Paul T. Brinkkoetter

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.

The role of the podocyte in albumin filtration

In the past decade, our understanding of the role of podocytes in the function of the glomerular filtration barrier, and of the role of podocyte injury in the pathogenesis of proteinuric kidney disease, has substantially increased. Landmark genetic studies identified mutations in genes expressed by podocytes as a cause of albuminuria and nephrotic syndrome, leading to breakthrough discoveries from many laboratories. These discoveries contributed to a dramatic change in our view of the glomerular filtration barrier of the kidney and of the role of podocyte injury in the development of albuminuria and progressive kidney disease. In the past several years, studies have demonstrated that podocyte injury is a major cause of marked albuminuria and nephrotic syndrome, and have confirmed that podocytes are important for the maintenance of an intact glomerular filtration barrier. An essential role of loss of these cells in the pathogenesis of glomerulosclerosis and progressive proteinuric kidney disease has also been identified. In this Review, we discuss the importance of podocytes for the maintenance of an intact glomerular filtration barrier and their role in albumin handling.

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.

Epithelial phenotype confers resistance of ovarian cancer cells to oncolytic adenoviruses

We studied the susceptibility of primary ovarian cancer cells to oncolytic adenoviruses. Using gene expression profiling of cancer cells either resistant or susceptible to viral oncolysis, we discovered that the epithelial phenotype of ovarian cancer represents a barrier to infection by commonly used oncolytic adenoviruses targeted to coxsackie-adenovirus receptor or CD46. Specifically, we found that these adenovirus receptors were trapped in tight junctions and not accessible for virus binding. Accessibility to viral receptors was critically linked to depolarization and the loss of tight and adherens junctions, both hallmarks of epithelial-to-mesenchymal transition (EMT). We showed that specific, thus far little-explored adenovirus serotypes (Ad3, Ad7, Ad11, and Ad14) that use receptor(s) other than coxsackie-adenovirus receptor and CD46 were able to trigger EMT in epithelial ovarian cancer cells and cause efficient oncolysis. Our studies on ovarian cancer cultures and xenografts also revealed several interesting cancer cell biology features. Tumors in situ as well as tumor xenografts in mice mostly contained epithelial cells and cells that were in a hybrid stage where they expressed both epithelial and mesenchymal markers (epithelial/mesenchymal cells). These epithelial/mesenchymal cells are the only xenograft-derived cells that can be cultured and with passaging undergo EMT and differentiate into mesenchymal cells. Our study provides a venue for improved virotherapy of cancer as well as new insights into cancer cell biology.

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.

Rosuvastatin protects against podocyte apoptosis in vitro

BACKGROUND Clinical studies suggest that statins reduce proteinuria and slow the decline in kidney function in chronic kidney disease. Given a rich literature identifying podocyte apoptosis as an early step in the pathophysiological progression to proteinuria and glomerulosclerosis, we hypothesized that rosuvastatin protects podocytes from undergoing apoptosis. Regarding a potential mechanism, our lab has shown that the cell cycle protein, p21, has a prosurvial role in podocytes and there is literature showing statins upregulate p21 in other renal cells. Therefore, we queried whether rosuvastatin is prosurvival in podocytes through a p21-dependent pathway. METHODS Two independent apoptotic triggers, puromycin aminonucleoside (PA) and adriamycin (ADR), were used to induce apoptosis in p21 +/+ and p21 -/- conditionally immortalized mouse podocytes with or without pre-exposure to rosuvastatin. Apoptosis was measured by two methods: Hoechst 33342 staining and fluorescence-activated cell sorting (FACS). To establish a role for p21, p21 levels were measured by western blotting following rosuvastatin exposure and p21 was stably transduced into p21 -/- mouse podocytes. RESULTS Rosuvastatin protects against ADR- and PA-induced apoptosis in podocytes. Further, exposure to rosuvastatin increases p21 levels in podocytes in vitro. ADR induces apoptosis in p21 -/- mouse podocytes, but rosuvastatins protective effect is not seen in the absence of p21. Reconstituting p21 in p21 -/- podocytes restores rosuvastatins prosurvival effect. CONCLUSION Rosuvastatin is prosurvival in injured podocytes. Rosuvastatin exerts its protective effect through a p21-dependent antiapoptotic pathway. These findings suggest that statins decrease proteinuria by protecting against podocyte apoptosis and subsequent podocyte depopulation.

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.

Breaking the chain at the membrane: paraoxonase 2 counteracts lipid peroxidation at the plasma membrane

Lipid peroxidation through electrophilic molecules of extracellular origin is involved in the pathogenesis of many inflammatory conditions. To counteract free radical actions at the plasma membrane, cells host a variety of antioxidative enzymes. Here we analyzed localization, membrane topology, and trafficking of PON2 a member of the paraoxonase family of 3 enzymatically active proteins (PON1–3) found to have antiatherogenic properties. Immunohistochemistry localized PON2 to the villous tip of human intestinal epithelial cells. Employing membrane preparations, surface biotinylation experiments, and mutational analyses in HEK 293T and HeLa cells, we demonstrate that PON2 is a type II transmembrane protein. A hydrophobic stretch in the N terminus was identified as single transmembrane domain of PON2. The enzymatically active domain faced the extracellular compartment, where it suppressed lipid peroxidation (P<0.05) and regulated the glucosylceramide content, as demonstrated by mass spectrometry (P<0.05). PON2 translocation to the plasma membrane was dependent on intracellular calcium responses and could be induced to >10‐fold as compared to baseline (P=0.0001) by oxidative stress. Taken together, these data identify the paraoxonase protein PON2 as a type II transmembrane protein, which is dynamically translocated to the plasma membrane in response to oxidative stress to counteract lipid peroxidation.—Hagmann, H., Kuczkowski, A., Ruehl, M., Lamkemeyer, T., Brodesser, S., Horke, S., Dryer, S., Schermer, B., Benzing, T., Brinkkoetter, P. T. Breaking the chain at the membrane: paraoxonase 2 counteracts lipid peroxidation at the plasma membrane. FASEB J. 28, 28–1769 (1779). www.fasebj.org

Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry-based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.

Single and transient Ca2+ peaks in podocytes do not induce changes in glomerular filtration and perfusion

Chronic alterations in calcium (Ca2+) signalling in podocytes have been shown to cause proteinuria and progressive glomerular diseases. However, it is unclear whether short Ca2+ peaks influence glomerular biology and cause podocyte injury. Here we generated a DREADD (Designer Receptor Exclusively Activated by a Designer Drug) knock-in mouse line to manipulate intracellular Ca2+ levels. By mating to a podocyte-specific Cre driver we are able to investigate the impact of Ca2+ peaks on podocyte biology in living animals. Activation of the engineered G-protein coupled receptor with the synthetic compound clozapine-N-oxide (CNO) evoked a short and transient Ca2+ peak in podocytes immediately after CNO administration in vivo. Interestingly, this Ca2+ peak did neither affect glomerular perfusion nor filtration in the animals. Moreover, no obvious alterations in the glomerular morphology could be observed. Taken together, these in vivo findings suggest that chronic alterations and calcium overload rather than an induction of transient Ca2+ peaks contribute to podocyte disease.