Frontiers in Research Reivew:
ISH Early Origins of Hypertension Workshop: Early Life Exposure and Development Estimating glomerular number: Why we do it and how John F Bertram Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University, Melbourne, Vic., Australia
SUMMARY 1. There is currently much interest in determining the number of glomeruli, and thereby nephrons, in the kidney. 2. Researchers have been trying to count glomeruli since the 19th century and currently four general approaches are available: (i) acid maceration; (ii) counting glomerular profiles in histological sections; (iii) model-based stereology; and (iv) design-based stereology. 3. Although design-based stereological methods are generally considered the gold-standard method, all current methods have limitations. A new approach using magnetic resonance imaging has recently been described and may ultimately enable glomerular imaging and quantification in vivo. 4. This report considers the advantages and disadvantages of current methods for counting glomeruli and describes the new magnetic resonance approach. In addition, a method for counting glomeruli in developing kidneys is described. Key words: glomerulus, kidney, magnetic resonance imaging, nephron number, stereology.
INTRODUCTION There is currently much interest in determining the number of glomeruli, and thereby nephrons, in the kidney. There are three main reasons for this. First, nephron number provides a quantitative index of the success of nephrogenesis during kidney development. It is well known that total nephron number varies widely in normal human kidneys. In the largest study to date of
420 human kidneys obtained at autopsy (the Monash Series), total glomerular number ranged from approximately 210 000–2.7 million, a 13-fold range.1 Other studies of human kidneys obtained at autopsy have also reported wide variations in glomerular number, albeit smaller due to the smaller sample sizes.2–4 Much of this variation in adult nephron number is “Nephron likely due to variability in nephron number varies endowment, this being the number of nephrons present at the conclusion of widely in nephrogenesis, which ends in humans around 36 weeks gestation.5 The complex human process of nephrogenesis is regulated by kidneys” numerous gene products and can also be influenced by factors in the fetomaternal environment.6,7 Therefore, the ability to count nephrons is integral to identifying those genetic and environmental factors that regulate nephron endowment. The second reason for wanting to count nephrons concerns the relationships between nephron number and adult disease. Brenner et al.8 were the first to propose a link between low nephron number and hypertension in adulthood, and subsequently identified associations between low nephron number and renal disease.9–11 At about the same time, Barker et al.12 identified links between birth weight and adult disease, including cardiovascular disease. Given the fact that human birth weight and nephron number are directly correlated,13 it seems likely that in some settings at least, low birth weight results in low nephron number, which, in turn, increases susceptibility to cardiovascular and renal disease in adulthood. The third reason for counting nephrons is that, with quantitative microanatomical data, we are able to examine correlations with
Correspondence: Professor JF Bertram, Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University, Vic. 3800, Australia. Email: [email protected]. This paper has been peer reviewed. Received 12 May 2013; revision 12 May 2013; accepted 31 May 2013. © 2013 Wiley Publishing Asia Pty Ltd doi: 10.1111/1440-1681.12133
JF Bertram
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quantitative physiological, biochemical and molecular data. For example, relationships between nephron number and glomerular filtration rate and renal blood flow can be analysed quantitatively in settings of development, growth and disease.
CURRENT METHODS FOR COUNTING GLOMERULI Four methods are currently used to count glomeruli and thereby nephrons. All have advantages and disadvantages, as described briefly below. Acid maceration Acid maceration involves digestion of kidney tissue in a weak acid solution, followed by mechanical dissociation of the kidney, after which the glomeruli are counted in “The disector aliquots of known volume. This method has clear advantages in terms of time method efficiency and has provided important renal physiology and revolutionized insights into pathology.3,14 However, estimates from stereology” laboratory to laboratory often vary widely, and the kidney has to be destroyed in order for glomerular counts to be obtained.
