Ronald D. Krofft

Inducible rodent models of acquired podocyte diseases

Glomerular diseases remain the leading cause of chronic and end-stage kidney disease. Significant advances in our understanding of human glomerular diseases have been enabled by the development and better characterization of animal models. Diseases of the glomerular epithelial cells (podocytes) account for the majority of proteinuric diseases. Rodents have been extensively used experimentally to better define mechanisms of disease induction and progression, as well as to identify potential targets and therapies. The development of podocyte-specific genetically modified mice has energized the research field to better understand which animal models are appropriate to study acquired podocyte diseases. In this review we discuss inducible experimental models of acquired nondiabetic podocyte diseases in rodents, namely, passive Heymann nephritis, puromycin aminonucleoside nephrosis, adriamycin nephrosis, liopolysaccharide, crescentic glomerulonephritis, and protein overload nephropathy models. Details are given on the model backgrounds, how to induce each model, the interpretations of the data, and the benefits and shortcomings of each. Genetic rodent models of podocyte injury are excluded.

Cyclin I activates Cdk5 and regulates expression of Bcl-2 and Bcl-XL in postmitotic mouse cells

Cyclin I is an atypical cyclin because it is most abundant in postmitotic cells. We previously showed that cyclin I does not regulate proliferation, but rather controls survival of podocytes, terminally differentiated epithelial cells that are essential for the structural and functional integrity of kidney glomeruli. Here, we investigated the mechanism by which cyclin I safeguards against apoptosis and found that cyclin I bound and activated cyclin-dependent kinase 5 (Cdk5) in isolated mouse podocytes and neurons. Cdk5 activity was reduced in glomeruli and brain lysates from cyclin I-deficient mice, and inhibition of Cdk5 increased in vitro the susceptibility to apoptosis in response to cellular damage. In addition, levels of the prosurvival proteins Bcl-2 and Bcl-XL were reduced in podocytes and neurons from cyclin I-deficient mice, and restoration of Bcl-2 or Bcl-XL expression prevented injury-induced apoptosis. Furthermore, we found that levels of phosphorylated MEK1/2 and ERK1/2 were decreased in cyclin I-deficient podocytes and that inhibition of MEK1/2 restored Bcl2 and Bcl-XL protein levels. Of interest, this pathway was also defective in mice with experimental glomerulonephritis. Taken together, these data suggest that a cyclin I-Cdk5 complex forms a critical antiapoptotic factor in terminally differentiated cells that functions via MAPK signaling to modulate levels of the prosurvival proteins Bcl-2 and Bcl-XL.

Establishment of Conditionally Immortalized Mouse Glomerular Parietal Epithelial Cells in Culture

Parietal epithelial cells (PEC) are major constituents of crescents in crescentic glomerulonephritis. The purpose of these studies was to establish an immortalized PEC cell line with similar characteristics to PEC in vivo for use in future mechanistic studies. Glomeruli were isolated from H-2Kb tsA58 transgenic mice (ImmortoMouse) by standard differential sieving, and several candidate PEC cell lines were obtained by subcloning outgrowths of cells from capsulated glomeruli. One clone, designated mouse PEC (mPEC), was extensively characterized. mPEC exhibited a compact cell body with typical epithelial morphology when grown in permissive conditions, but the cell shape changed to polygonal after 14 d in growth-restrictive conditions. mPEC but not podocytes used as a negative control expressed claudin-1, claudin-2, and protein gene product 9.5, which are proteins specific to PEC in vivo, and did not express the podocyte-specific proteins synaptopodin and nephrin. The junctional proteins zonula occludens-1 and beta-catenin stained positively in both mPEC and podocytes, but the staining pattern at cell-cell contacts was intermittent in mPEC and linear in podocytes. Finally, mPEC had thin bundled cortical F-actin filaments and no F-actin projections compared with podocytes, which exhibited thick bundled cortical F-actin filaments and interdigitating F-actin projections at cell-cell contacts. We conclude that immortalized mPEC in culture exhibit specific features of PEC in vivo and that these cells are distinct from podocytes, despite having the same mesenchymal origin. This mPEC line will assist in future mechanistic studies of PEC and enhance our understanding of glomerular injury.

