Prostanoid receptors in hyperfiltration-mediated glomerular injury: Novel agonists and antagonists reveal opposing roles for EP2 and EP4 receptors.

Increased fluid-flow shear stress (FFSS) contributes to hyperfiltration-induced podocyte and glomerular injury resulting in progression of chronic kidney disease (CKD). We reported that increased FFSS in vitro and in vivo upregulates PGE2 receptor EP2 (but not EP4 expression), COX2-PGE2 -EP2 axis, and EP2-linked Akt-GSK3ß-ß-catenin signaling pathway in podocytes. To understand and use the disparities between PGE2 receptors, specific agonists, and antagonists of EP2 and EP4 were used to assess phosphorylation of Akt, GSK3ß and ß-catenin in podocytes using Western blotting, glomerular filtration barrier function using in vitro albumin permeability (Palb ) assay, and mitigation of hyperfiltration-induced injury in unilaterally nephrectomized (UNX) mice at 1 and 6 months. Results show an increase in Palb by PGE2 , EP2 agonist (EP2AGO ) and EP4 antagonist (EP4ANT ), but not by EP2 antagonist (EP2ANT ) or EP4 agonist (EP4AGO ). Pretreatment with EP2ANT blocked the effect of PGE2 or EP2AGO on Palb . Modulation of EP2 and EP4 also induced opposite effects on phosphorylation of Akt and ß-Catenin. Individual agonists or antagonists of EP2 or EP4 did not induce significant improvement in albuminuria in UNX mice. However, treatment with a combination EP2ANT + EP4AGO for 1 or 6 months caused a robust decrease in albuminuria. EP2ANT + EP4AGO combination did not impact adaptive hypertrophy or increased serum creatinine. Observed differences between expression of EP2 and EP4 on the glomerular barrier highlight these receptors as potential targets for intervention. Safe and effective mitigating effect of EP2ANT + EP4AGO presents a novel opportunity to delay the progression of hyperfiltration-associated CKD as seen in transplant donors.

A mouse model of prenatal exposure to Interleukin-6 to study the developmental origin of health and disease.

Systemic inflammation in pregnant obese women is associated with 1.5- to 2-fold increase in serum Interleukin-6 (IL-6) and newborns with lower kidney/body weight ratio but the role of IL-6 in increased susceptibility to chronic kidney (CKD) in adult progeny is not known. Since IL-6 crosses the placental barrier, we administered recombinant IL-6 (10 pg/g) to pregnant mice starting at mid-gestation yielded newborns with lower body (p < 0.001) and kidney (p < 0.001) weights. Histomorphometry indicated decreased nephrogenic zone width (p = 0.039) with increased numbers of mature glomeruli (p = 0.002) and pre-tubular aggregates (p = 0.041). Accelerated maturation in IL-6 newborns was suggested by early expression of podocyte-specific protein podocin in glomeruli, increased 5-methyl-cytosine (LC-MS analysis for CpG DNA methylation) and altered expression of certain genes of cell-cycle and apoptosis (RT-qPCR array-analysis). Western blotting showed upregulated pJAK2/pSTAT3. Thus, treating dams with IL-6 as a surrogate provides newborns to study effects of maternal systemic inflammation on future susceptibility to CKD in adulthood.

Transcription Factor ß-Catenin Plays a Key Role in Fluid Flow Shear Stress-Mediated Glomerular Injury in Solitary Kidney.

Increased fluid flow shear stress (FFSS) in solitary kidney alters podocyte function in vivo. FFSS-treated cultured podocytes show upregulated AKT-GSK3ß-ß-catenin signaling. The present study was undertaken to confirm (i) the activation of ß-catenin signaling in podocytes in vivo using unilaterally nephrectomized (UNX) TOPGAL mice with the ß-galactosidase reporter gene for ß-catenin activation, (ii) ß-catenin translocation in FFSS-treated mouse podocytes, and (iii) ß-catenin signaling using publicly available data from UNX mice. The UNX of TOPGAL mice resulted in glomerular hypertrophy and increased the mesangial matrix consistent with hemodynamic adaptation. Uninephrectomized TOPGAL mice showed an increased ß-galactosidase expression at 4 weeks but not at 12 weeks, as assessed using immunofluorescence microscopy (p < 0.001 at 4 weeks; p = 0.16 at 12 weeks) and X-gal staining (p = 0.008 at 4 weeks; p = 0.65 at 12 weeks). Immunofluorescence microscopy showed a significant increase in phospho-ß-catenin (Ser552, p = 0.005) at 4 weeks but not at 12 weeks (p = 0.935) following UNX, and the levels of phospho-ß-catenin (Ser675) did not change. In vitro FFSS caused a sustained increase in the nuclear translocation of phospho-ß-catenin (Ser552) but not phospho-ß-catenin (Ser675) in podocytes. The bioinformatic analysis of the GEO dataset, #GSE53996, also identified ß-catenin as a key upstream regulator. We conclude that transcription factor ß-catenin mediates FFSS-induced podocyte (glomerular) injury in solitary kidney.