tissue processing and embedding. Tissue dimensions also change during sectioning. These dimensional changes affect kidney volume and section area, and therefore any parameter that relates glomerular profile number or glomerular number to kidney volume or section area must be corrected for these changes in dimensions. These shrinkage and swelling artefacts also likely vary with age and disease (e.g. oedema, interstitial fibrosis).3 Finally, knowing the number of glomerular profiles per unit area of section tells us nothing about the total number of glomeruli in the kidney. Model-based stereological methods To overcome the problems with glomerular profiles described above, several stereological methods for counting particles (such as glomeruli) were developed in the 20th century.17–22 These methods provide estimates of the number of glomeruli per unit volume of cortex or kidney. Multiplication of these numerical densities by kidney volume provides estimates of total glomerular number. Although these methods represent a major advance on glomerular profile counting, all require knowledge of glomerular geometry, such as glomerular calliper diameter, the glomerular size distribution and/or glomerular shape, and are therefore known as model-based methods. These parameters are not easy to measure and therefore most researchers assume values for these parameters, rather than measure them. To the extent that these assumptions are incorrect, the final estimates of glomerular number are biased.
Number of glomerular profiles per unit area of histological section
Design-based stereological methods
On the face of it, this would appear the ideal method for counting glomeruli. In short, glomeruli seen in histological sections (typically paraffin sections) are counted and this count is expressed as glomerular number per unit area of section. Despite the fact that this parameter is frequently measured and reported, it is unfortunately seriously flawed in many ways, as now described.1 Although parts of glomeruli can definitely be seen in histological sections, these are not whole glomeruli, but rather thin sections (or samples) of glomeruli (glomerular bits and pieces), which are known as glomerular ‘profiles’. It has been known for many years that the number of glomerular profiles seen in a histological section depends not only on the number of glomeruli in the kidney, but also on glomerular size and shape.15,16 Indeed, a single histological section is a biased sample of glomerular number, because large glomeruli have a greater chance than small glomeruli of being sampled. The number of glomeruli seen in a section is also influenced by section thickness. Thus, the relationship between glomerular profile number in a section and the number of glomeruli in a kidney is far from straightforward.2 It is well known that the dimensions of tissues such as the kidney change with fixation,
The disector method of Sterio23 provides estimates of total glomerular number and requires no knowledge or assumptions of glomerular geometry. Counting approaches based on the disector principle are considered design based and, if used correctly, the resulting estimates are unbiased. Two general disector-based approaches are available for counting glomeruli, namely the disector/Cavalieri principle and the disector/fractionator principle.16,24 These approaches have been used to count glomeruli in a range of species, including humans.1,2,4,13,25,26 They are considered the current gold-standard techniques, but unfortunately also have a number of limitations, including: (i) most studies have used glycolmethacrylate embedding and sectioning for dimensional stability, but relatively few histology laboratories are equipped for glycolmethacrylate; (ii) the approaches involve exhaustive sectioning, which is time consuming and therefore expensive; and (iii) counting glomeruli with disectors is time consuming and tedious (up to 6 h for a rat kidney and 8 h for a human kidney, following all tissue processing, sectioning and staining). Thus, although publication of the disector method revolutionized stereology and for the first time facilitated counting of glomeruli of any size or shape, the
© 2013 Wiley Publishing Asia Pty Ltd
Estimating glomerular number
method is relatively slow, tedious and expensive. For these reasons, relatively few laboratories have adopted this counting approach, most preferring to report glomerular profile counts or biased estimates. A new method is required.
A NEW APPROACH FOR COUNTING GLOMERULI A new glomerular counting method using magnetic resonance (MR) imaging has recently been described.27,28 To date, this method has been used to estimate the total number of glomeruli in the rat kidney.27,28 The method also provides estimates for the volume of every glomerulus in the kidney and thus, for the first time, the glomerular size distribution for an entire kidney. With this method, cationic ferritin is perfused intravenously and the kidneys are then dissected and imaged whole. The MR estimates of glomerular number and size were then validated using the gold-standard disector/fractionator approach. Agreement between the MR and disector/fractionator estimates were excellent for glomerular counts, but more variable for volume estimates.27,28 Advantages of this new MR approach “Glomeruli for counting and sizing glomeruli include: (i) for the first time we can be obtain the glomerular size distribution, counted providing a new and powerful technique ex vivo using for assessing glomerular growth, hypertrophy and shrinkage; (ii) the MRI” kidney is imaged whole ex vivo and therefore does not need to be sliced and diced into pieces; and (iii) the estimates can be obtained in approximately one-sixth of the hands-on time of estimates obtained using design-based stereology. A major disadvantage of course is that one needs access to a high field strength MR scanner. Whether MR can ever be used to count and size glomeruli in vivo remains to be determined, but this would provide significant promise in terms of longitudinal studies in laboratory models of kidney disease, for example, and in clinical nephrology. The current reliance on an exogenous contrast agent (cationic ferritin) is clearly an issue and further work is needed to prove efficacy and safety. Bennett et al.29 have discussed in greater detail the technical and regulatory challenges involved in developing MR techniques for glomerular imaging and quantification in vivo.