Podocyte repopulation by renal progenitor cells following glucocorticoids treatment in experimental FSGS

Prednisone is a mainstay of treatment for patients with focal segmental glomerulosclerosis (FSGS), a disease characterized by reduced podocyte number and glomerulosclerosis. Although the systemic immune-modulatory effects of prednisone are well-known, direct tissue effects on glomerular cells are poorly understood. Experimental FSGS was induced in mice with a cytotoxic anti-podocyte antibody, resulting in an abrupt decrease in podocyte number by day 3, proteinuria, and the development of glomerulosclerosis. Administering daily prednisone to mice with FSGS, beginning at day 3, significantly increased podocyte number at weeks 2 and 4. Podocyte number did not increase in control mice with FSGS given DMSO. The increase in podocyte number in prednisone-treated mice correlated significantly with reduced glomerulosclerosis. Prednisone reduced podocyte apoptosis measured by synaptopodin⁺/caspase-3⁺ double staining. Additionally, the number of podocyte progenitors, defined as cells expressing both a parietal epithelial cell protein and a podocyte protein, was significantly increased in prednisone-treated mice with FSGS at weeks 2 and 4. This was associated with increased phospho-ERK staining in both parietal epithelial cells (PAX2⁺/p-ERK⁺) and in podocyte progenitors (WT-1⁺/p-ERK⁺ lining Bowmans capsule). These data show that in this model of experimental FSGS, prednisone augments glomerular repair by increasing podocyte number through direct effects on both glomerular epithelial cells. Prednisone limits podocyte loss by reducing apoptosis, and it increases regeneration by augmenting the number of podocyte progenitors. The data support a direct glomerular cell action for prednisone in improving outcomes in FSGS.

The enigmatic parietal epithelial cell is finally getting noticed: a review

Although the normal glomerulus comprises four resident cell types, least is known about the parietal epithelial cells (PECs). This comprehensive review addresses the cellular origin of PECs, discusses the normal structure and protein makeup of PECs, describes PEC function, and defines the responses to injury in disease and how these events lead to clinical events. The data show that PECs have unique properties and that new functions are being recognized such as their role in differentiating into podocytes during disease.

Retinoids augment the expression of podocyte proteins by glomerular parietal epithelial cells in experimental glomerular disease.

Background/Aims: A decrease in glomerular podocyte number in membranous nephropathy and focal segmental glomerulosclerosis (FSGS) ultimately underlines glomerulosclerosis and the decrease in kidney function. Recent studies have shown that in these diseases, glomerular parietal epithelial cells begin to express proteins considered unique to podocytes, and that these glomerular epithelial transition cells might serve as podocyte progenitors. Because retinoids improve many forms of experimental glomerular disease characterized by podocyte injury and loss, we asked if all-trans retinoic acid (ATRA) induces parietal epithelial cells to express podocyte proteins. Methods: ATRA or vehicle was administered to rats with experimental membranous nephropathy (passive Heymann nephritis model) and mice with experimental FSGS (anti-glomerular antibody model) following the onset of proteinuria. Immunohistochemistry staining of PAX2 (parietal epithelial cell marker), WT-1 (podocyte cell marker), and Ki-67 (proliferation marker) were performed on kidney tissues. Results: Compared to diseased animals receiving vehicle, ATRA statistically significantly increased the number of glomerular transition cells, defined as cells double-staining for PAX2 and WT-1, in membranous nephropathy at weeks 2, 5 and 16, and in FSGS at weeks 1 and 2. This was accompanied by an increase in the number of podocytes compared to diseased controls receiving vehicle. Conclusion: ATRA increases the number of glomerular epithelial transition cells in experimental proteinuric glomerular diseases. Thus, ATRA may provide a useful pharmacologic approach to decipher the mechanisms underlying the possible progenitor role of parietal epithelial cells.

Novel siRNA delivery system to target podocytes in vivo.

Podocytes are injured in several glomerular diseases. To alter gene expression specifically in podocytes in vivo, we took advantage of their active endocytotic machinery and developed a method for the targeted delivery of small interfering ribonucleic acids (siRNA). We generated an anti-mouse podocyte antibody that binds to rat and mouse podocytes in vivo. The polyclonal IgG antibody was cleaved into monovalent fragments, while preserving the antigen recognition sites. One Neutravidin molecule was linked to each monovalent IgG via the available sulfohydryl group. Protamine, a polycationic nuclear protein and universal adaptor for anionic siRNA, was linked to the neutravidin via biotin. The delivery system was named shamporter (s heep anti mouse podocyte transporter). Injection of shamporter coupled with either nephrin siRNA or TRPC6 siRNA via tail vein into normal rats substantially reduced the protein levels of nephrin or TRPC6 respectively, measured by western blot analysis and immunostaining. The effect was target specific because other podocyte-specific genes remained unchanged. Shamporter + nephrin siRNA induced transient proteinuria in rats. Control rats injected with shamporter coupled to control-siRNA showed no changes. These results show for the first time that siRNA can be delivered efficiently and specifically to podocytes in vivo using an antibody-delivery system.

