John F. Bertram

Analyzing Renal Glomeruli with the New Stereology

The highly specialized architecture of the renal glomerulus is altered in a variety of disease states. Morphometric methods, including stereological methods, have been widely used to analyze these changes in both animal and human glomeruli. However, many of the methods available until recently were biased and provided incomplete information. The past few years have witnessed the development of a new generation of unbiased stereological methods. Another advantage of these new methods and strategies is that they are less influenced by technical artifacts than the traditional methods. This chapter describes how these new stereological methods can be used to quantify glomerular morphology. Parameters considered include glomerular number and volume; glomerular cell number and size; and the length, surface area, and number of glomerular capillaries. Methods for obtaining data for average glomeruli as well as individual glomeruli are described. Technical details are included wherever possible.

Total numbers of glomeruli and individual glomerular cell types in the normal rat kidney

SummaryAlterations in numbers of glomeruli and glomerular cells occur in various renal disorders. Although values for these parameters have previously been reported for several species, the estimates have often been biased due to assumptions regarding glomerular and/or nuclear size and shape. Other studies have used tedious serial-section reconstruction methods. In the present study, unbiased stereological methods were used to estimate total numbers of glomeruli and individual glomerular cell types in normal rats. The kidneys of seven adult Sprague-Dawley rats were perfused with 4% paraformaldehyde and 1% glutaraldehyde in phosphate buffer and embedded in either glycolmethacrylate (for light microscopy, LM) or Epon/Araldite (for transmission electron microscopy, TEM). Total glomerular number was estimated using an LM physical disector/fractionator combination; the total number of cells per average glomerulus was estimated using an LM optical disector/ Cavalieri combination; and TEM physical disectors were used to count individual cell types. The normal rat kidney was found to contain 31764±3667 (mean±SD) glomeruli. An average glomerulus contained 674±129 cells, of which 181±53 were epithelial cells (podocytes), 248±53 were endothelial cells, and 245±45 were mesangial cells. An average renal corpuscle contained 117±27 parietal epithelial cells. Following sectioning and staining, less than 6.5 h was needed to obtain the above estimates for a single animal, with coefficients of variation (SD as a percent of the mean) ranging from 10% to 25%. The unbiased stereological methods used in the present study constitute an unbiased, precise and cost-efficient set of quantitative tools for assessing glomerular morphology in health and disease.

Factors influencing mammalian kidney development : implications for health in adult life

There are many reasons why it is timely to review the development of the mammalian kidney. Perhaps the most important of these is the increasing amount of evidence to demonstrate that factors which impinge on/alter the normal developmental processes of this organ can have lifelong consequences for the health of the adult. TheDevelopmental Origins of Health and Adult Disease (DOHaD) hypothesis, proposes that changes in the environment during the development of an organ or system, can have permanent deleterious effects leading to increased risk of cardiovascular and/or metabolic disease. The permanent metanephric kidney has been shown to be very vulnerable to such influences with many factors shown to alter both the permanent structure and the level of expression of important functional genes. Thus it is important to understand the precise timing of kidney development in terms of both structure and the genes involved at each stage. Such knowledge has been gained by significant advances in technology, which allow quantification of the number of nephrons by unbiased stereology, detections of both levels and site of gene expression,knock-out and knock-in of genes in animal (mainly mouse) models and by the ability to examine nephron development, in real time, in culture systems.