COUNTING GLOMERULI IN THE DEVELOPING KIDNEY Most studies to date of glomerular (nephron) number have been conducted in kidneys in which nephrogenesis has finished. However, on certain occasions we may be interested in knowing glomerular number in a kidney still undergoing nephrogenesis.
787
For example, we may want to: (i) know the impact of genetic abnormalities or environmental perturbations on nephrogenesis at certain stages of nephrogenesis; (ii) identify periods of susceptibility during nephrogenesis; or (iii) compare and contrast the impact of prenatal and postnatal factors on nephrogenesis, including the effects of premature birth and lactation. Cullen-McEwen et al.30 described a combined histochemical/ stereological technique that provides design-based estimates of nephron number (from the early S-shaped body stage to the mature glomerulus) in the developing rat kidney. Histochemistry (in this case using the lectin peanut agglutinin (PNA)) was used to provide unambiguous identification of developing nephrons, which are not always easily identified in standard histological sections. The PNA-positive structures were counted using the disector/fractionator principle. The method was used to estimate glomerular number in rats from Embryonic Day 17.25 to adulthood. Cullen-McEwen et al.30 also applied a curve-fitting procedure to generate nephron number growth curves for the laboratory rat. This approach could be modified, by the use of appropriate specific markers, to count numbers of vesicles and comma-shaped bodies during nephrogenesis.
CONCLUSION Design-based stereological methods provide accurate and precise estimates of the number of glomeruli (nephrons) in developing and adult kidneys. These approaches are the current gold-standard techniques, but have disadvantages in terms of time and costefficiency. Recently described methods using MR imaging have advantages in terms of time efficiency and also provide the glomerular size distribution. Further efforts to improve and expand MR techniques for estimation of total glomerular number and the glomerular size distribution ex vivo and in vivo are warranted.
DISCLOSURE The author declares no conflicts of interest.
REFERENCES 1. Bertram JF, Douglas-Denton RN, Diouf B, Hughson MD, Hoy WE. Human nephron number: Implications for health and disease. Pediatr. Nephrol. 2011; 26: 1529–33. 2. Nyengaard JR, Bendtsen TF. Glomerular number and size in relation to age, kidney weight, and body surface in normal man. Anat. Rec. 1992; 232: 194–201. 3. Merlet-Benichou C, Gilbert T, Vilar J, Moreau E, Freund N, Lelievre-Pegorier M. Nephron number: Variability is the rule causes and consequences. Lab. Invest. 1999; 79: 515–27. 4. Keller G, Zimmer G, Mall G, Ritz E, Amann K. Nephron number in patients with primary hypertension. N. Engl. J. Med. 2003; 348: 101–8.
