Glomerular podocytes are highly specialized cells with a complex cytoarchitecture. Their most prominent features are interdigitated foot processes with filtration slits in between. These are bridged by the slit diaphragm, which plays a major role in establishing the selective permeability of the glomerular filtration barrier. Injury to podocytes leads to proteinuria, a hallmark of most glomerular diseases. New technical approaches have led to a considerable increase in our understanding of podocyte biology including protein inventory, composition and arrangement of the cytoskeleton, receptor equipment, and signaling pathways involved in the control of ultrafiltration. Moreover, disturbances of podocyte architecture resulting in the retraction of foot processes and proteinuria appear to be a common theme in the progression of acquired glomerular disease. In hereditary nephrotic syndromes identified over the last 2 years, all mutated gene products were localized in podocytes. This review integrates our recent physiological and molecular understanding of the role of podocytes during the maintenance and failure of the glomerular filtration barrier.

Cell Biology of the Glomerular Podocyte

Administration of mesenchymal stem cells (MSCs) improves the recovery from acute kidney injury (AKI). The mechanism may involve paracrine factors promoting proliferation of surviving intrinsic epithelial cells, but these factors remain unknown. In the current study, we found that microvesicles derived from human bone marrow MSCs stimulated proliferation in vitro and conferred resistance of tubular epithelial cells to apoptosis. The biologic action of microvesicles required their CD44- and beta1-integrin-dependent incorporation into tubular cells. In vivo, microvesicles accelerated the morphologic and functional recovery of glycerol-induced AKI in SCID mice by inducing proliferation of tubular cells. The effect of microvesicles on the recovery of AKI was similar to the effect of human MSCs. RNase abolished the aforementioned effects of microvesicles in vitro and in vivo, suggesting RNA-dependent biologic effects. Microarray analysis and quantitative real time PCR of microvesicle-RNA extracts indicate that microvesicles shuttle a specific subset of cellular mRNA, such as mRNAs associated with the mesenchymal phenotype and with control of transcription, proliferation, and immunoregulation. These results suggest that microvesicles derived from MSCs may activate a proliferative program in surviving tubular cells after injury via a horizontal transfer of mRNA.

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Mesenchymal Stem Cell-Derived Microvesicles Protect Against Acute Tubular Injury

Recent molecular insights have established the podocyte as a key component of the glomerular filtration barrier, and hence an important common pathway in proteinuric diseases. A conditionally immortalized human podocyte cell line has been developed by transfection with the temperature-sensitive SV40-T gene. These cells proliferate at the "permissive" temperature (33 degrees C). After transfer to the "nonpermissive" temperature (37 degrees C), they entered growth arrest and expressed markers of differentiated in vivo podocytes, including the novel podocyte proteins, nephrin, podocin, CD2AP, and synaptopodin, and known molecules of the slit diaphragm ZO-1, alpha-, beta-, and gamma-catenin and P-cadherin. The differentiation was accompanied by a growth arrest and the upregulation of cyclin-dependent kinase inhibitors, p27 and p57, as well as cyclin D(1), whereas cyclin A was downregulated. These data are consistent with cell cycle protein expression during podocyte maturation in vivo. In conclusion, the development of this cell line provides a new tool in the study of podocyte biology, which will enable accurate assessment of the behavior of these complex cells in health and disease.

A Conditionally Immortalized Human Podocyte Cell Line Demonstrating Nephrin and Podocin Expression

Ischemic acute renal failure: an inflammatory disease?

The possibility that proteinuria may accelerate kidney disease progression to end-stage renal failure has received support from the results of increasing numbers of experimental and clinical studies. Evidence indicating that this process occurs through multiple pathways, including induction of tubular chemokine expression and complement activation that lead to inflammatory cell infiltration in the interstitium and sustained fibrogenesis, is reviewed. Macrophages are prominent in the interstitial inflammatory infiltrate. This cell type mediates progression of renal injury to the extent that macrophage numbers in renal biopsy predict renal survival in patients with chronic renal disease. Chemoattractants and adhesive molecules for inflammatory cells are upregulated by excess ultrafiltered protein load of proximal tubular cells via activation of NF-kappaB-dependent and NF-kappaB-independent pathways. This mechanism is a potential target for therapeutic approaches, as shown by beneficial effects of manipulations with inhibitory molecules of NF-kappaB activation or of chemokine receptors in experimental studies. Targeting complement synthesis or activation in proximal tubule might offer novel therapeutic opportunities. Finally, proximal tubular cell receptors for uptake of plasma proteins that are under investigation may provide activation signals on excess tubular protein handling.

