The immunosuppressive action of the calcineurin inhibitor cyclosporine A (CsA) stems from the inhibition of nuclear factor of activated T cells (NFAT) signaling in T cells. CsA is also used for the treatment of proteinuric kidney diseases. As it stands, the antiproteinuric effect of CsA is attributed to its immunosuppressive action. Here we show that the beneficial effect of CsA on proteinuria is not dependent on NFAT inhibition in T cells, but rather results from the stabilization of the actin cytoskeleton in kidney podocytes. CsA blocks the calcineurin-mediated dephosphorylation of synaptopodin, a regulator of Rho GTPases in podocytes, thereby preserving the phosphorylation-dependent synaptopodin–14-3-3β interaction. Preservation of this interaction, in turn, protects synaptopodin from cathepsin L–mediated degradation. These results represent a new view of calcineurin signaling and shed further light on the treatment of proteinuric kidney diseases. Novel calcineurin substrates such as synaptopodin may provide promising starting points for antiproteinuric drugs that avoid the serious side effects of long-term CsA treatment.

The integrin CD11b/CD18 (also known as Mac-1), which is a heterodimer of the αM (CD11b) and β2 (CD18) subunits, is critical for leukocyte adhesion and migration and for immune functions. Blocking integrin-mediated leukocyte adhesion, although beneficial in experimental models, has had limited success in treating inflammatory diseases in humans. Here, we used an alternative strategy of inhibiting leukocyte recruitment by activating CD11b/CD18 with small-molecule agonists, which we term leukadherins. These compounds increased the extent of CD11b/CD18-dependent cell adhesion of transfected cells and of primary human and mouse neutrophils, which resulted in decreased chemotaxis and transendothelial migration. Leukadherins also decreased leukocyte recruitment and reduced arterial narrowing after injury in rats. Moreover, compared to a known integrin antagonist, leukadherins better preserved kidney function in a mouse model of experimental nephritis. Leukadherins inhibited leukocyte recruitment by increasing leukocyte adhesion to the inflamed endothelium, which was reversed with a blocking antibody. Thus, we propose that pharmacological activation of CD11b/CD18 offers an alternative therapeutic approach for inflammatory diseases.

The actin-based foot processes of kidney podocytes and the interposed slit diaphragm form the final barrier to proteinuria. Mutations affecting several podocyte proteins cause disruption of the filtration barrier and rearrangement of the highly dynamic podocyte actin cytoskeleton. Proteins regulating the plasticity of the podocyte actin cytoskeleton are therefore of critical importance for sustained kidney barrier function. Synaptopodin is an actin-associated protein essential for the integrity of the podocyte actin cytoskeleton because synaptopodin-deficient mice display impaired recovery from protamine sulfate-induced foot process effacement and lipopolysaccharide-induced nephrotic syndrome. Moreover, bigenic heterozygosity for synaptopodin and CD2AP is sufficient to induce spontaneous proteinuria and focal segmen-

This study examined the response of human keratinocytes in different stages of transformation to exogenous TGF-beta 1 and EGF as well as their receptor and growth-factor expression. Cells of the spontaneously immortalized HaCaT cell line and c-Ha-ras transfected clones (I-6, I-7, II-3, II-4) exhibited different tumorigenic potentials when transplanted to athymic mice. HaCaT- and I-6 cells were non-tumorigenic, I-7 cells formed persisting epidermal cysts (benign tumours) and II-3 and II-4 cells developed into invasive squamous-cell carcinomas. TGF-beta 1 inhibited thymidine uptake in a dose-dependent manner, a progressive decrease in response being associated with an increasing malignant potential (HaCaT greater than I-6 greater than I-7 = II-4). HaCaT-cells and ras-clones expressed TGF-beta 1 mRNA at similar levels, but cells of increasing malignant potential secreted markedly less receptor-binding TGF-beta (HaCaT greater than I-6 = I-7 greater than II-3 greater than II-4) into the culture medium. Whilst ras-transfected cells expressed fewer TGF-beta receptors than HaCaT cells, there was little difference between TGF-beta receptor number or affinity between the 4 transfected cell clones. The same was true for the TGF-beta receptor types, but Type-II receptors were expressed at lower levels by the malignant clones II-3 and II-4. When HaCaT and ras-transfected cells were investigated for their response to exogenous EGF, cells were refractory (I-7, II-4), partially stimulated (I-6) or fully stimulated (HaCaT). Cells with increasing malignant potential produced increasing amounts of endogenous TGF-alpha (II-4 = II-3 greater than I-7 = I-6 greater than HaCaT). All tumorigenic ras clones expressed higher mRNA levels than HaCaT-cells. Ras-transfected clones expressed fewer high- and low-affinity EGF receptors than HaCaT cells with a tendency toward increased numbers of high-affinity EGF receptors associated with increasing malignant potential (II-4 = II-3 greater than I-7 greater than I-6) but these changes were associated with a progressive decrease in receptor affinity. The results indicate that tumour progression in human epidermal keratinocytes transfected with c-Ha-ras is associated with a progressive abrogation of TGF-beta 1 and EGF growth control. They suggest that the increased autonomous growth potential associated with advanced stages of epithelial tumour progression can be defined more closely using a cellular profile of TGF-beta and EGF.