© 2013 Wiley Publishing Asia Pty Ltd
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5. Hinchliffe SA, Sargent PH, Howard CV, Chan YF, van Velzen D. Human intrauterine renal growth expressed in absolute number of glomeruli assessed by the disector method and Cavalieri principle. Lab. Invest. 1991; 64: 777–84. 6. Moritz K, Wintour EM, Black MJ, Bertram JF, Caruana G. Factors influencing mammalian kidney development: Implications for health in adult life. Adv. Anat. Embryol. Cell Biol. 2008; 196: 1–78. 7. Hoy WE, Ingelfinger JR, Hallan S, Hughson MD, Mott SA, Bertram JF. The early development of the kidney and implications for future health. J. Dev. Origins Health Dis. 2010; 1: 216–33. 8. Brenner B, Garcia DL, Anderson S. Glomeruli and blood pressure. Less of one, more of the other? Am. J. Hypertens. 1988; 1: 335–47. 9. Brenner BM. The etiology of adult hypertension and progressive renal injury: An hypothesis. Bull. Mem. Acad. R. Med. Belg. 1994; 149: 121–5. 10. Brenner BM, Mackenzie HS. Nephron mass as a risk factor for progression of renal disease. Kidney Int. Suppl. 1997; 63: S124–7. 11. Luyckx VA, Brenner BM. The clinical importance of nephron mass. J. Am. Soc. Nephrol. 2010; 21: 898–910. 12. Barker DJ, Osmond C, Law CM. The intrauterine and early postnatal origins of cardiovascular disease and chronic bronchitis. J. Epidemiol Community Health 1989; 43: 237–40. 13. Hughson MD, Douglas-Denton R, Bertram JF, Hoy WE. Hypertension, glomerular number, and birth weight in African Americans and White subjects in the southeastern United States. Kidney Int. 2006; 69: 671–8. 14. Lelievre-Pegorier M, Vilar J, Ferrier ML et al. Mild vitamin A deficiency leads to inborn nephron deficit in the rat. Kidney Int. 1998; 54: 1455–62. 15. Weibel ER. Stereological Methods, Vol. 1. Practical Methods for Biological Morphometry. Academic Press, London. 1979. 16. Bertram JF. Analyzing renal glomeruli with the new stereology. Int. Rev. Cytol. 1995; 161: 111–72. 17. Wicksell SD. The corpuscle problem I. Biometrica 1925; 17: 84–99. 18. Wicksell SD. The corpuscle problem II. Biometrica 1926; 18: 152. 19. Floderus S. Untersuchungen uber den Bau der menschlichen hypophse mit besondener Berucksichtigung der quantitativen
20. 21.
22. 23. 24.
25.
26.
27.
28.
29.
30.
mikromorphologischen Verhaltnisse. Acta Pathol. Microbiol. Scand. 1944; 53: 1–26. Abercrombie M. Estimation of nuclear population from microtomic sections. Anat. Rec. 1946; 94: 239–47. DeHoff R, Rhines F. Determination of number of particles per unit volume from measurements made on random plane sections: The general cylinder and the ellipsoid. Trans. Metal. Soc. AIME 1961; 221: 975–82. Weibel ER, Gomez DM. A principle for counting tissue structures on random sections. J. Appl. Physiol. 1962; 17: 343–8. Sterio DC. The unbiased estimation of number and sizes of arbitrary particles using the disector. J. Microsc. 1984; 134: 127–36. Bertram JF, Soosaipillai M, Ricardo SD, Ryan GB. Total numbers of glomeruli and individual glomerular cell types in the normal rat kidney. Cell Tissue Res. 1992; 270: 37–45. Hoy WE, Hughson M, Cass A, Johnson K, Douglas-Denton R, Bertram JF. A stereological study of glomerular number and volume: Preliminary studies in a multiracial study of kidneys at autopsy. Kidney Int. 2003; 63(Suppl. 83): S31–7. Hoy WE, Hughson MD, Singh GR, Douglas-Denton R, Bertram JF. Reduced nephron number and glomerulomegaly in Australian Aborigines. A group at high risk for renal disease and hypertension. Kidney Int. 2006; 70: 104–10. Beeman SC, Zhang M, Gubhaju L et al. Measuring glomerular number and size in perfused kidneys using MRI. Am. J. Physiol. Renal Physiol. 2011; 300: F1454–7. Heilmann M, Neudecker S, Wolf I et al. Quantification of glomerular number and size distribution in normal rat kidneys using magnetic resonance imaging. Nephrol. Dial. Transplant. 2012; 27: 100–7. Bennett KM, Bertram JF, Beeman SC, Gretz N. The emerging role of MRI in quantitative renal glomerular morphology. Am. J. Physiol. Renal Physiol. 2013 May; 304: F1252–7. Epub 20 March 2013; doi: 10.1152/ajprenal.007142012 Cullen-McEwen LA, Armitage JA, Nyengaard JR, Moritz KM, Bertram JF. A design-based method for estimating glomerular number in the developing kidney. Am. J. Physiol. Renal Physiol. 2011; 300: F1448–53.
