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Semaphorin 7A (SEMA7A) is an immunomodulating protein implicated in lung and liver fibrosis. In autosomal-dominant polycystic kidney disease (ADPKD), the progressive expansion of renal cysts, inflammation, and subsequent renal fibrosis leads to end-stage renal disease (ESRD). SEMA7A may play a role in renal fibrosis and in ADPKD. We evaluated Sema7a in a mouse model of renal fibrosis and determined the expression of SEMA7A in human ADPKD kidney. We analyzed SEMA7A expression on peripheral blood mononuclear cells (PBMCs), including CD45+ (leukocyte), CD14+(monocyte), CD4+ (T lymphocytes) and CD4+Foxp3+CD25+ [regulatory T lymphocytes (Tregs)] from 90 ADPKD patients (11 tolvaptan treated and 79 tolvaptan naive), and 21 healthy volunteers, using a Fluorescence-Activated Cell Sorting (FACS). Sema7a is required for renal fibrosis. SEMA7A shows robust expression in ADPKD kidneys, localizing to cysts derived from distal tubules. SEMA7A is higher in circulating monocytes, but unchanged in CD4+ lymphocytes in ADPKD patients. The SEMA7A increase was detected early (stage 1 CKD) and seemed more prominent in patients with smaller kidneys (p = 0.09). Compared to tolvaptan-naive ADPKD patients, those treated with tolvaptan showed reduced SEMA7A expression on monocytes, T lymphocytes, and Tregs, although the number of PBMCs was unchanged. After 1 month of tolvaptan treatment, SEMA7A expression on Tregs decreased. SEMA7A shows potential as both a therapeutic target in mammalian kidney fibrosis and as a marker of inflammation in ADPKD patients. SEMA7A expression was lower after tolvaptan treatment, which may reflect drug efficacy.

Polycystic kidney disease (PKD) exhibits an inflammatory component, but the contribution of inflammation to cyst progression is unknown. Macrophages promote the proliferation of tubular cells following ischemic injury, suggesting that they may have a role in cystogenesis. Furthermore, cultured Pkd1-deficient cells express the macrophage chemoattractants Mcp1 and Cxcl16 and stimulate macrophage migration. Here, in orthologous models of both PKD1 and PKD2, abnormally large numbers of alternatively activated macrophages surrounded the cysts. To determine whether pericystic macrophages contribute to the proliferation of cyst-lining cells, we depleted phagocytic cells from Pkd1(fl/fl);Pkhd1-Cre mice by treating with liposomal clodronate from postnatal day 10 until day 24. Compared with vehicle-treated controls, macrophage-depleted mice had a significantly lower cystic index, reduced proliferation of cyst-lining cells, better-preserved renal parenchyma, and improved renal function. In conclusion, these data suggest that macrophages home to cystic areas and contribute to cyst growth. Interruption of these homing and proliferative signals could have therapeutic potential for PKD.

Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD.

Cyst growth in patients with autosomal dominant polycystic kidney disease is thought to be due to increased tubular cell proliferation. One model to explain this altered proliferation suggests that the polycystin proteins PC1 and PC2 localize to apical cilia and serve as an integral part of the flow-sensing pathway thus modulating the proliferative response. We measured proliferation and apoptosis in proximal tubule derived cell lines lacking PC1. These cells showed increased rates of proliferation, a decreased rate of apoptosis, compared to control heterozygous cell lines, and spontaneously formed cysts rather than tubules in an in vitro tubulogenesis assay. Addition of neutrophil gelatinase associated lipocalin (NGAL), a small secreted protein that binds diverse ligands, to the cells lacking PC1 inhibited proliferation and increased apoptosis leading to slower cyst growth in vitro. Sustained over-expression at low level of NGAL by an adenoviral delivery system suppressed cyst enlargement without improving renal function in the Pkd1 mutant mice. Our studies show that renal epithelial cells lacking PC1 have an inherent tendency to hyper-proliferate forming cysts in vitro independent of a flow stimulus. The potential benefit of attenuating cyst growth with NGAL remains to be determined.

