Glomerular cell number in normal subjects and in type 1 diabetic patients.

BACKGROUND: The number of cells in glomeruli has been a challenging measure, especially in human kidneys, with only a small amount of tissue obtained by biopsy. However, the number of cells and their function are important determinants of renal function in health and disease. METHODS: Modern morphometric techniques have now provided the means to determine the numerical density (Nv) and number (with a measure of glomerular volume) of endothelial cells, mesangial cells, and podocytes in plastic-embedded renal tissue biopsied from nondiabetic subjects (N = 36) and type 1 diabetic patients (N = 46) over an extended age range from childhood through late adult. RESULTS: Nv values for all glomerular cells varied only slightly with age and did not change within the range of glomerular lesions of diabetes studied. Thus, the increase in glomerular volume during childhood to a steady level thereafter was the primary determinant of total glomerular cell number. The number of mesangial cells and endothelial cells increased with age, reflecting the increase in all cells, while the podocytes remained unchanged in number over all ages studied (10 to 69 years). Numbers of total glomerular cells, mesangial cells, and endothelial cells were not changed with diabetes, while podocytes were fewer in number in diabetic patients of all ages, with reduced podocyte numbers even in diabetes of short duration. CONCLUSIONS: The essentially constant glomerular cell density in nondiabetic and diabetic subjects under different circumstances possibly indicates an underlying propensity for the glomerulus to regulate its architecture to maintain a constant number of cells per volume, no matter the size of the glomerulus or the severity of diabetic nephropathy studied in this set of patients. The reductions in podocyte numbers in both younger and older diabetic patients indicate a significant risk for functional abnormalities as diabetic nephropathy progresses. Moreover, these observations do not support the suggestion of marked increases in glomerular cell number (and especially mesangial cells) with the development and progression of diabetic nephropathy.

Proximal tubular basement membrane width in insulin-dependent diabetes mellitus.

Although glomerular structure has been studied, careful evaluation of tubular basement membrane (TBM) structure in diabetes in humans has not been done. We measured proximal TBM width, glomerular basement membrane (GBM) width, mesangial fractional volume [Vv(Mes/glom)], mesangial matrix fractional volume [Vv(MM/glom)], and cortical interstitial fractional volume [Vv(Int/cortex)] in 35 insulin-dependent diabetic (IDDM) patients and 20 controls. The patients' mean age was 28 +/- 10 years (X +/- SD) and IDDM duration was 17 +/- 8 years. Twenty-five patients were normoalbuminuric, four microalbuminuric, and six had overt proteinuria. Tubular basement membrane and GBM widths were measured by the orthogonal intercept method and mesangial and interstitial parameters by point counting. The TBM width was 915 +/- 320 nm in IDDM patients and 558 +/- 116 nm in controls (P = 0.0005); the TBM width was also increased in normoalbuminuric patients (849 +/- 297 nm, P = 0.0005). The TBM width was strongly directly related to GBM width (r = 0.67, P < 0.001), Vv(Mes/glom) (r = 0.52, P < 0.01), and Vv(MM/glom) (r = 0.61, P < 0.001), but only weakly to Vv(Int/cortex) (r = 0.29, NS). The TBM width (r = 0.65, P < 0.001) and GBM width (r = 0.65, P < 0.001) were strongly related to hemoglobin A1C (HbA1C), while the Vv(Mes/glom) (r = 0.35, P < 0.05) and Vv(Int/cortex) (r = 0.30, NS) were only weakly related to HbA1C. Thus, increased proximal TBM width is an integral component of early nephropathology in IDDM patients. This study suggests that the metabolic disturbances of diabetes are strong determinants of the constellation of structural abnormalities occurring in human diabetic nephropathy.

Estimating glomerular number in situ using magnetic resonance imaging and biopsy.

In the past researchers have used an estimate of one million as the number of glomeruli in each human kidney. However, recent work on excised kidneys has demonstrated a large variation in glomerular number from one person to another (330,000 to 1,400,000) per kidney. Theoretically an in situ estimate of glomerular number could be obtained if renal cortical volume, volume density of glomeruli per cortex [Vv(glom/cortex)] and mean glomerular volume are known. We used a dog model to demonstrate that an accurate estimate of cortical volume could be obtained in situ using magnetic resonance imaging (MRI). Vv(glom/cortex) and mean glomerular volume were obtained from needle biopsies. An independent and more direct method (the fractionator) was used to validate the estimate of glomerular number obtained using MRI and renal biopsy. On average there was very good agreement between the fractionator method (379,000 +/- 40,000) and the MRI/renal biopsy method (376,000 +/- 108,000) for the 10 dog kidneys measured; however we found up to a 36% difference between the two methods in an individual kidney. Nonetheless, the estimate from the MRI/renal biopsy method has more precision than the assumption that there are one million glomeruli per human kidney.

Quantitation of antigen in tissue by immunofluorescence image analysis.

A method is described to measure antigen(s) in tissue section using an image analysis system to quantitate immunofluorescence following staining with fluorescein isothiocyanate (FITC)-conjugated antibody. The antigen utilized was an 125I-labeled goat anti-glomerular basement membrane antibody (1409 cpm/micrograms) administered intravenously to each of 11 Sprague-Dawley rats in doses ranging from 1.09 to 34.72 mg IgG. 24 h later, both kidneys were obtained for quantitative immunofluorescence following staining of tissue sections with FITC-labeled rabbit anti-goat IgG and for determination of radioactivity which reflects the amount of goat IgG present in isolated glomeruli. A linear correlation (r = 0.97) was observed between the dose of administered goat 125I-IgG and the amount bound to isolated glomeruli over the entire dosage range. A highly significant correlation (r = 0.98) was also seen between the mean brightness per glomerulus as determined by quantitative immunofluorescence and the amount of 125I-IgG bound per glomerulus but only at values less than 500 pg of IgG per glomerulus. Above these levels no correlation was observed, suggesting the presence of hidden epitopes in the bound goat IgG or the lack of availability of the FITC-labeled rabbit antibody.