How Does Proteinuria Cause Progressive Renal Damage

A single i.v. injection of a mAb 5-1-6 to rats was found to cause massive though transient proteinuria. This mAb 5-1-6, IgG1 was produced by immunization of BALB/c mice with collagenase-treated Wistar rat glomeruli and was highly organ and species specific. Immunoelectron microscopy using immunoperoxidase with the avidin-biotin complex and immunogold staining indicated mAb 5-1-6 to bind in vitro to the surface of glomerular epithelial foot processes, mainly to slit diaphragms. The recognized antigenic molecule was not susceptible to neuraminidase treatment and its Mr was about 51 kDa by immunoprecipitation. A one-shot i.v. injection of this mAb induced proteinuria in rats starting immediately, reaching the peak on day 8 (mean value of 150 mg/24 h), then gradually decreasing to normal level on day 18. The in vivo localization of administrated mAb 5-1-6 changed with time. Linear binding along glomerular capillary walls was observed 2 h after injection. However, 3 days later, it partially shifted to a fine granular pattern. The linear pattern disappeared and the size as well as intensity of the fluorescent granules decreased on day 12 to trace positive on day 18. Immunoelectron microscopy revealed the binding pattern of in vivo injected mAb 5-1-6 after 2 h to be similar to that in vitro. Three days later, injected mAb was observed within multivesicular bodies in glomerular epithelial cells as well as along the surface of foot processes and around slit diaphragms. Twelve days after injection, mAb along the surface of the foot processes and around slit diaphragms decreased but those in multivesicular bodies were observed more frequently. Rat IgG and C3 could not be detected throughout the period of observation. No histologic abnormalities were noted except for partial retraction of epithelial foot processes at the peak of proteinuria on day 8. This mAb thus provides a valuable means for examining the mechanism of proteinuria.

Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody.

Cloning of rat nephrin: Expression in developing glomeruli and in proteinuric states

SUMMARY: Although the role of glomerular basement membrane has been emphasised as the barrier for retaining plasma proteins in the past three decades, some recent studies have demonstrated that the slit diaphragm of the glomerular epithelial cell (podocyte) is the structure likely to be the barrier in the glomerular capillary wall. Nephrin and podocin were identified as gene products mutated in Finnish-type congenital nephrotic syndrome and autosomal recessive steroid-resistant nephrotic syndrome, respectively. Nephrin s located at the outer leaflet of plasma membranes of the slit diaphragm. Podocin is reported to have an interaction with nephrin. The anti-nephrin antibody is capable of inducing massive proteinuria, which indicates that nephrin is a key functional molecule in the slit diaphragm. The expression of nephrin and podocin was reduced in glomeruli of minimal change nephrotic syndrome, which suggested that the altered expression of these molecules contributes to the development of proteinuria also in acquired diseases. Some recent studies demonstrated that CD2-associated protein (CD2AP) is also a functional molecule in the slit diaphragm, and its expression is altered in membranous nephropathy. These observations suggested that alteration of the molecular arrangement in the slit diaphragm is involved in the development of proteinuria in several kinds of glomerular diseases.

Role of podocyte slit diaphragm as a filtration barrier.

A MoAb 1-22-3 (IgG3) was produced in mice immunized with rat glomeruli. A blotting study indicated the antigen molecule recognized by this MoAb has a molecular weight of about 25 kD, which is the same as that of the Thy 1.1 molecule. This MoAb is capable of inducing the morphological changes similar to those induced by anti-thymocyte serum or the anti-Thy 1.1 MoAb, ER4 and massive proteinuria in rats by a single i.v. injection. Proteinuria started immediately after MoAb 1-22-3 injection and peaked on day 5. Reaction products were detected by immunoelectron microscopy in vitro on the limited mesangial cell surface facing endothelial cells and in vivo in partially lysed mesangial cells 30 min after injection. Unlike the proteinuria-inducing MoAb ER4, reactivity of MoAb 1-22-3 was detected neither on endothelial cells, epithelial cells, nor along the glomerular basement membrane in vivo and in vitro. There is a difference in reactivity toward guinea-pig and rabbit materials between MoAb 1-22-3 and the commercial anti-Thy 1.1 MoAb (OX7). The antigenic determinant of MoAb 1-22-3 is concluded to be a new epitope and only the binding of MoAb 1-22-3 to this epitope proved to lead to an abnormal increase of glomerular capillary wall permeability.

Epitope-specific induction of mesangial lesions with proteinuria by a MoAb against mesangial cell surface antigen.

Monoclonal antibody (MAb) 5-1-6 identifies a 51-kDa protein (p51) on rat podocyte foot processes and causes severe complement- and leukocyte-independent proteinuria when injected into rats. In the ...

Michiaki Orikasa

Papers

Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody.

Cloning of rat nephrin: Expression in developing glomeruli and in proteinuric states

Role of podocyte slit diaphragm as a filtration barrier.

Epitope-specific induction of mesangial lesions with proteinuria by a MoAb against mesangial cell surface antigen.

Slit diaphragm-reactive nephritogenic MAb 5-1-6 alters expression of ZO-1 in rat podocytes.