Vascular endothelial growth factor (VEGF) is well characterized for its role in endothelial cell differentiation and vascular tube formation. Alternate splicing of the VEGF gene in mice results in various VEGF-A isoforms, including VEGF-121 and VEGF-165. VEGF-165 is the most abundant isoform in the kidney and has been implicated in glomerulogenesis. However, its role in the tubular epithelium is not known. We demonstrate that VEGF-165 but not VEGF-121 induces single-cell branching morphogenesis and multicellular tubulogenesis in mouse renal tubular epithelial cells and that these morphogenic effects require activation of the phosphatidylinositol 3-kinase (PI 3-K) and, to a lesser degree, the extracellular signal-regulated kinase and protein kinase C signaling pathways. Further, VEGF-165-stimulated sheet migration is dependent only on PI 3-K signaling. These morphogenic effects of VEGF-165 require activation of both VEGF receptor 2 (VEGFR-2) and neuropilin-1 (Nrp-1), since neutralizing antibodies to either of these receptors or the addition of semaphorin 3A (which blocks VEGF-165 binding to Nrp-1) prevents the morphogenic response and the phosphorylation of VEGFR-2 along with the downstream signaling. We thus conclude that in addition to endothelial vasculogenesis, VEGF can induce renal epithelial cell morphogenesis in a Nrp-1-dependent fashion.

The paradigm for recovery of the renal tubule from acute tubular necrosis is that surviving cells from the areas bordering the injury must migrate into the regions of tubular denudation and proliferate to re-establish the normal tubular epithelium. However, therapies aimed at stimulating these events have failed to alter the course of acute renal failure in human trials. In the present study, we demonstrate that Lin-Sca-1+ cells from the adult mouse bone marrow are mobilized into the circulation by transient renal ischemia and home specifically to injured regions of the renal tubule. There they differentiate into renal tubular epithelial cells and appear to constitute the majority of the cells present in the previously necrotic tubules. Loss of stem cells following bone marrow ablation results in a greater rise in blood urea nitrogen after renal ischemia, while stem cell infusion after bone marrow ablation reverses this effect. Thus, therapies aimed at enhancing the mobilization, propagation, and/or delivery of bone marrow stem cells to the kidney hold potential as entirely new approaches for the treatment of acute tubular necrosis.

Ureteric bud (UB) branching during kidney development determines the final number of nephrons. Although hepatocyte growth factor and its receptor Met have been shown to stimulate branching morphogenesis in explanted embryonic kidneys, loss of Met expression is lethal during early embryogenesis without obvious kidney abnormalities. Metfl/fl;HoxB7-Cre mice,which lack Met expression selectively in the UB, were generated and found to have a reduction in final nephron number. These mice have increased Egf receptor expression in both the embryonic and adult kidney, and exogenous Egf can partially rescue the branching defect seen in kidney explants. Metfl/fl;HoxB7-Cre;wa-2/wa-2 mice, which lack normal Egfr and Met signaling, exhibit small kidneys with a marked decrease in UB branching at E14.5 as well as a reduction in final glomerular number. These mice developed progressive interstitial fibrosis surrounding collecting ducts with kidney failure and death by 3-4 weeks of age. Thus, in support of previous in vitro findings, Met and the Egf receptor can act cooperatively to regulate UB branching and mediate maintenance of the normal adult collecting duct.

There is growing evidence that vascular endothelial growth factor (Vegf), a well-recognized angiogenic factor, plays a regulatory role in non-endothelial tissues such as neurons and epithelial cells. In the kidney Vegf receptors have been detected in proximal tubule cells of the adult kidney and Vegf has been show to stimulate branching morphogenesis of the developing kidney. In this study, using laser-microdissection as well as manual separation of the UB, we demonstrate that Vegf receptors are present in the ureteric bud (UB). Furthermore, we determine that Vegf stimulates UB branching in whole kidney explant that is mediated directly by signaling through Vegfr2. In addition, Vegf also induced branching response in isolated UBs that are free of the surrounding mesenchyme. These responses seem to be strictly dependent on the dose of Vegf such that higher doses are inhibitory while lower dose are stimulatory. These data place Vegf in a unique position of being able to modulate vascular as well as epithelial development in the embryonic kidney.