P35, the non-cyclin activator of Cdk5, protects podocytes against apoptosis in vitro and in vivo

Cyclin-dependent kinase-5 is widely expressed and predominantly regulated by the non-cyclin activator p35. Since we recently showed that expression of p35 in the kidney is restricted to podocytes, we examined here its function in mice in which p35 was genetically deleted. The mice did not exhibit kidney abnormalities during glomerular development or during adult life. Conditionally immortalized cultured podocytes, derived from these null mice, did not have any change in their morphology, differentiation, or proliferation. However, when these cultured podocytes were exposed to UV-C irradiation, serum depletion, puromycin aminonucleoside, or transforming growth factor-beta-1, they showed increased apoptosis compared to those from wild-type mice. Levels of Bcl-2 were decreased in these null podocytes but increased after transduction with human p35. Restoration of p35 or the ectopic expression of Bcl-2 reduced the susceptibility of p35-null podocytes to apoptosis. Experimental glomerulonephritis, characterized by podocyte apoptosis and subsequent crescent formation, was utilized to test these findings in vivo. Podocyte apoptosis was significantly increased in diseased p35-null compared with wild-type mice, accompanied by increased glomerulosclerosis and decreased renal function. Our study shows that p35 does not affect glomerulogenesis but controls podocyte survival following injury, in part, by regulating Bcl-2 expression.

Cells of Renin lineage are adult pluripotent progenitors in experimental glomerular disease

Modified vascular smooth muscle cells of the kidney afferent arterioles have recently been shown to serve as progenitors for glomerular epithelial cells in response to glomerular injury. To determine whether such cells of renin lineage (CoRL) serve as progenitors for other cells in kidney disease characterized by both glomerular and tubulointerstitial injury, permanent genetic cell fate mapping of adult CoRL using Ren1cCreER × Rs-tdTomato-R reporter mice was performed. TdTomato-labeled CoRL were almost completely restricted to the juxtaglomerular compartment in healthy kidneys. Following 2 wk of antibody-mediated focal segmental glomerulosclerosis (FSGS) or 16 wk of ⅚ nephrectomy-induced chronic kidney diseases, tdTomato-mapped CoRL were identified in both interstitial and glomerular compartments. In the interstitium, PDGFβ receptor (R)-expressing cells significantly increased, and a portion of these expressed tdTomato. This was accompanied by a decrease in native pericyte number, but an increase in the number of tdTomato cells that coexpressed the pericyte markers PDGFβ-R and NG2. These cells surrounded vessels and coexpressed the pericyte markers CD73 and CD146, but not the endothelial marker ERG. Within glomeruli of reporter mice with the ⅚ nephrectomy model, a subset of labeled CoRL migrated to the glomerular tuft and coexpressed podocin and synaptopodin. By contrast, labeled CoRL were not detected in glomerular or interstitial compartments following uninephrectomy. These observations indicate that in addition to supplying new adult podocytes to glomeruli, CoRL have the capacity to become new adult pericytes in the setting of interstitial disease. We conclude that CoRL have the potential to function as progenitors for multiple adult cell types in kidney disease.

Cells of renin lineage take on a podocyte phenotype in aging nephropathy

Aging nephropathy is characterized by podocyte depletion accompanied by progressive glomerulosclerosis. Replacement of terminally differentiated podocytes by local stem/progenitor cells is likely a critical mechanism for their regeneration. Recent studies have shown that cells of renin lineage (CoRL), normally restricted to the kidneys extraglomerular compartment, might serve this role after an abrupt depletion in podocyte number. To determine the effects of aging on the CoRL reserve and if CoRL moved from an extra- to the intraglomerular compartment during aging, genetic cell fate mapping was performed in aging Ren1cCre × Rs-ZsGreen reporter mice. Podocyte number decreased and glomerular scarring increased with advanced age. CoRL number decreased in the juxtaglomerular compartment with age. There was a paradoxical increase in CoRL in the intraglomerular compartment at 52 and 64 wk of age, where a subset coexpressed the podocyte proteins nephrin, podocin, and synaptopodin. Transmission electron microscopy studies showed that a subset of labeled CoRL in the glomerulus displayed foot processes, which attached to the glomerular basement membrane. No CoRL in the glomerular compartment stained for renin. These results suggest that, despite a decrease in the reserve, a subpopulation of CoRL moves to the glomerulus after chronic podocyte depletion in aging nephropathy, where they acquire a podocyte-like phenotype. This suggests that they might serve as adult podocyte stem/progenitor cells under these conditions, albeit in insufficient numbers to fully replace podocytes depleted with age.