Characterization of an animal model of hepatic metastasis

The experimental study of possible therapies for control of the growth of liver metastases requires the availability of a model which is technically feasible and appears to exhibit growth characteristics similar to human tumours. We report on the development of an intrasplenic injection model of liver metastases, and describe the histology, growth pattern and blood flow demonstrated by light microscopy, stereology and laser Doppler flowmetry. The hepatic metastases were induced in mice by intrasplenic injection of dimethylhydrazine (DMH) induced primary colonic carcinoma cells (106 cells in 1 mL). The growth and development of metastases was studied over a period of 3 weeks at predetermined time points. Tumour cells were visible in the hepatic sinusoids by day 7 by light microscopy. Macroscopically visible tumours with a diameter of 0.18 ± 0.02 cm (mean ± s.d.) were seen by day 10. By this time the tumours had derived a blood supply from the hepatic sinusoids adjacent to the tumour periphery. With further vascularization the tumours reached a diameter of 0.96 ± 0.50 cm by day 22. Metastatic growth was quantitated by stereological analysis of tumour volume in relation to non‐diseased hepatic tissue. Normal mouse liver had a mean volume of 1.13 ± 0.14 cm3. Tumour growth occurred in three phases. During the initial slow phase the volume of metastases increased from 0.03 ± 0.02 cm3 at day 10 to 0.22 ± 0.24 cm3 by day 16. Rapid tumour growth, occurring over the next 3 days, constituted the intermediate phase with metastatic volume reaching 1.21 ± 0.74 cm3 by day 19 (P= 0.0003 compared with day 16). This growth was followed by a plateau phase when the metastatic volume was 1.40 ± 0.55 cm3 at day 22. The volume of total liver and of tumour necrosis followed a similar growth pattern. A necrotic tumour volume of 0.004 ± 0.006 cm3 first seen on day 10 increased to 0.05 ± 0.06 cm3 by day 16, and to 0.25 ± 0.20 cm3 by day 22 (P=0.0022 compared with day 16). The blood flow in metastases measured by laser Doppler flowmetry was lower compared to the non‐diseased liver. Tumour blood flow, expressed as a percentage of normal liver blood flow, was 63.31 ± 26.28% at day 10 and diminished to 27.91 ± 8.99% by day 22, with an increase in tumour size and age. The decrease in flow was significant between days 13 and 16 (P= 0.0015). This intrasplenic mouse model of metastases is reproducible and should prove useful in the study of treatment of hepatic metastases.

Molecular regulation of nephron endowment.

Recent data suggests that the number of nephrons in normal adult human kidneys ranges from approximately 300,000 to more than 1 million. There is increasing evidence that reduced nephron number, either inherited or acquired, is associated with the development of essential hypertension, chronic renal failure, renal disease in transitional indigenous populations, and possibly the long-term success of renal allografts. Three processes ultimately govern the number of nephrons formed during the development of the permanent kidney (metanephros): branching of the ureteric duct in the metanephric mesenchyme; condensation of mesenchymal cells at the tips of the ureteric branches; and conversion of the mesenchymal condensates into epithelium. This epithelium then grows and differentiates to form nephrons. In recent years, we have learned a great deal about the molecular regulation of these three central processes and hence the molecular regulation of nephron endowment. Data has come from studies on cell lines, isolated ureteric duct epithelial cells, isolated metanephric mesenchyme, and whole metanephric organ culture, as well as from studies of heterozygous and homozygous null mutant mice. With accurate and precise methods now available for estimating the total number of nephrons in kidneys, more advances in our understanding of the molecular regulation of nephron endowment can be expected in the near future.

Enalapril Does Not Prevent Renal Arterial Hypertrophy in Spontaneously Hypertensive Rats

Angiotensin-converting enzyme inhibitors prevent the development of vessel wall hypertrophy in some vascular beds in spontaneously hypertensive rats (SHR), but their effects on hypertrophy of renal arterial vessels have not been studied. We therefore used stereological techniques to study wall and lumen dimensions of the interlobular (cortical radial) and arcuate arteries in the kidneys of SHR (n = 7), SHR treated from 4 to 10 weeks of age with enalapril (25 to 30 mg/kg per day; SHR-E, n = 7), and Wistar-Kyoto rats (WKY, n = 7). All kidneys were perfusion-fixed at 10 weeks. Systolic blood pressure was 199 +/- 9, 139 +/- 11, and 156 +/- 8 mm Hg in the SHR, SHR-E, and WKY groups, respectively. For the interlobular arteries, the volume density of artery wall, wall-to-lumen ratio, and wall thickness in the untreated SHR were significantly greater than in the WKY (0.84 +/- 0.09 versus 0.69 +/- 0.07 x 10(-3), 0.75 +/- 0.20 versus 0.53 +/- 0.08, and 13.6 +/- 3.3 versus 10.6 +/- 0.8 microns, respectively), but values in the SHR-E were similar to those in the untreated SHR (1.10 +/- 0.20 x 10(-3), 0.88 +/- 0.22, and 14.0 +/- 2.6 microns, respectively). For the arcuate arteries, wall thickness and volume density were significantly greater in SHR than WKY (17.3 +/- 3.0 versus 13.9 +/- 1.7 microns and 1.63 +/- 0.51 versus 1.14 +/- 0.27 x 10(-3), respectively), and values in the SHR-E (15.7 +/- 1.7 microns and 1.69 +/- 0.50 x 10(-3), respectively) were not significantly different from those in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of volumes of individual glomeruli in kidneys at autopsy: association with age, nephron number, birth weight and body mass index.