The vasculature consists of endothelial cells (ECs) lined by pericyte/vascular smooth muscle cells (vSMCs). Pericyte/vSMCs provide support to the mature vasculature but are also essential for normal blood vessel development. To determine how pericyte-EC communication influences vascular development, we used the well-established in vitro model of TGFbeta-stimulated differentiation of 10T1/2 cells into pericyte/vSMCs. Microarray analysis was performed to identify genes that were differentially expressed by induced vs. uninduced 10T1/2 cells. We discovered that these cells show an angiogenic program of gene expression, with up-regulation of several genes previously implicated in angiogenesis, including VEGF, IL-6, VEGF-C, HB-EGF, CTGF, tenascin C, integrin alpha5, and Eph receptor A2. Up-regulation of some genes was validated by Western blots and immunocytochemistry. We also examined the functional significance of these gene expression changes. VEGF and IL-6 alone and in combination were important in 10T1/2 cell differentiation. Furthermore, we used a coculture system of 10T1/2 and human umbilical vein ECs (HUVECs), resulting in the formation of cordlike structures by the HUVECs. This cordlike structure formation was disrupted when neutralizing antibodies to VEGF or IL-6 were added to the coculture system. The results of these studies show that factors produced by pericytes may be responsible for recruiting ECs and promoting angiogenesis. Therefore, a further understanding of the genes involved in pericyte differentiation could provide a novel approach for developing anti-angiogenic therapies.

Endostatin (ES) inhibits endothelial cell migration and has been found to bind to glypicans (Gpcs) on both endothelial cells and renal epithelial cells. We examined the possibility that ES might regulate epithelial cell morphogenesis. The addition of ES to cultured epithelial cells causes an inhibition of both hepatocyte growth factor- and epidermal growth factor-dependent process formation and migration. In contrast, ES does not inhibit epidermal growth factor-dependent morphogenesis in renal epithelial cells derived from Gpc-3 −/mice, whereas expression of Gpc-1 in these cells reconstitutes ES responsiveness. Gpc-3 −/mice have been shown to display enhanced ureteric bud (UB) branching early in development, and cultured UB cells release ES into the media, suggesting that ES binding to Gpcs may regulate UB branching. The addition of ES inhibits branching of the explanted UB, whereas a neutralizing Ab to ES enhances UB outgrowth and branching. Thus, local expression of ES at the tips of the UB may play a role in the regulation of UB arborization.

Endostatin, a collagen XVIII fragment, is a potent anti-angiogenic protein. We sought to identify its endothelial cell surface receptor(s). Alkaline phosphatase- tagged endostatin bound endothelial cells revealing two binding affinities. Expression cloning identified glypican, a cell surface proteoglycan as the lower-affinity receptor. Biochemical and genetic studies indicated that glypicans' heparan sulfate glycosaminoglycans were critical for endostatin binding. Furthermore, endostatin selected a specific octasulfated hexasaccharide from a sequence in heparin. We have also demonstrated a role for endostatin in renal tubular cell branching morphogenesis and show that glypicans serve as low-affinity receptors for endostatin in these cells, as in endothelial cells. Finally, antisense experiments suggest the critical importance of glypicans in mediating endostatin activities.

Background Previous work in our laboratory demonstrated that hyperoxia suppressed the expression of vascular endothelial growth factor (VEGF) by the embryonic lung, leading to increased epithelial cell apoptosis and failure of explant airway growth and branching that was rescued by the addition of Vegf165. The aims of this study were to determine protective pathways by which VEGF isoforms attenuate hyperoxic lung growth retardation and to identify the target cell for VEGF action. Methods Timed pregnant CD-1 or fetal liver kinase (FLK1)-eGFP lung explants cultured in 3% or 50% oxygen were treated ± Vegf121, VEGF164/Vegf165 or VEGF188 in the presence or absence of anti-rat neuropilin-1 (NRP1) antibody or GO6983 (protein kinase C (PKC) pan-inhibitor) and lung growth and branching quantified. Immunofluorescence studies were performed to determine apoptosis index and location of FLK1 phosphorylation and western blot studies of lung explants were performed to define the signaling pathways that mediate the protective effects of VEGF. Results Heparin-binding VEGF isoforms (VEGF164/Vegf165 and VEGF188) but not Vegf121 selectively reduced epithelial apoptosis and partially rescued lung bud branching and growth. These protective effects required NRP1-dependent FLK1 activation in endothelial cells. Analysis of downstream signaling pathways demonstrated that the VEGF-mediated anti-apoptotic effects were dependent on PKC activation. Conclusions Vegf165 activates FLK1-NRP1 signaling in endothelial cells, leading to a PKC-dependent paracrine signal that in turn inhibits epithelial cell apoptosis.