© 2013 Wiley Publishing Asia Pty Ltd
ISH Early Origins of Hypertension Workshop: Early Life Exposure and Development Estimating glomerular number: Why we do it and how John F Bertram Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University, Melbourne, Vic., Australia
SUMMARY 1. There is currently much interest in determining the number of glomeruli, and thereby nephrons, in the kidney. 2. Researchers have been trying to count glomeruli since the 19th century and currently four general approaches are available: (i) acid maceration; (ii) counting glomerular profiles in histological sections; (iii) model-based stereology; and (iv) design-based stereology. 3. Although design-based stereological methods are generally considered the gold-standard method, all current methods have limitations. A new approach using magnetic resonance imaging has recently been described and may ultimately enable glomerular imaging and quantification in vivo. 4. This report considers the advantages and disadvantages of current methods for counting glomeruli and describes the new magnetic resonance approach. In addition, a method for counting glomeruli in developing kidneys is described. Key words: glomerulus, kidney, magnetic resonance imaging, nephron number, stereology.
INTRODUCTION There is currently much interest in determining the number of glomeruli, and thereby nephrons, in the kidney. There are three main reasons for this. First, nephron number provides a quantitative index of the success of nephrogenesis during kidney development. It is well known that total nephron number varies widely in normal human kidneys. In the largest study to date of
420 human kidneys obtained at autopsy (the Monash Series), total glomerular number ranged from approximately 210 000–2.7 million, a 13-fold range.1 Other studies of human kidneys obtained at autopsy have also reported wide variations in glomerular number, albeit smaller due to the smaller sample sizes.2–4 Much of this variation in adult nephron number is “Nephron likely due to variability in nephron number varies endowment, this being the number of nephrons present at the conclusion of widely in nephrogenesis, which ends in humans around 36 weeks gestation.5 The complex human process of nephrogenesis is regulated by kidneys” numerous gene products and can also be influenced by factors in the fetomaternal environment.6,7 Therefore, the ability to count nephrons is integral to identifying those genetic and environmental factors that regulate nephron endowment. The second reason for wanting to count nephrons concerns the relationships between nephron number and adult disease. Brenner et al.8 were the first to propose a link between low nephron number and hypertension in adulthood, and subsequently identified associations between low nephron number and renal disease.9–11 At about the same time, Barker et al.12 identified links between birth weight and adult disease, including cardiovascular disease. Given the fact that human birth weight and nephron number are directly correlated,13 it seems likely that in some settings at least, low birth weight results in low nephron number, which, in turn, increases susceptibility to cardiovascular and renal disease in adulthood. The third reason for counting nephrons is that, with quantitative microanatomical data, we are able to examine correlations with
Correspondence: Professor JF Bertram, Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University, Vic. 3800, Australia. Email: [email protected]. This paper has been peer reviewed. Received 12 May 2013; revision 12 May 2013; accepted 31 May 2013. © 2013 Wiley Publishing Asia Pty Ltd doi: 10.1111/1440-1681.12133
JF Bertram
786
quantitative physiological, biochemical and molecular data. For example, relationships between nephron number and glomerular filtration rate and renal blood flow can be analysed quantitatively in settings of development, growth and disease.
CURRENT METHODS FOR COUNTING GLOMERULI Four methods are currently used to count glomeruli and thereby nephrons. All have advantages and disadvantages, as described briefly below. Acid maceration Acid maceration involves digestion of kidney tissue in a weak acid solution, followed by mechanical dissociation of the kidney, after which the glomeruli are counted in “The disector aliquots of known volume. This method has clear advantages in terms of time method efficiency and has provided important renal physiology and revolutionized insights into pathology.3,14 However, estimates from stereology” laboratory to laboratory often vary widely, and the kidney has to be destroyed in order for glomerular counts to be obtained.