BACKGROUNDnGlomerular hypertrophy occurs in a number of normal and pathological states. Glomerular volume in kidneys at autopsy is usually indirectly derived from estimates of total glomerular mass and nephron number, and provides only a single value per kidney, with no indication of the range of volumes of glomeruli within the kidney of any given subject. We review findings of the distribution of volumes of different glomeruli within subjects without kidney disease, and their correlations with age, nephron number, birth weight and body mass index (BMI).nnnMETHODSnThe study describes findings from autopsy kidneys of selected adult white males from the Southeast USA who had unexpected deaths, and who did not have renal scarring or renal disease. Total glomerular (nephron) number and total glomerular volume were estimated using the disector/fractionator combination, and mean glomerular volume (Vglom) was derived. The volumes of 30 individual glomeruli (IGV) in each subject were determined using the disector/Cavalieri method. IGV values were compared by categories of age, nephron number, birth weight and BMI.nnnRESULTSnThere was substantial variation in IGV within subjects. Older age, lower nephron number, lower birth weight and gross obesity were associated with higher mean IGV and with greater IGV heterogeneity. High Vglom and high IGVs were associated with more glomerulosclerosis. However, amongst the generally modest numbers of sclerosed glomeruli, the pattern was uniformly of ischemic collapse of the glomerular tuft. There was no detectable focal segmental glomerular tuft injury.nnnDISCUSSIONnIn this series of people without overt renal disease, greater age, nephron deficit, lower birth weight and obesity were marked by glomerular enlargement and greater glomerular volume heterogeneity within individuals.

Glomerulomegaly in Australian Aborigines

Summary: Idiopathic glomerular enlargement has previously been described in a number of indigenous populations, including Australian Aborigines. This study had three aims: (1) evaluate three methods for estimating mean glomerular tuft and renal corpuscle volume; (2) assess the effects of fixation on glomerular dimensions; and (3) estimate glomerular tuft and renal corpuscle volume in clinical biopsies from Australian non‐Aboriginals, Aboriginals and Aboriginal inhabitants of the Tiwi Islands (Bathurst Island and Melville Island, Northern Territory, Australia). First, glomerular volume was determined in initial transplant biopsies from 17 non‐Aboriginal males (30–50 years) using three methods: the Cavalieri method, a stereological method that requires serial sectioning of glomeruli and knowledge of section thickness, but requires no knowledge or assumptions of glomerular size or shape (the gold‐standard method); the stereological method of Weibel and Gomez that employs a single section but requires assumptions of glomerular size distribution and shape; and the maximal profile method, with which the largest glomerular profile in a single section is identified, and used to calculate the volume of the parent glomerulus (assuming glomerular sphericity). Estimates for glomerular tuft volume were (mean ± SD): Cavalieri method (2.08 ± 0.37 × 106μm3); Weibel and Gomez (2.55 ± 0.63 × 106μ3); maximal profile method (3.09 + 0.66106μ3). Taking the Cavalieri estimate to be accurate, the maximal profile method is seen to grossly overestimate mean glomerular tuft volume, whereas the Weibel and Gomez method overestimated tuft volume by 23%. Both methods considerably overestimated mean renal corpuscle volume. In the study of fixation and glomerular dimensions, we found that glomeruli in clinical biopsies fixed in formalin were larger (47% for glomerular tuft and 25% for renal corpuscle) than the glomeruli in biopsies fixed in formol mercury/Dubosq Brazil. This result emphasizes the importance of standardizing the histological technique in quantitative studies of glomeruli. Finally, the Weibel and Gomez method was used to estimate mean glomerular volume in formalin‐fixed clinical biopsies from 80 non‐Aboriginal Australians, 78 non‐Tiwi Aboriginals and 72 Tiwi Aboriginals. Mean glomerular tuft volumes were: 3.12 ± 1.46 × 106μm3, 4.91 ± 2.59 × 106μm3 and 4.79 ± 2.08 × 106μm3, respectively, (for biopsies with four or more profiles). Mean glomerular tuft volume in the two Aboriginal populations was significantly (P < 0.001 in each case) greater than that in the non‐Aboriginals. These data indicate that there is pronounced glomerulomegaly in Australian Aborigines.

