Interconnected network motifs control podocyte morphology and kidney function

Abstract

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3',5'-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element-binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a β-adrenergic receptor-driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease.

Fig. 1. Proteomic analysis indicates that PKA is important for differentiated podocyte morphology in vivo

(A) Transmission electron microscopy images of glomerular podocytes from control [phosphate-buffered saline (PBS)–injected] and puromycin-induced nephropathy rats (×5000). Interdigitating foot processes in the control images are marked with a cyan arrow and are absent from the electron micrographs of puromycin-injected animals. Scale bar, 2 μm. (B) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)–normalized expression of podocyte differentiation markers WT-1, nephrin, and synaptopodin in control (black) and puromycin-injected (red) rats, as quantified by reverse transcription polymerase chain reaction [RT-PCR; all values are means ± SEM; P < 0.05, one-way analysis of variance (ANOVA); n = 3 rats]. (C) Loss of podocyte morphology is associated with functional impairment as quantified by the significant increase of protein in the urine (P < 0.05, two-way ANOVA; n = 3 rats). Insets: Urinalysis results with Chemstrip indicate clinical proteinuria when top strips turn from yellow to deep green. (D) Heatmap of changes in the abundance of known podocyte-associated markers as determined by proteomics. (E) Negative log of P values for the top 10 highest ranked upstream protein kinases computationally identified by X2K using differentially expressed proteins as input nodes.

Fig. 2. Construction of sign-specified directed cAMP signaling network from receptor to CREB

(A) Directed graphs of initial three models (models 1, 2, and 3) with increasing number of components used in the iterative model-building process. (B) The final right-sized model (model 4) with labels on individual reactions tested using a leave-one-out scheme for their effects on CREB and MAPK activity or MAPK and PKA translocation shown in (C). Deviation from experimental observations was quantified at each time point individually. Roman numerals represent the edges (reactions) that were tested. (C) Heatmap of RMS errors between individual computational models and the experimental observations. Each voxel represents the RMS error at an independent spatial or temporal data point of a given model. CREB (pCREB) and MAPK (pMAPK) activities were experimentally tested by immunoblotting; PKA activity in the nucleus (Nuc) and cytoplasm (Cyto) by PKA enzymatic assay (biochemistry); relative amount of pMAPK to total MAPK and the activity in the nucleus versus the cytoplasm were measured by immunofluorescence. Rows represent the different models, and the columns represent different data points. Triangles represent time course experiments (0 to 45 min). (D) Total error of all computational models from experimental observations as quantified by the sum of RMS errors in a given model divided by that of the right-sized model.

Fig. 3. Minimally complex directed graph of the cAMP signaling network that controls CREB phosphorylation in the context of podocyte synaptopodin expression and actin bundling

The right-sized model from Fig. 2B is marked with color-coded boxes to highlight the four key regulatory motifs; these motifs are shown separately on the right for clarity.

Fig. 4. Computational modeling and experiments show that interconnected feedforward loops control duration and extent of CREB activation

(A) Top group represents the control condition of cells stimulated with isoproterenol (ISO), middle group represents the response in the presence of isoproterenol and PKA inhibition (Rp-cAMPs or PKA-DN), and bottom group represents the response in the presence of isoproterenol and MEK inhibition (MEKi or U0126). Upper line graphs on the left represent the computationally determined system response at the level of CREB (dashed black line) compared with the experimental observations (solid black line). Color-coded lower graphs represent the computationally determined activity of the indicated motifs over time. Network diagrams on the right show a schematic representation of the signal flow from cAMP to CREB upon isoproterenol stimulation at 3, 10, or 30 min. Activities of the simulated FFMs (diamonds) are depicted with color-coded scales; cAMP and CREB activities are depicted in grayscale. The intensity of the relationship lines represents the percent of the activity through that reaction, with dashed lines representing indirect and solid lines representing direct interactions (reaction flow). Interactive version of these dynamic graphs is available as fig. S7. (B) Schematic of the temporal contribution of the regulatory motifs in the maintenance of CREB activation as determined by motif lifetime analysis.

