correlation between nephron number and glomerular size (r2= 0.49, P= 0.0043). CONCLUSION: There is not a direct corollary between nephron number or renal FSA and level of blood pressure in this rat model.

Age Factors↗

PubMed2003

Glomerular number and size in autopsy kidneys: the relationship to birth weight.

BACKGROUND: In the Southeast United States, African Americans have an estimated incidence of hypertension and end-stage renal disease (ESRD) that is five times greater than Caucasians. Higher rates of low birth weight (LBW) among African Americans is suggested to predispose African Americans to the higher risk, possibly by reducing the number of glomeruli that develop in the kidney. This study investigates the relationships between age, race, gender, total glomerular number (Nglom), mean glomerular volume (Vglom), body surface area (BSA), and birth weight. METHODS: Stereologic estimates of Nglom and Vglom were obtained using the physical disector/fractionator combination for autopsy kidneys from 37 African Americans and 19 Caucasians. RESULTS: Nglom was normally distributed and ranged from 227,327 to 1,825,380, an 8.0-fold difference. A direct linear relationship was observed between Nglom and birth weight (r = 0.423, P = 0.0012) with a regression coefficient that predicted an increase of 257,426 glomeruli per kilogram increase in birth weight (alpha = 0.050:0.908). Among adults there was a 4.9-fold range in Vglom, and in adults, Vglom was strongly and inversely correlated with Nglom (r =-0.640, P = 0.000002). Adult Vglom showed no significant correlation with BSA for males (r = -0.0150, P = 0.936), although it did for females (r = 0.606, P = 0.022). No racial differences in average Nglom or Vglom were observed. CONCLUSION: Birth weight is a strong determinant of Nglom and thereby of glomerular size in the postnatal kidney. The findings support the hypothesis that LBW by impairing nephron development is a risk factor for hypertension and ESRD in adulthood.

Adolescent↗

PubMed2003

A stereological study of glomerular number and volume: preliminary findings in a multiracial study of kidneys at autopsy.

BACKGROUND: This report describes preliminary results of a study of glomerular number and volume and their associations, in kidneys of people coming to autopsy. METHODS: Both kidneys were weighed at autopsy and the right kidney was perfusion-fixed and sub-sampled for stereological estimation of total glomerular number, and of mean renal corpuscle volume, using the physical disector/fractionator combination. RESULTS: The 78 kidneys studied so far were from Australian Aborigines, Australian non-Aborigines, US blacks and US whites, ages newborn to 84 years. Glomerular number ranged almost ninefold (from 210,332 to 1,825,380), with mean (SD) of 784,909 (314,686); it decreased throughout adult life (r=-0.32, P=0.009). Mean renal corpuscle volume varied 5.6-fold in adults and was inversely correlated with glomerular number (r=-0.38, P=0.001). Total renal corpuscle volume varied in adults by a factor of 15.8. Kidney weight correlated with body surface area (BSA) at all ages (r=0.76, P < 0.001); it varied 3.4-fold among adults, while kidney weight/m2 varied 3.7-fold. The percentage of sclerosed glomeruli varied from 0 to 23%, and it correlated strongly with age (r=0.58, P < 0.001). Females had smaller kidneys than males, and, marginally, fewer glomeruli. There were no significant variations by ethnic group. CONCLUSIONS: These extraordinary ranges of glomerular number and size among ostensibly "normal" people, and their inverse relationship, probably have important implications for susceptibility to renal insufficiency. People with low glomerular (nephron) numbers are likely to be particularly predisposed, with the process marked by compensatory hypertrophy of residual nephrons, which, in turn, accelerates their obsolescence. Much, however, remains to be done, including evaluation of history, clinical features, accompanying pathology, detailed renal morphology, and further pursuit of potentially defining characteristics in high risk groups.

Adolescent↗

PubMed2003

Organisation of bone morphogenetic proteins in renal development.

Bone morphogenetic proteins (BMPs) comprise the largest subfamily of the transforming growth factor-beta (TGF-beta) superfamily of secreted proteins. Evidence for the involvement of BMPs in metanephric development emerged recently when renal phenotypes were observed in BMP7 null mutant mice. Since then, several other BMPs, BMP receptor serine/threonine kinases and BMP signal transduction molecules (Smads) have been implicated in mammalian metanephric development. It appears that particular BMPs have pivotal roles in specific aspects of metanephric development. Current knowledge and evidence of specific roles of particular BMPs are presented in this review.