Upregulated proteoglycan-related signaling pathways in fluid flow shear stress-treated podocytes.

The ultrafiltrate flow over the major processes and cell body generates fluid flow shear stress (FFSS) on podocytes. Hyperfiltration-associated increase in FFSS can lead to podocyte injury and detachment. Previously, we showed that FFSS-induced upregulation of the cyclooxygenase 2 (COX2)-PGE2-prostaglandin E receptor 2 (EP2) axis in podocytes activates Akt-glycogen synthase kinase-3ß-ß-catenin and MAPK/ERK signaling in response to FFSS. Integrative MultiOmics Pathway Resolution (IMPRes) is a new bioinformatic tool that enables simultaneous time-series analysis of more than two groups to identify pathways and molecular connections. In the present study, we used previously characterized COX2 [prostaglandin-endoperoxide synthase 2 (Ptgs2)], EP2 (Ptger2), and ß1-catenin (Ctnnb1) as "seed genes" from an array data set of four groups analyzed over a time course. The 3 seed genes shared 7 pathways and 50 genes of 14 pathways and 89 genes identified by IMPRes. A composite of signaling pathways highlighted the temporal molecular connections during mechanotransduction signaling in FFSS-treated podocytes. We investigated the "proteoglycans in cancer" and "galactose metabolism" pathways predicted by IMPRes. A custom-designed PCR array validated 60.7% of the genes predicted by IMPRes analysis, including genes for the above-named pathways. Further validation using Western blot analysis showed increased expression of phosho-Erbb2, phospho-mammalian target of rapamycin (mTOR), CD44, and hexokinase II (Hk2); decreased total Erbb2, galactose mutarotase (Galm), and ß-1,4-galactosyltransferase 1 (B4galt1); and unchanged total mTOR and AKT3. These findings corroborate our previously reported results. This study demonstrates the potential of the IMPRes method to identify novel pathways. Identifying the "proteoglycans in cancer" and "galactose metabolism" pathways has generated a lead to study the significance of FFSS-induced glycocalyx remodeling and possible detachment of podocytes from the glomerular matrix.

Serum glomerular albumin permeability activity: association with rapid progression to end-stage renal disease in focal segmental glomerulosclerosis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a major cause of renal failure. Sera of some FSGS patients increase glomerular albumin permeability (Palb) during in vitro testing and cause proteinuria in experimental animals. OBJECTIVES: To determine whether permeability activity of FSGS serum (Palb activity) is associated with rate of progression to renal replacement therapy (RRT). DESIGN: This is an observational study based on medical and demographic information and Palb activity testing. SETTING: Studies were performed at Medical College of Wisconsin. PATIENTS: Serum was submitted by patients' nephrologists for measurement of Palb activity. Each patient had had a biopsy diagnosis of FSGS, had reached ESRD and was on dialysis or had a functioning transplant. MEASUREMENTS: Palb activity, clinical characteristics and time between biopsy diagnosis and RRT (T-RRT) were recorded for each patient. METHODS: Palb activity was measured using established in vitro techniques. RESULTS: Palb and T-RRT were inversely correlated. Neither Palb nor T-RRT varied with demographics or medications. Kaplan-Meier survival curves showed that patients with Palb ≥ 0.5 progressed to RRT more rapidly than others. LIMITATIONS: Only patients who had reached RRT were included. Limited clinical information was available for each patient. Central verification of biopsy characteristics was not performed and detailed descriptions of renal histology were not available. CONCLUSIONS: Palb activity is associated with the rate of progression to RRT in patients with FSGS. Additional observations will be needed to verify that Palb activity predicts prognosis and is useful in stratifying patients for clinical decision making or treatment trials.

Do ASARM peptides play a role in nephrogenic systemic fibrosis?

