Silja K. Sanden

Glomerular injury and proteinuria in diabetes (types 1 and 2) and IgA nephropathy is related to the degree of podocyte depletion in humans. For determining the causal relationship between podocyte depletion and glomerulosclerosis, a transgenic rat strain in which the human diphtheria toxin receptor is specifically expressed in podocytes was developed. The rodent homologue does not act as a diphtheria toxin (DT) receptor, thereby making rodents resistant to DT. Injection of DT into transgenic rats but not wild-type rats resulted in dose-dependent podocyte depletion from glomeruli. Three stages of glomerular injury caused by podocyte depletion were identified: Stage 1, 0 to 20% depletion showed mesangial expansion, transient proteinuria and normal renal function; stage 2, 21 to 40% depletion showed mesangial expansion, capsular adhesions (synechiae), focal segmental glomerulosclerosis, mild persistent proteinuria, and normal renal function; and stage 3, >40% podocyte depletion showed segmental to global glomerulosclerosis with sustained high-grade proteinuria and reduced renal function. These pathophysiologic consequences of podocyte depletion parallel similar degrees of podocyte depletion, glomerulosclerosis, and proteinuria seen in diabetic glomerulosclerosis. This model system provides strong support for the concept that podocyte depletion could be a major mechanism driving glomerulosclerosis and progressive loss of renal function in human glomerular diseases.

We report a transgenic mouse line that expresses Cre recombinase exclusively in podocytes. Twenty- four transgenic founders were generated in which Cre recombinase was placed under the regulation of a 2.5-kb fragment of the human NPHS2 promoter. Previously, this fragment was shown to drive beta-galactosidase (beta-gal) expression exclusively in podocytes of transgenic mice. For analysis, founder mice were bred with ROSA26 mice, a reporter line that expresses beta-gal in cells that undergo Cre recombination. Eight of 24 founder lines were found to express beta-gal exclusively in the kidney. Histological analysis of the kidneys showed that beta-gal expression was confined to podocytes. Cre recombination occurred during the capillary loop stage in glomerular development. No evidence for Cre recombination was detected in any of 14 other tissues examined.

Under physiologic conditions, significant amounts of plasma protein pass the renal filter and are reabsorbed by proximal tubular cells, but it is not clear whether the endocytosed protein, particularly albumin, is degraded in lysosomes or returned to the circulatory system intact. To resolve this question, a transgenic mouse with podocyte-specific expression of doxycycline-inducible tagged murine albumin was developed. To assess potential glomerular backfiltration, two types of albumin with different charges were expressed. On administration of doxycycline, podocytes expressed either of the two types of transgenic albumin, which were secreted into the primary filtrate and reabsorbed by proximal tubular cells, resulting in serum accumulation. Renal transplantation experiments confirmed that extrarenal transcription of transgenic albumin was unlikely to account for these results. Genetic deletion of the neonatal Fc receptor (FcRn), which rescues albumin and IgG from lysosomal degradation, abolished transcytosis of both types of transgenic albumin and IgG in proximal tubular cells. In summary, we provide evidence of a transcytosis within the kidney tubular system that protects albumin and IgG from lysosomal degradation, allowing these proteins to be recycled intact.

Transgenic manipulation of the glomerular visceral epithelial cell offers a powerful approach for studying the biology of this morphologically complex cell type. It has been previously demonstrated that an 8.3-kb and a 5.4-kb fragment of the murine Nphs1 (nephrin) promoter-enhancer drives lacZ expression in podocytes, brain, and pancreas of transgenic mice, recapitulating the expression pattern of the endogenous nephrin gene. In this present study, two truly podocyte-specific promoters were identified that drive transgene expression in podocytes without expression in extrarenal tissues in adult or embryonic mice. A 1.25-kb fragment driving a lacZ reporter gene (p1.25N-nlacF) was derived from murine Nphs1 promoter similar to a human NPHS1 promoter fragment previously reported. Transgenic mice were generated and beta-galactosidase (beta-gal) expression was analyzed. Four of twelve founder mice were found to express beta-gal in podocytes (33% penetrance). Expression in brain and pancreas was absent in all animals, suggesting that nephrin expression in these organs might be driven by distinct cis-regulatory elements that can be removed to obtain podocyte-specific expression. A 2.5-kb fragment derived from the human NPHS2 (podocin) gene was designed in a similar fashion to drive lacZ expression in transgenic mice (p2.5P-nlacF). Twelve of twlve NPHS2 mouse founder lines expressed beta-gal exclusively in podocytes (100% penetrance). Beta-gal activity was not observed extrinsic to the kidney in p1.25N-nlacF or p2.5P-nlacF mouse embryos at gestational time points between 8.5 d post coitus and birth. In conclusion, the 2.5-kb NPHS2 promoter fragment may be useful for podocyte-specific transgenic expression when extrarenal expression of a transgene is problematic.

Podocyte loss and glomerular hypertrophy are associated with development of glomerulosclerosis, suggesting that there may be a maximal area for each podocyte in terms of its capacity to support and maintain the glomerular filter. This study hypothesized that exceeding this maximal threshold will result in mesangial expansion and glomerulosclerosis. It may therefore be useful to measure podocyte number, glomerular volume, and glomerular volume per podocyte in clinical biopsy samples. An approach that uses thick and thin histologic sections cut from paraffin-embedded tissue to measure Wilms' tumor-1 protein-positive podocyte nuclear number and glomerular tuft area was studied. A rat model of aging has been used to track changes in glomerular podocyte number, glomerular volume per podocyte, and glomerular volume. Implications for clinical use of these variables are discussed.