Animals↗

PubMed2003

Nephron number, renal function, and arterial pressure in aged GDNF heterozygous mice.

The loss of one allele for glial cell line-derived neurotrophic factor (GDNF) results in approximately 30% fewer but normal sized glomeruli in young mice. Low nephron number, inherited or acquired, has been linked to increased risk of development of hypertension and renal failure. This study examines whether GDNF heterozygous mice, with an inherent reduction in nephron number, demonstrate a deterioration in renal structure and function and rise in arterial pressure in later life. Fourteen-month-old male GDNF heterozygous (n=7) and wild-type (n=6) mice were anesthetized and prepared for measurement of mean arterial pressure, glomerular filtration rate (GFR), and renal blood flow. After measurement of renal function, kidneys were fixed for stereological determination of total glomerular number and mean glomerular volume. Mean arterial pressure was, on average, 18 mm Hg higher in GDNF heterozygous (98+/-4 mm Hg) than wild-type mice (80+/-2 mm Hg; P<0.01). However, GFR (0.656+/-0.054 versus 0.688+/-0.076 mL/min per g kidney wt) and renal blood flow (5.29+/-0.42 versus 4.70+/-0.34 mL/min per g kidney wt) were not different between groups. Fourteen-month-old GDNF heterozygous mice had approximately 30% fewer glomeruli than wild-type mice (9206+/-934 versus 13440+/-1275; P<0.01) and significantly larger glomeruli (4.51+/-0.39 versus 3.72+/-0.63x10(-4)mm(3); P<0.01). Thus, aged GDNF heterozygous mice maintained a normal GFR and renal blood flow despite reduced nephron numbers. The elevated arterial pressure, glomerular hypertrophy, and hyperfiltration demonstrated in the GDNF heterozygous mice at this age may indicate a compensatory mechanism whereby GFR is maintained in the presence of a reduced nephron endowment.

Aging↗

PubMed2002

Nephron number and blood pressure in rat offspring with maternal high-protein diet.

This study investigated the effects of a high-protein diet during pregnancy on nephron endowment and subsequent levels of blood pressure in the offspring. Female WKY rats were fed either a normal (20%, NPD) or a high (54%, HPD) protein diet during pregnancy. Male offspring were paired at birth. At 4 weeks of age, 1 of the pair was randomly chosen for perfusion fixation, and total glomerular number, and thereby nephron number, was estimated using an unbiased stereological technique. The other rat of the pair was allowed to grow to 30 weeks of age, during which time tail cuff systolic blood pressure was monitored twice weekly. There was no effect of the HPD on birth weight (NPD 4.23+/-0.53 g, HPD 4.26+/-0.45 g, mean+/-SD), kidney weight (NPD 0.372+/-0.049 g, HPD 0.337+/-0.090 g), or total nephron number (NPD 27,191+/-3,512, HPD 26,738+/-4,735). Systolic blood pressure at 30 weeks was 170+/-14 mmHg in NPD and 169+/-14 in HPD offspring. These findings show that a HPD during pregnancy did not lead to an increase in birth weight, kidney weight, or nephron endowment, nor did the HPD affect adult blood pressure.

Animals↗

PubMed2002

Glomerular size and glomerulosclerosis: relationships to disease categories, glomerular solidification, and ischemic obsolescence.

In focal segmental glomerulosclerosis (FSGS), a biphasic change in glomerular size is described in which glomeruli are enlarged with early glomerulosclerosis. Hyperperfusion of larger glomeruli is believed to contribute to progressive glomerular injury. This study was undertaken to investigate whether similar alterations in glomerular size can be found in other renal diseases. Volumes of sclerotic and nonsclerotic glomeruli were estimated in renal biopsy specimens using the Weibel and Gomez method (1962). Glomerulosclerosis was graded on individual glomeruli from 0 to 4, with 0 as no sclerosis and 4 as 76% to 100% sclerosis. Primary and secondary FSGS showed a biphasic change in which grade 2 glomeruli were 50% larger than grade 0 glomeruli and grades 3 and 4 glomeruli were solidified and smaller than grade 0 glomeruli. In essential hypertension, no increase in glomerular size was seen with early glomerulosclerosis, and the latter stages consisted of ischemic obsolescence in which collapsed tufts were 50% smaller than solidified glomeruli of FSGS. Grade 0 glomeruli of membranous glomerulonephritis (MGN) were significantly larger than grade 0 glomeruli of FSGS, and no significant difference in size was seen between grades 0 and 2 glomerulosclerosis. Solidified diabetic glomeruli maintained a large size with grades 3 and 4 sclerosis. Glomeruloscleroses of FSGS, hypertension, MGN, and diabetes have stereologically distinct features. A biphasic change in glomerular size is characteristic of primary and secondary FSGS, but not hypertension, in which tuft collapse supports reduced rather than increased perfusion in the pathogenesis of its glomerular obsolescence.