Nephrogenic systemic fibrosis (NSF) is a devastating condition associated with gadolinium (Gd3+)-based contrast agents (GBCAs) in patients with kidney disease. The release of toxic Gd3+ from GBCAs likely plays a major role in NSF pathophysiology. The cause and etiology of Gd3+ release from GBCAs is unknown. Increased Acidic Serine Aspartate Rich MEPE-associated peptides (ASARM peptides) induce bone mineralization abnormalities and contribute to renal phosphate-handling defects in inherited hypophosphatemic rickets and tumor-induced osteomalacia. The proteolytic cleavage of related bone matrix proteins with ASARM motifs results in release of ASARM peptide into bone and circulation. ASARM peptides are acidic, reactive, phosphorylated inhibitors of mineralization that bind Ca2+ and hydroxyapatite. Since the ionic radius of Gd3+ is close to that of Ca2+, we hypothesized that ASARM peptides increase the risk of NSF by inducing release of Gd3+ from GBCAs. Here, we show 1) ASARM peptides bind and induce release of Gd3+ from GBCAs in vitro and in vivo; 2) A bioengineered peptide (SPR4) stabilizes the Gd3+-GBCA complex by specifically binding to ASARM peptide in vitro and in vivo; and 3) SPR4 peptide infusion prevents GBCA-induced NSF-like pathology in a murine model with increased ASARM peptide (Hyp mouse). We conclude ASARM peptides may play a role in NSF and SPR4 peptide is a candidate adjuvant for preventing or reducing risk of disease.

Polyomavirus nephropathy of the native kidney in a patient with rheumatoid arthritis and pulmonary fibrosis.

Polyomavirus nephropathy is commonly seen in the renal allograft setting but is uncommon in native kidneys. This paper describes polyomavirus nephropathy that developed in the native kidneys of a patient following immunosuppressive therapy for rheumatoid arthritis/Sjögren's syndrome associated lung disease. The patient presented with dyspnoea and a slow steady rise in serum creatinine. Owing to chronic immunosuppression, calcineurin-inhibitor toxicity was suspected. However, renal biopsy revealed polyomavirus nephropathy. The treatment of choice, lowered immunosuppression, was complicated by exacerbation of the patient's lung disease. This case highlights features of polyomavirus nephropathy in the native kidney, as well as the difficulty in its treatment when immunosuppressive treatment is necessary for medical comorbidities.

Renal and Hematological Effects of CLCF-1, a B-Cell-Stimulating Cytokine of the IL-6 Family.

CLCF-1 is a cytokine known for B-cell stimulation and for neurotrophic properties. We have identified CLCF-1 as a potential injurious factor in the human renal disease focal segmental glomerulosclerosis (FSGS). We investigated its effects on renal cells and renal function in in vitro and in vivo studies. Methods include measurement of the effect of CLCF-1 on phosphorylation of target molecules of the JAK/STAT pathway, on cytoskeleton and cell morphology in cultured podocytes, on albumin permeability of isolated rat glomeruli, and on tissue phosphorylation and urine albumin after acute or chronic CLCF-1 injection. In addition, cell sorting was performed to determine the presence of cells expressing CLCF-1 in spleen and bone marrow of normal mice and the effect of CLCF-1 infusion on splenic B-cell populations. CLCF-1 increased phosphorylation of STAT3 in multiple cell types, activated podocytes leading to formation of lamellipodia and decrease in basal stress fibers, increased glomerular albumin permeability, and increased STAT3 phosphorylation of peripheral blood cells and renal cortex. CLCF-1 increased urine albumin/creatinine ratio in mice and increased B-cell expression of IgG in mouse spleen. We conclude that CLCF-1 has potentially important systemic effects, alters podocyte function, and may contribute to renal dysfunction and albuminuria.

Antibodies to protein tyrosine phosphatase receptor type O (PTPro) increase glomerular albumin permeability (P(alb)).

Glomerular capillary filtration barrier characteristics are determined in part by the slit-pore junctions of glomerular podocytes. Protein tyrosine phosphatase receptor-O (PTPro) is a transmembrane protein expressed on the apical surface of podocyte foot processes. Tyrosine phosphorylation of podocyte proteins including nephrin may control the filtration barrier. To determine whether PTPro activity is required to maintain glomerular macromolecular permeability, albumin permeability (P(alb)) was studied after incubation of glomeruli from normal animals with a series of monoclonal (mAb) and polyclonal antibodies. Reagents included mAbs to rabbit and rat PTPro and polyclonal rabbit immune IgG to rat PTPro. mAb 4C3, specific to the amino acid core of PTPro, decreased its phosphatase activity and increased P(alb) of rabbit glomeruli in a time- and concentration-dependent manner. In contrast, mAb P8E7 did not diminish phosphatase activity and did not alter P(alb). Preincubation of 4C3 with PTPro extracellular domain fusion protein blocked glomerular binding and abolished permeability activity. In parallel experiments, P(alb) of rat glomeruli was increased by two mAbs (1B4 and 1D1) or by polyclonal anti-rat PTPro. We conclude that PTPro interaction with specific antibodies acutely increases P(alb). The identity of the normal ligand for PTPro and of its substrate, as well as the mechanism by which phosphatase activity of this receptor affects the filtration barrier, remain to be determined.

Serum permeability activity in steroid-resistant minimal change nephrotic syndrome is abolished by treatment of Hodgkin disease.

