Immunomorphometric Studies of Proteinuria in Individual Deep and Superficial Nephrons of Rats

Abstract

SUMMARY:Heterogeneity of structure and function among nephrons is a well-recognized feature of chronic renal diseases.However, only a small number of superficial nephrons per kidney are accessible for micropuncture analysis and relationships of proteinuria to structural change in individual nephrons of experimental models are not clearly established. To directly evaluate proteinuria in many individual nephrons, we developed an immunomorphometric method of analysis. This method is based on the uniformly abundant renal synthesis of Tamm-Horsfall protein (THP) in the thick ascending limb of Henle's loop (TAL). Luminal rabbit immunoglobulin G (IgG) deposits are formed in TALs of proteinuric nephrons in rats injected with heterologous IgG anti-THP antibodies. This immunomorphometric luminal deposit method of assessing proteinuria was previously validated through analysis of heterologous immune complex nephropathy. Glomerular dysfunction in several models-spontaneously hypertensive rats (SHR), aging Sprague-Dawley (SD) rats, rats with adriamycin nephropathy (ADR), and rats subjected to subtotal nephrectomy (NX)-was characterized by immunomorphometric analysis after injection of anti-THP antibodies. Luminal IgG deposits were used to identify nephrons with increased proteinuria. Nephrons were identified histologically as either long looped (LL) or short looped (SL), and frequency of luminal deposits in these nephrons was determined. Glomerular size and sclerosis in deep and superficial zones of renal cortex were determined. Luminal deposits in LL nephrons were more frequent than luminal deposits in SL nephrons in SHRs (p Ͻ .001) and aging rats (p Ͻ .001) and SL nephrons in ADR rats (p Ͻ .02). Whole kidney levels of albuminuria correlated closely with the frequency of luminal deposits in both LL and SL nephrons of SHRs and ADR rats and in LL nephrons of aging rats (p Ͻ .005). In contrast, LL and SL deposits were equal in NX rats and did not correlate with albuminuria. A majority of luminal deposits extended beyond the first medullary TAL zone of NX rats, but was confined to this zone in the other 3 models. Deep cortical glomeruli were larger with more glomerulosclerosis than superficial cortical glomeruli. Albuminuria correlated with sclerosis of both deep (p Ͻ .002) and superficial (p Ͻ .01) glomeruli in NX rats, but not in the other three models. These studies provide a detailed characterization of a new method that allows comparison of proteinuria derived from deep and superficial nephrons. They also provide evidence that pathogenesis of the glomerulosclerosis in NX rats differs from that of the other three models. Glomerulosclerosis was closely linked to the overall level of albuminuria in NX rats, but not to luminal deposits. In the other three models, albuminuria and luminal deposits were closely linked but did not correlate with glomerulosclerosis. Furthermore, LL and SL nephron proteinuria of NX rats was comparable while LL proteinuria was markedly greater than SL proteinuria in the other three models. The lumina...

Podocytes that detach in experimental membranous nephropathy are viable11See Editorial by Mundel, p. 1529.

“…Adherent cells were fixed at -20ЊC in methanol:acetone (1:1) for 20 minutes, air dried for 15 minutes, rehydrated with phosphate-buffered saline (PBS), and permeabilized with 0.1% NP40. Cells were incubated overnight at 4ЊC with the following podocyte-specific antibodies: (1) WT-1 (mouse monoclonal; clone sc-7385) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:100 dilution); (2) To determine if cells other than podocytes were growing ex vivo, immunostaining was also performed with antibodies to (1) Thy1.1, a mesangial cell specific antigen (clone MRC OX-7) (Serotec, Ltd., Oxford, England); (2) RECA-1, an endothelial cell-specific antibody (clone HIS52) (Serotec Ltd.); (3) Tamm-Horsfall protein, specific for thick ascending limb (gift of Dr. J. Hoyer, Department of Pediatrics, University of Pennsylvania, Philadelphia, PA, USA) [21]; and (4) a fluorescein isothiocyanate (FITC)-labeled lectin from Tetragonolobus purpureas, specific for proximal tubular cells (Sigma Chem-ical Co., St. Louis, MO, USA) [22]. We tested the specificity of all the antibodies on frozen kidney sections by immunoflourescence as described above for cultured cells.…”

Viable Podocytes Detach in Experimental Diabetic Nephropathy: Potential Mechanism Underlying Glomerulosclerosis

“…To determine if cells other than podocytes were growing ex vivo, immunostaining was also performed with antibodies to (a) Thy1.1, a mesangial cell-specific antigen (clone MRC OX-7; Serotec Ltd, Oxford, UK), (b) RECA-1, an endothelial cell-specific antibody (clone HIS52; Serotec Ltd), (c) Tamm-Horsfall protein, specific for thick ascending limb (gift of Dr. J. Hoyer; Department of Pediatrics, Philadelphia, Pa., USA) [18], and (d) a FITC-labeled lectin from Tetragonolobus purpureas , specific for proximal tubular cells (Sigma) [19]. We also tested the specificity of all the antibodies on frozen kidney sections by immunoflourescence as described above for cultured cells.…”

Measuring the intrarenal distribution of glomerular volumes from histological sections

“…Glomerular hypertrophy is manifested in a number of diseases, such as diabetes, obesity, pregnancy, autosomal dominant polycystic kidney disease, and focal segmental glomerulosclerosis, and following acute kidney injury (13). Although many types of glomerular hyperfiltration lead to glomerular hypertrophy and glomerulosclerosis, the specific mechanisms depend on the pathology (18), and the damage is distributed heterogeneously throughout the kidney (15). Importantly, disease progression is often observed heterogeneously in individual kidneys, particularly at the earliest stages, and focal changes may not present in estimates of average glomerular volume.…”