Adult↗

PubMed2002

Angiogenesis occurs by vessel elongation in proliferative phase human endometrium.

BACKGROUND: Angiogenesis occurs by at least three mechanisms: sprouting, intussusception and elongation. Studies to date have failed to identify the mechanisms or timing of endometrial angiogenesis during the menstrual cycle. The aim of this study was to determine if vessel elongation plays a role in human endometrial angiogenesis. METHODS: Forty-nine full thickness endometrial sections from 27 hysterectomy samples were immunostained for CD34 to identify blood vessels, and analysed using an interactive computerized stereological program. Based on counts from 9746 individual microscope fields, blood vessel length density (L(v)), branch point density (N(v)) and mean vessel length per branch point (L(v)/N(v)) were calculated for three endometrial zones during five phases of the menstrual cycle. RESULTS: There was an increase in L(v)/N(v) in the mid-late proliferative compared with early proliferative, early-mid secretory and late secretory phases of the menstrual cycle in the functionalis (mean +/- SEM: 174.5 +/- 20.1 versus 76.6 +/- 8.4, 118.6 +/- 9.4 and 104.2 +/- 4.1 microm respectively, P < 0.001) and between the mid-late proliferative and the menstrual phases in the basalis (158.0 +/- 18.2 versus 95.4 +/- 10.0 microm, P = 0.025). An increase in L(v) occurred in the subepithelial capillary plexus in the mid-late proliferative and early-mid secretory phases compared with the early proliferative phase (316.7 +/- 32.4 and 338.8 +/- 45.3 versus 178.5 +/- 8.9 mm/mm(3), P = 0.027). CONCLUSIONS: These data are the first evidence that vessel elongation is a major angiogenic mechanism in mid-late proliferative phase human endometrium.

Adult↗

PubMed2002

Correlation of histopathological features and renal impairment in autosomal dominant Alport syndrome in Bull terriers.

BACKGROUND: Bull terrier hereditary nephritis represents a model for autosomal dominant Alport syndrome, as affected dogs have the characteristically lamellated glomerular basement membrane and demonstrate vertical male-to-male disease transmission. METHODS: This study compared the histopathological features in kidneys from affected Bull terrier neonates, puppies, and adult dogs with normal or impaired renal function, with the histopathological appearance of kidneys from age- and size-matched normal dogs. RESULTS: There were fewer glomeruli per unit area of cortex in kidneys from affected neonatal kidneys (P<0.05), increased numbers of fetal glomeruli in affected puppy kidneys (P<0.05), and a separate population of glomeruli with larger renal corpuscles and glomerular tufts in kidneys from affected adult dogs with normal renal function (both P<0.0001) compared with normal dogs. Other histological features that are characteristic of human X-linked and autosomal recessive Alport syndrome and that were present included hypercellular glomeruli, occasional crescents, segmental and global glomerular sclerosis, periglomerular fibrosis, interstitial fibrosis without significant cellular infiltrates and cystic dilatation of Bowman's capsular space and tubules. In dogs with renal impairment, the tubular index was the best predictor of increased urinary protein:creatinine (r=0.92) compared with glomerular, interstitial and vascular indices (r=0.77, 0.88 and 0.81), and medullary fibrosis correlated best with serum creatinine (r=0.72, P=0.0002). CONCLUSIONS: The demonstration in Bull terrier kidneys of fewer nephrons in neonates increased fetal glomeruli, and a separate population of glomeruli with larger corpuscles and tufts reflects the effects of the underlying genetic mutation that are first manifest antenatally. The major determinant of renal impairment in adult affected Bull terriers is, however, progressive tubulointerstitial damage after birth.

Animals↗

PubMed2002

Nephron endowment and renal filtration surface area in young spontaneously hypertensive rats.