Glomerular dimensions in spontaneously hypertensive rats: effects of AT1 antagonism.

Objective A reduction in glomerular number and/or size has been implicated in the development of hypertension. This study investigated whether differences in glomerular number and/or size occur during the development of hypertension in the spontaneously hypertensive rat (SHR) and whether angiotensin II is responsible for any glomerular differences. Methods SHR (n=6) and Wistar–Kyoto (WKY) rats (n=6) were administered the angiotensin II type I receptor antagonist TCV-116 from 4 to 10 weeks of age. At 10 weeks of age, the kidneys from these rats and those from untreated SHR (n=6) and WKY rats (n=6) controls were perfusion fixed at physiological pressures and analysed using unbiased stereological techniques. Results There were no significant differences in glomerular number, glomerular volume or total glomerular volume between SHR and WKY rats. Treatment of SHR with TCV-116 significantly lowered systolic blood pressure but had no significant effect on glomerular number or volume or total glomerular volume. Treatment of WKY rats with TCV-116 reduced systolic blood pressure, body weight, glomerular volume and total glomerular volume; however, total glomerular volume per body weight of treated WKY rats was not significantly different from that of untreated WKY rats. Conclusion There were no differences in glomerular number or volume in SHR compared with WKY rats at 10 weeks of age. We therefore conclude that glomerular changes are not responsible for the development of hypertension in SHR. Angiotensin II, via the type 1 receptor, does not contribute to glomerular growth during the development of hypertension in the SHR.

Cardiovascular hypertrophy in one-kidney, one clip renal hypertensive rats : a role for angiotensin II ?

Objective To investigate the role of angiotensin II (Ang II) in cardiovascular hypertrophy in the Goldblatt one-kidney, one clip (1-K,1C) renal hypertensive rat. Methods Six-week-old Wistar-Kyoto (WKY) rats underwent uninephrectomy and left renal artery clipping. After surgery, rats were treated with perindopril, an angiotensin converting enzyme (ACE) inhibitor, or losartan, an Ang II type 1 (AT1) receptor antagonist, for 4 weeks. Untreated 1-K,1C rats and uninephrectomized (sham) rats served as controls. Results The rise in systolic blood pressure in the perindopril-treated and losartan-treated rats was not significantly different from that in the untreated 1-K,1C group throughout the treatment period. At 4 weeks after surgery the heart weight:body weight ratios of the untreated 1-K,1C and losartan-treated 1-K,1C groups were significantly greater than for sham-operated normotensive rats and hypertensive perindopril-treated rats. The total number of smooth muscle cells in the thoracic aortae of the 1-K,1C untreated, losartan-treated 1-K,1C and sham groups were similar. However, after treatment the aortae of the perindopril-treated group contained significantly fewer smooth muscle cells. The medial cross-sectional wall area and wall: lumen ratio were similar in the 1-K,1C untreated and perindopril-treated 1-K,1 C groups. Conclusion These results suggest that Ang II, via its effects on cardiac and vascular AT1 receptors, does not contribute to the development of cardiovascular hypertrophy in the 1-K,1C rat. Attenuation of cardiac and vascular growth after ACE inhibition appears to be mediated by mechanisms independent of the actions of the renin-angiotensin system.