Fig. 5. Expression and functional localization of synaptopodin in cultured podocytes is controlled by PKA

(A) Podocytes were treated with Rp-cAMPs or U0126, or transfected with PKA-insensitive PTP-SL-S231A mutant, and then stimulated with 10 μM isoproterenol (ISO). Synaptopodin mRNA abundance was quantified using RT-PCR and normalized against the amount of tubulin mRNA (all values are means ± SEM; *P < 0.001, one-way ANOVA; n = 3). (B) Cells transfected with dominant-negative K-CREB (or empty vector) and stimulated with 10 μM isoproterenol. Isoproterenol stimulation led to no change in synaptopodin expression in the K-CREB group, where significant increase was observed in vector-transfected cells (*P < 0.001, one-way ANOVA; n = 3). (C) Representative immunofluorescence images of cultured podocytes that have been treated with 10 μM isoproterenol. Colocalization of synaptopodin (green pseudocolor) with actin bundles (red pseudocolor) is indicated with a yellow arrow. Red arrow indicates loss of bundling in cells treated with isoproterenol and Rp-cAMPs. Scale bar, 50 μm. (D) Quantification of immunofluorescence images in (C) (*P < 0.05, one-way ANOVA; n = 18 cells over five slides in two experiments).

Fig. 6. Regulatory motifs within cAMP network are spatially specified

(A) Schematic representation of FFM2. (B) Immunofluorescence images of cultured podocytes at baseline (Control) and 30 min of isoproterenol stimulation under basal (30′ ISO) conditions or with wild-type (WT) PTP-SL transfection, S231 mutant PTP-SL transfection, or preincubated with 100 μm Rp-cAMPs. Cells are stained for MAPK1,2 (red), phospho-MAPK (green), and actin (white), and nuclei-labeled with DAPI (4′,6-diamidino-2-phenylindole) (blue). Scale bar, 50 μm. (C) Time course of MAPK1,2 activation as measured by ratiometric quantification of phospho-MAPK and total MAPK immunofluorescence images. Values are means ± SEM; n = 24 cells over seven slides in three experiments. (D) Time course of MAPK1,2 translocation to the nucleus as measured by ratiometric quantification of immunofluorescence images. Values are means ± SEM; n = 24 cells over seven slides in three experiments. (E) Schematic representation of FFM3. (F) Time course of activated PKA in the cytoplasm and nucleus as measured by enzymatic assays after subcellular fractionation (*P < 0.05, two-way ANOVA; n = 3).

Fig. 7. Overall network response to motif modulation predicted by the computational model can be experimentally validated in vitro using pharmacologic agents

(A) Schematic diagram of FFM2, FBL1, and loci of pharmacological inhibition. (B) Effect of disruption of FBL1 with PDE-4 inhibitor rolipram (ISO + Roli) or disruption of FFM2 with the MEK inhibitor U0126 (ISO + Roli + U0126) during β-adrenergic stimulation with low-dose isoproterenol (0.1 μM; ISO Only) on CREB activity. Upper panel: Model predictions. Lower panel: Experimental observations.

Fig. 8. Compensatory modulation of a regulatory motif by pharmacologic agents restores podocyte morphology and function in vivo

(A) Modulation of FBL1 and FFM2 in vivo recapitulates computationally predicted behavior in puromycin-induced nephropathy. Loss of expression of podocyte differentiation markers nephrin, synaptopodin, and WT-1, as measured by RT-PCR in rats subjected to the indicated treatments. Values are means ± SEM; *P < 0.05 versus control, one-way ANOVA; n = 4 rats for each condition. (B) Transmission electron microscopy images of glomerular podocytes from healthy animals (black border), puromycin-induced nephropathy (red border) animals, nephropathy animals with FBL1 inhibition by rolipram (green border), or nephropathy animals with simultaneous FBL1 and FFM2 inhibition by rolipram plus AZD6244 (blue border). Arrows point to interdigitating foot process morphology (cyan arrows) that was disrupted in untreated and rolipram plus AZD6244–treated puromycin animals (magenta arrows). Scale bar, 5 μm. (C) Blinded quantification of foot process morphology shown in (B). All groups are significantly different from each of the other groups (P < 0.05, one-way ANOVA; n = 60, 15 images from four rats for each condition). (D) Glomerular pathophysiology as measured by protein in urine. Glomerular function in animals treated with puromycin and animals treated with rolipram plus AZD6244 (after puromycin) was significantly different from control (P < 0.05, two-way ANOVA with post hoc Tukey test; n = 4 rats). There was no statistical difference between control animals and animals treated with rolipram (after puromycin).