BACKGROUND/AIMS: A reduction in nephron endowment leading to reduced renal filtration surface area has been implicated in the development of hypertension. The aim of this study was to compare glomerular (and thereby nephron) number and renal filtration surface area in young Wistar-Kyoto rats (WKY) with young spontaneously hypertensive rats (SHR), prior to the development of hypertension in this model. METHODS: Using unbiased stereological methods the number and size of glomeruli, as well as total renal filtration surface area were determined in perfusion-fixed kidneys of 4-week-old WKY and SHR. RESULTS: At 4 weeks of age, in weight-matched animals, there was no significant difference in the number of glomeruli in the kidneys of SHR compared to WKY (28,620 +/- 1,643 and 25,670 +/- 1,263 glomeruli/kidney, respectively). Similarly, there was no difference in mean glomerular volume (SHR: 4.70 +/- 0.31 x 10(-4) mm(3); WKY: 4.28 +/- 0.20 x 10(-4) mm(3)). Surprisingly, total renal filtration surface area was significantly greater in SHR than WKY (3,867 +/- 116 and 3,176 +/- 83 mm(2), respectively). CONCLUSION: The renal abnormality underlying the development of hypertension in the SHR is not due to inborn deficits in nephron endowment and/or filtration surface area.

Aging↗

PubMed2002

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.

Animals↗

PubMed2002

BMPs and BMP receptors in mouse metanephric development: in vivo and in vitro studies.

BMPs have recently emerged as likely regulators of development of the permanent kidney (metanephros). Transcripts for BMPs and their receptors have been localised in the developing metanephros. In vitro, BMPs 2, 4 and 7 have direct or indirect roles in regulation of ureteric branching morphogenesis and branch formation. In vivo, renal phenotypes have been reported in BMP7 homozygous null mutant mice and BMP4 heterozygous null mutant mice. In the present study, in vivo and in vitro roles of BMPs and BMP receptors in metanephric development were further analysed. Stereology and histology were used to analyse kidneys from mice heterozygous for mutations in either BMP2, BMPR-IA or ActR-IA. Roles of BMPs 2 and 4 in mouse metanephric development in vitro were analysed by culturing whole metanephroi in the presence of BMP2, BMP4, the BMP inhibitor noggin, and BMP4 plus noggin. Ureteric branching morphogenesis and nephrogenesis were analysed. By qualitative histology, kidneys from BMP2, BMPR-IA and ActR-IA heterozygous null mutant mice were found to be the same as those from wild type mice. The kidneys of the heterozygous mice contained the normal complement of nephrons. In vitro, high concentrations of BMP4 inhibited branching of the ureteric epithelium and changed its morphology, while nephrogenesis was inhibited by 50%. A range of concentrations of BMP2 did not alter ureteric or mesenchyme morphology, or the number of glomeruli formed. Noggin did not alter metanephric development in vitro, but did block the effect of BMP4. The experiments described in this study have shown that BMP4 has distinct roles from BMP2 in metanephric development.

Animals↗

PubMed2002

Quantitation of 3D ureteric branching morphogenesis in cultured embryonic mouse kidney.

The growth and branching of the epithelial ureteric tree is critical for development of the permanent kidney (metanephros). Current methods of analysis of ureteric branching are mostly qualitative. We have developed a method for measuring the length of individual branches, and thereby the total length of the ureteric tree in 3 dimensions (3D). The method involves confocal microscopy of whole-mount immunostained metanephroi and computer-based image segmentation, skeletonisation and measurement. The algorithm performs semi-automatic segmentation of a set of confocal images and skeletonisation of the resulting binary object. Length measurements and number of branch points are automatically obtained. The final representation can be reconstructed providing a fully rotating 3D perspective of the skeletonised tree. After 36 h culture of E12 mouse metanephroi, the total length of the ureteric tree was 6103 +/- 291 microm (mean +/- SD), a four-fold increase compared with metanephroi cultured for just 6 h (1522 +/- 149 microm). Ureteric duct length increased at a rate of 153 microm/h over the first 30 h period and was maximal between 18 and 24 h at 325 microm/h. The distribution of branch lengths at the six time points studied was similar, suggesting tight control of ureteric lengthening and branching. This method will be of use in analysing ureteric growth in kidneys cultured in the presence of specific molecules suspected of regulating ureteric growth. The method can also be used to analyse in vivo kidneys and to quantify branching morphogenesis in other developing organs.

Algorithms↗