tissue processing and embedding. Tissue dimensions also change during sectioning. These dimensional changes affect kidney volume and section area, and therefore any parameter that relates glomerular profile number or glomerular number to kidney volume or section area must be corrected for these changes in dimensions. These shrinkage and swelling artefacts also likely vary with age and disease (e.g. oedema, interstitial fibrosis).3 Finally, knowing the number of glomerular profiles per unit area of section tells us nothing about the total number of glomeruli in the kidney. Model-based stereological methods To overcome the problems with glomerular profiles described above, several stereological methods for counting particles (such as glomeruli) were developed in the 20th century.17–22 These methods provide estimates of the number of glomeruli per unit volume of cortex or kidney. Multiplication of these numerical densities by kidney volume provides estimates of total glomerular number. Although these methods represent a major advance on glomerular profile counting, all require knowledge of glomerular geometry, such as glomerular calliper diameter, the glomerular size distribution and/or glomerular shape, and are therefore known as model-based methods. These parameters are not easy to measure and therefore most researchers assume values for these parameters, rather than measure them. To the extent that these assumptions are incorrect, the final estimates of glomerular number are biased.
Number of glomerular profiles per unit area of histological section
Design-based stereological methods
On the face of it, this would appear the ideal method for counting glomeruli. In short, glomeruli seen in histological sections (typically paraffin sections) are counted and this count is expressed as glomerular number per unit area of section. Despite the fact that this parameter is frequently measured and reported, it is unfortunately seriously flawed in many ways, as now described.1 Although parts of glomeruli can definitely be seen in histological sections, these are not whole glomeruli, but rather thin sections (or samples) of glomeruli (glomerular bits and pieces), which are known as glomerular ‘profiles’. It has been known for many years that the number of glomerular profiles seen in a histological section depends not only on the number of glomeruli in the kidney, but also on glomerular size and shape.15,16 Indeed, a single histological section is a biased sample of glomerular number, because large glomeruli have a greater chance than small glomeruli of being sampled. The number of glomeruli seen in a section is also influenced by section thickness. Thus, the relationship between glomerular profile number in a section and the number of glomeruli in a kidney is far from straightforward.2 It is well known that the dimensions of tissues such as the kidney change with fixation,
The disector method of Sterio23 provides estimates of total glomerular number and requires no knowledge or assumptions of glomerular geometry. Counting approaches based on the disector principle are considered design based and, if used correctly, the resulting estimates are unbiased. Two general disector-based approaches are available for counting glomeruli, namely the disector/Cavalieri principle and the disector/fractionator principle.16,24 These approaches have been used to count glomeruli in a range of species, including humans.1,2,4,13,25,26 They are considered the current gold-standard techniques, but unfortunately also have a number of limitations, including: (i) most studies have used glycolmethacrylate embedding and sectioning for dimensional stability, but relatively few histology laboratories are equipped for glycolmethacrylate; (ii) the approaches involve exhaustive sectioning, which is time consuming and therefore expensive; and (iii) counting glomeruli with disectors is time consuming and tedious (up to 6 h for a rat kidney and 8 h for a human kidney, following all tissue processing, sectioning and staining). Thus, although publication of the disector method revolutionized stereology and for the first time facilitated counting of glomeruli of any size or shape, the
© 2013 Wiley Publishing Asia Pty Ltd
Estimating glomerular number
method is relatively slow, tedious and expensive. For these reasons, relatively few laboratories have adopted this counting approach, most preferring to report glomerular profile counts or biased estimates. A new method is required.
A NEW APPROACH FOR COUNTING GLOMERULI A new glomerular counting method using magnetic resonance (MR) imaging has recently been described.27,28 To date, this method has been used to estimate the total number of glomeruli in the rat kidney.27,28 The method also provides estimates for the volume of every glomerulus in the kidney and thus, for the first time, the glomerular size distribution for an entire kidney. With this method, cationic ferritin is perfused intravenously and the kidneys are then dissected and imaged whole. The MR estimates of glomerular number and size were then validated using the gold-standard disector/fractionator approach. Agreement between the MR and disector/fractionator estimates were excellent for glomerular counts, but more variable for volume estimates.27,28 Advantages of this new MR approach “Glomeruli for counting and sizing glomeruli include: (i) for the first time we can be obtain the glomerular size distribution, counted providing a new and powerful technique ex vivo using for assessing glomerular growth, hypertrophy and shrinkage; (ii) the MRI” kidney is imaged whole ex vivo and therefore does not need to be sliced and diced into pieces; and (iii) the estimates can be obtained in approximately one-sixth of the hands-on time of estimates obtained using design-based stereology. A major disadvantage of course is that one needs access to a high field strength MR scanner. Whether MR can ever be used to count and size glomeruli in vivo remains to be determined, but this would provide significant promise in terms of longitudinal studies in laboratory models of kidney disease, for example, and in clinical nephrology. The current reliance on an exogenous contrast agent (cationic ferritin) is clearly an issue and further work is needed to prove efficacy and safety. Bennett et al.29 have discussed in greater detail the technical and regulatory challenges involved in developing MR techniques for glomerular imaging and quantification in vivo.