A circulating permeability factor is present in some patients with minimal change nephrotic syndrome (MCNS) or focal segmental glomerulosclerosis. Nephrotic syndrome occurs in less than 1% of patients with Hodgkin disease. A substance derived from T lymphocytes may be responsible for proteinuria in these patients, but a circulating permeability factor was not shown. Serum permeability activity (P(alb)) of a young man who presented with MCNS was tested over 11 years. He first was treated with oral prednisone, then cyclosporine (CsA; 4 mg/kg/d). Two years after the initial diagnosis, during CsA-induced remission of nephrotic syndrome, Hodgkin disease was diagnosed and he underwent systemic chemotherapy with doxorubicin hydrochloride, bleomycin, vinblastine, and dacarbazine and radiation therapy. P(alb) was 0.67 before CsA therapy. Although CsA treatment decreased proteinuria to protein less than 100 mg/d, P(alb) did not change. P(alb) decreased to 0.19 within 2 weeks of initiation of chemotherapy for Hodgkin disease and has remained at less than 0.17 for the last 9 years. The patient, in remission from Hodgkin disease, has normal renal function and no detectable proteinuria. This is the first demonstration of the presence of P(alb) in a patient with MCNS and subsequent Hodgkin disease. It also is the first report that aggressive chemotherapy abolishes P(alb). Although the potential causal relationship between nephrotic syndrome and Hodgkin disease in this patient is not clear, the immediate decrease in P(alb) during treatment suggests that aggressive chemotherapy may be an effective treatment for patients with high P(alb) in steroid-resistant MCNS or focal segmental glomerulosclerosis.

Arachidonic acid metabolites mediate the radiation-induced increase in glomerular albumin permeability.

Radiation-induced renal injury is characterized by proteinuria, hypertension, and progressive decline in renal function. We have previously shown that in vivo or in vitro irradiation of glomeruli with a single dose of radiation (9.5 Gy) increases glomerular albumin permeability (P(alb)) within 1 hr. The current studies tested the hypothesis that this early radiation-induced increase in P(alb) is caused by the release of arachidonic acid and by the generation of specific arachidonic acid metabolites. Glomeruli obtained from WAG/Rij/MCW rats and cultured rat glomerular epithelial and mesangial cells were studied after irradiation (9.5 Gy, single dose). Arachidonic acid release and eicosanoid synthesis by glomeruli or cultured glomerular cells were measured after irradiation, and the effect of inhibitors of phospholipase A2 (PLA2) and cyclooxygenase (COX) on the irradiation-induced increase in P(alb) was assessed. Arachidonic acid release was demonstrated within 10 mins of irradiation of isolated glomeruli and monolayer cultures of glomerular epithelial and mesangial cells. Prostaglandin F(2alpha) (PGF(2alpha)) and PGE2 release was increased after irradiation of isolated glomeruli. Blocking arachidonic acid release or COX activity before irradiation completely prevented the increase in P(alb). COX inhibition immediately after irradiation also diminished the radiation-induced increase in P(alb). We conclude that arachidonic acid and its COX metabolites play an essential role in the early cellular changes that lead to the radiation-induced increase in P(alb). Understanding of the early epigenetic effects of irradiation may lead to new intervention strategies against radiation-induced injury of normal tissues.

Nitric oxide preserves the glomerular protein permeability barrier by antagonizing superoxide.

BACKGROUND: The interaction of nitric oxide with superoxide (O2-) is a major O2- scavenging mechanism that can minimize O2 (-)-mediated oxidative stress. Glomeruli produce both nitric oxide and O2- and generation of both radicals is increased in various forms of glomerular disease. O2- increases glomerular capillary permeability to albumin (P(alb)). The present studies tested the hypothesis that nitric oxide opposes this effect, thereby preserving the glomerular protein permeability barrier. METHODS: P(alb) was determined in isolated rat glomeruli by measuring the change in glomerular volume in response to an experimental oncotic gradient. Changes in P(alb) in response to O2- generated by tumor necrosis factor-alpha (TNF-alpha) or xanthine/xanthine oxidase (X/XO) was assessed under conditions of nitric oxide depletion and repletion. RESULTS: Incubation of rat glomeruli with the nitric oxide synthase (NOS) inhibitor L-N(G)-monomethyl arginine (L-NMMA) increased P(alb.) This effect was reversed by the nitric oxide donor diethylenetriamine NONOate (DETA-NONOate) and by the superoxide dismutase (SOD) mimetic Tempol. O2- generated after incubation with TNF-alpha or X/XO increased P(alb). This effect was blocked by DETA-NONOate. CONCLUSION: We demonstrate that nitric oxide protects the glomerular filtration barrier from injury caused by O2- and suggest that inhibition of nitric oxide synthesis could enhance O2(-)-mediated oxidative injury under pathologic conditions.