Measuring the volume density of the glomerular mesangium.

Volume density (Vv) is a morphometric parameter used to detect pathological changes within renal tissue. To establish the most efficient technique, Vv of the mesangium in renal biopsies was studied. First, a digitizing tablet was used to trace mesangial and total glomerular areas, subsequently used to calculate Vv of the mesangium. Secondly, a point counting technique was tested using either a 16-point or an 81-point grid; the number of grid points falling on the mesangium and glomerulus was used to calculate mesangial Vv. The digitizing tablet, the 81-point grid and the 16-point grid all gave equivalent Vv for the 15 biopsies. The 81-point grid and the 16-point grid were, respectively, 1.9 and 3.2-fold faster than the digitizing tablet. Since all techniques gave comparable results, the 16-point grid was the most efficient.

Quantitative indexes of aminonucleoside-induced nephrotic syndrome.

Aminonucleoside of puromycin (PAN) is known to cause altered glomerular permeability, resulting in a nephrotic syndrome in rats. The early sequence of this lesion was studied quantitatively, with the application of a new morphometric technique for determining epithelial foot process widths and a sensitive assay for quantifying urinary albumin excretion. Twenty-four hours following a single intraperitoneal injection of PAN, significant widening of foot processes was documented. Within 36 hours significant increases in urinary albumin excretion were observed. When control rats were examined, there was no clear correlation between epithelial foot process width and quantitative albumin excretion. However, in the PAN-treated animals, abnormal albuminuria only appeared in association with appreciable foot process expansion. These studies indicate that quantitative alterations occur in the rat glomerular capillary wall as early as 24 hours after PAN. Further studies of altered glomerular permeability may use these sensitive measures to more precisely define the temporal sequence and elucidate possible subgroups of experimental glomerular injury.

Quantitative glomerular morphology of the normal human kidney.

We determined glomerular basement membrane (GBM) width (as the harmonic mean) and relative volumes of the glomerular mesangium and of its cellular and matrix components in 59 male (73% living-related) and 59 female (93% living-related) donors of kidneys for transplantation. The GBM, consistently wider in male (mean, 373 nm.) versus female (mean, 326 nm.) donors, increased in width in all donors until the fourth decade of life when it appeared to decrease in width. The relative volume of the mesangium did not differ as to sex or age (mean, 14.2% of the glomerular volume), nor did either of its components (mean cellular mesangium, 7.1%; mean matrix mesangium, 7.1%). We found no correlations among renal index, GBM width, or the mesangium. No parameter differed in diabetic-related compared with nondiabetic-related donors. Results in cadavers for GBM width and the mesangium were no different from those of living-related donors. These observations yield insights into the development of the human kidney and its glomerular components, and in addition the GBM and mesangial measures will serve as normative values to which surgical or biopsy specimens can be related.

Diabetic glomerulopathy in the uninephrectomized rat resists amelioration following islet transplantation.

Uninephrectomy is known to accelerate the development of both functional and morphological changes seen with experimental diabetic nephropathy in the rat. The present experiments utilized electron and light microscopic morphometric techniques to assess glomerular basement membrane width and the volumes of the total mesangium and its cellular and matrix components of inbred Lewis rats made diabetic at 6 weeks of age and uninephrectomized 9 days later. Immunofluorescent microscopy was used to evaluate IgG and C3 in the mesangium. The reversibility of established diabetic glomerular lesions in uninephrectomized diabetic rats after 7 months of diabetes was studied by performing intraportal transplant of neonatal pancreatic tissue. Renal biopsies were taken 2 months later in transplanted and non-transplanted animals. Islet transplantation lowered plasma glucose to normal levels (29.6 to 7.3 mmol/l) and raised plasma insulin values (6.3 to 53 muU/I). Glomerular basement membrane width in transplanted rats (268 nm) still exceeded the same measure (226 nm) in nondiabetic uninephrectomized rats. In transplanted animals volumes of the mesangium (0.51 x 10(6) micrometers 3) and of its cellular (0.27 x 10(6) micrometers 3) and matrix (0.24 x 10(6) micrometers 3) components remained higher than similar measures in control rats (0.32 x 10(6), 0.17 x 10(6) and 0.15 x 10(6) micrometers 3, respectively). Mesangial IgG in treated animals approached normal, but mesangial C3 remained similar to levels in non-transplanted diabetic control animals. These observations in uninephrectomized-diabetic rats contrast with previous observations in intact diabetic rats in which mesangial volumes and localization of immunoglobulins and complement returned to normal levels following islet transplantation.

Glomerular basement membrane thickness following islet transplantation in the diabetic rat.

Glomerular basement membrane (GBM) thickness was measured in diabetic rats prior to and following islet transplantation. Over the course of the experiment (from 7 to 13 months of diabetes) GBM thickness in diabetic animals exceeded that of littermate controls. After 7 months of diabetes a group of animals received successful intraportal transplants of neonatal pancreatic tissue. GBM thickness at 2 and 6 months following islet transplantation matched the thickness in nontransplanted diabetic rats and exceeded that in control animals. Failure to reverse GBM thickening in diabetic rats following islet transplantation may be due to very slow rates of GBM tunover in the rat. Previous work has demonstrated normalization of urinary albumin excretion after islet transplantation, suggesting that GBM thickening, per se, is not a significant factor causing albuminuria in rats with longstanding diabetes.