COUNTING GLOMERULI IN THE DEVELOPING KIDNEY Most studies to date of glomerular (nephron) number have been conducted in kidneys in which nephrogenesis has finished. However, on certain occasions we may be interested in knowing glomerular number in a kidney still undergoing nephrogenesis.
787
For example, we may want to: (i) know the impact of genetic abnormalities or environmental perturbations on nephrogenesis at certain stages of nephrogenesis; (ii) identify periods of susceptibility during nephrogenesis; or (iii) compare and contrast the impact of prenatal and postnatal factors on nephrogenesis, including the effects of premature birth and lactation. Cullen-McEwen et al.30 described a combined histochemical/ stereological technique that provides design-based estimates of nephron number (from the early S-shaped body stage to the mature glomerulus) in the developing rat kidney. Histochemistry (in this case using the lectin peanut agglutinin (PNA)) was used to provide unambiguous identification of developing nephrons, which are not always easily identified in standard histological sections. The PNA-positive structures were counted using the disector/fractionator principle. The method was used to estimate glomerular number in rats from Embryonic Day 17.25 to adulthood. Cullen-McEwen et al.30 also applied a curve-fitting procedure to generate nephron number growth curves for the laboratory rat. This approach could be modified, by the use of appropriate specific markers, to count numbers of vesicles and comma-shaped bodies during nephrogenesis.
CONCLUSION Design-based stereological methods provide accurate and precise estimates of the number of glomeruli (nephrons) in developing and adult kidneys. These approaches are the current gold-standard techniques, but have disadvantages in terms of time and costefficiency. Recently described methods using MR imaging have advantages in terms of time efficiency and also provide the glomerular size distribution. Further efforts to improve and expand MR techniques for estimation of total glomerular number and the glomerular size distribution ex vivo and in vivo are warranted.
DISCLOSURE The author declares no conflicts of interest.
REFERENCES 1. Bertram JF, Douglas-Denton RN, Diouf B, Hughson MD, Hoy WE. Human nephron number: Implications for health and disease. Pediatr. Nephrol. 2011; 26: 1529–33. 2. Nyengaard JR, Bendtsen TF. Glomerular number and size in relation to age, kidney weight, and body surface in normal man. Anat. Rec. 1992; 232: 194–201. 3. Merlet-Benichou C, Gilbert T, Vilar J, Moreau E, Freund N, Lelievre-Pegorier M. Nephron number: Variability is the rule causes and consequences. Lab. Invest. 1999; 79: 515–27. 4. Keller G, Zimmer G, Mall G, Ritz E, Amann K. Nephron number in patients with primary hypertension. N. Engl. J. Med. 2003; 348: 101–8.
© 2013 Wiley Publishing Asia Pty Ltd
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JF Bertram
5. Hinchliffe SA, Sargent PH, Howard CV, Chan YF, van Velzen D. Human intrauterine renal growth expressed in absolute number of glomeruli assessed by the disector method and Cavalieri principle. Lab. Invest. 1991; 64: 777–84. 6. Moritz K, Wintour EM, Black MJ, Bertram JF, Caruana G. Factors influencing mammalian kidney development: Implications for health in adult life. Adv. Anat. Embryol. Cell Biol. 2008; 196: 1–78. 7. Hoy WE, Ingelfinger JR, Hallan S, Hughson MD, Mott SA, Bertram JF. The early development of the kidney and implications for future health. J. Dev. Origins Health Dis. 2010; 1: 216–33. 8. Brenner B, Garcia DL, Anderson S. Glomeruli and blood pressure. Less of one, more of the other? Am. J. Hypertens. 1988; 1: 335–47. 9. Brenner BM. The etiology of adult hypertension and progressive renal injury: An hypothesis. Bull. Mem. Acad. R. Med. Belg. 1994; 149: 121–5. 10. Brenner BM, Mackenzie HS. Nephron mass as a risk factor for progression of renal disease. Kidney Int. Suppl. 1997; 63: S124–7. 11. Luyckx VA, Brenner BM. The clinical importance of nephron mass. J. Am. Soc. Nephrol. 2010; 21: 898–910. 12. Barker DJ, Osmond C, Law CM. The intrauterine and early postnatal origins of cardiovascular disease and chronic bronchitis. J. Epidemiol Community Health 1989; 43: 237–40. 13. Hughson MD, Douglas-Denton R, Bertram JF, Hoy WE. Hypertension, glomerular number, and birth weight in African Americans and White subjects in the southeastern United States. Kidney Int. 2006; 69: 671–8. 14. Lelievre-Pegorier M, Vilar J, Ferrier ML et al. Mild vitamin A deficiency leads to inborn nephron deficit in the rat. Kidney Int. 1998; 54: 1455–62. 15. Weibel ER. Stereological Methods, Vol. 1. Practical Methods for Biological Morphometry. Academic Press, London. 1979. 16. Bertram JF. Analyzing renal glomeruli with the new stereology. Int. Rev. Cytol. 1995; 161: 111–72. 17. Wicksell SD. The corpuscle problem I. Biometrica 1925; 17: 84–99. 18. Wicksell SD. The corpuscle problem II. Biometrica 1926; 18: 152. 19. Floderus S. Untersuchungen uber den Bau der menschlichen hypophse mit besondener Berucksichtigung der quantitativen
20. 21.
22. 23. 24.
25.
26.
27.
28.
29.
30.
mikromorphologischen Verhaltnisse. Acta Pathol. Microbiol. Scand. 1944; 53: 1–26. Abercrombie M. Estimation of nuclear population from microtomic sections. Anat. Rec. 1946; 94: 239–47. DeHoff R, Rhines F. Determination of number of particles per unit volume from measurements made on random plane sections: The general cylinder and the ellipsoid. Trans. Metal. Soc. AIME 1961; 221: 975–82. Weibel ER, Gomez DM. A principle for counting tissue structures on random sections. J. Appl. Physiol. 1962; 17: 343–8. Sterio DC. The unbiased estimation of number and sizes of arbitrary particles using the disector. J. Microsc. 1984; 134: 127–36. Bertram JF, Soosaipillai M, Ricardo SD, Ryan GB. Total numbers of glomeruli and individual glomerular cell types in the normal rat kidney. Cell Tissue Res. 1992; 270: 37–45. Hoy WE, Hughson M, Cass A, Johnson K, Douglas-Denton R, Bertram JF. A stereological study of glomerular number and volume: Preliminary studies in a multiracial study of kidneys at autopsy. Kidney Int. 2003; 63(Suppl. 83): S31–7. Hoy WE, Hughson MD, Singh GR, Douglas-Denton R, Bertram JF. Reduced nephron number and glomerulomegaly in Australian Aborigines. A group at high risk for renal disease and hypertension. Kidney Int. 2006; 70: 104–10. Beeman SC, Zhang M, Gubhaju L et al. Measuring glomerular number and size in perfused kidneys using MRI. Am. J. Physiol. Renal Physiol. 2011; 300: F1454–7. Heilmann M, Neudecker S, Wolf I et al. Quantification of glomerular number and size distribution in normal rat kidneys using magnetic resonance imaging. Nephrol. Dial. Transplant. 2012; 27: 100–7. Bennett KM, Bertram JF, Beeman SC, Gretz N. The emerging role of MRI in quantitative renal glomerular morphology. Am. J. Physiol. Renal Physiol. 2013 May; 304: F1252–7. Epub 20 March 2013; doi: 10.1152/ajprenal.007142012 Cullen-McEwen LA, Armitage JA, Nyengaard JR, Moritz KM, Bertram JF. A design-based method for estimating glomerular number in the developing kidney. Am. J. Physiol. Renal Physiol. 2011; 300: F1448–53.
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