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.

Glomerular Biomechanical Stress and Lipid Mediators during Cellular Changes Leading to Chronic Kidney Disease.

Hyperfiltration is an important underlying cause of glomerular dysfunction associated with several systemic and intrinsic glomerular conditions leading to chronic kidney disease (CKD). These include obesity, diabetes, hypertension, focal segmental glomerulosclerosis (FSGS), congenital abnormalities and reduced renal mass (low nephron number). Hyperfiltration-associated biomechanical forces directly impact the cell membrane, generating tensile and fluid flow shear stresses in multiple segments of the nephron. Ongoing research suggests these biomechanical forces as the initial mediators of hyperfiltration-induced deterioration of podocyte structure and function leading to their detachment and irreplaceable loss from the glomerular filtration barrier. Membrane lipid-derived polyunsaturated fatty acids (PUFA) and their metabolites are potent transducers of biomechanical stress from the cell surface to intracellular compartments. Omega-6 and ω-3 long-chain PUFA from membrane phospholipids generate many versatile and autacoid oxylipins that modulate pro-inflammatory as well as anti-inflammatory autocrine and paracrine signaling. We advance the idea that lipid signaling molecules, related enzymes, metabolites and receptors are not just mediators of cellular stress but also potential targets for developing novel interventions. With the growing emphasis on lifestyle changes for wellness, dietary fatty acids are potential adjunct-therapeutics to minimize/treat hyperfiltration-induced progressive glomerular damage and CKD.

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.

Urinary prostaglandin E2 is a biomarker of early adaptive hyperfiltration in solitary functioning kidney.

INTRODUCTION: Hyperfiltration is a major contributor to progression of chronic kidney disease (CKD) in diabetes, obesity and in individuals with solitary functioning kidney (SFK). We have proposed hyperfiltration-induced injury as a continuum of overlapping glomerular changes caused by increased biomechanical forces namely, fluid flow shear stress (FFSS) and tensile stress. We have shown that FFSS is elevated in animals with SFK and, it upregulates prostaglandin E2 (PGE2), cyclooxygenase-2 and PGE2 receptor EP2 in cultured podocytes and in uninephrectomized mice. We conceptualized urinary PGE2 as a biomarker of early effects of hyperfiltration-induced injury preceding microalbuminuria in individuals with SFK. We studied children with SFK to validate our hypothesis. METHODS: Urine samples from children with SFK and controls were analyzed for PGE2, albumin (glomerular injury biomarker) and epidermal growth factor (EGF, tubular injury biomarker). Age, gender, and Z-scores for height, weight, BMI, and blood pressure were obtained. RESULTS: Children with SFK were comparable to controls except for lower BMI Z-scores. The median values were elevated in SFK compared to control for urine PGE2 [9.1 (n = 57) vs. 5.7 (n = 72), p = 0.009] ng/mgCr and albumin [7.6 (n = 40) vs. 7.0 (n = 41), p = 0.085] µg/mgCr, but not for EGF [20098 (n = 44) vs. 18637 (n = 44), p = 0.746] pg/mgCr. Significant increase in urinary PGE2 (p = 0.024) and albumin (p = 0.019) but not EGF (p = 0.412) was observed using additional regression modeling. These three urinary analytes were independent of each other. CONCLUSION: Increased urinary PGE2 from elevated SNGFR and consequently increased FFSS during early stage of CKD precedes overt microalbuminuria and is a biomarker for early hyperfiltration-induced injury in individuals with SFK.

Hyperfiltration-mediated Injury in the Remaining Kidney of a Transplant Donor.

Kidney donors face a small but definite risk of end-stage renal disease 15 to 30 years postdonation. The development of proteinuria, hypertension with gradual decrease in kidney function in the donor after surgical resection of 1 kidney, has been attributed to hyperfiltration. Genetic variations, physiological adaptations, and comorbidities exacerbate the hyperfiltration-induced loss of kidney function in the years after donation. A focus on glomerular hemodynamics and capillary pressure has led to the development of drugs that target the renin-angiotensin-aldosterone system (RAAS), but these agents yield mixed results in transplant recipients and donors. Recent work on glomerular biomechanical forces highlights the differential effects of tensile stress and fluid flow shear stress (FFSS) from hyperfiltration. Capillary wall stretch due to glomerular capillary pressure increases tensile stress on podocyte foot processes that cover the capillary. In parallel, increased flow of the ultrafiltrate due to single-nephron glomerular filtration rate elevates FFSS on the podocyte cell body. Although tensile stress invokes the RAAS, FFSS predominantly activates the cyclooxygenase 2-prostaglandin E2-EP2 receptor axis. Distinguishing these 2 mechanisms is critical, as current therapeutic approaches focus on the RAAS system. A better understanding of the biomechanical forces can lead to novel therapeutic agents to target FFSS through the cyclooxygenase 2-prostaglandin E2-EP2 receptor axis in hyperfiltration-mediated injury. We present an overview of several aspects of the risk to transplant donors and discuss the relevance of FFSS in podocyte injury, loss of glomerular barrier function leading to albuminuria and gradual loss of renal function, and potential therapeutic strategies to mitigate hyperfiltration-mediated injury to the remaining kidney.

Mechanotransduction signaling in podocytes from fluid flow shear stress.

Recently, we and others have found that hyperfiltration-associated increase in biomechanical forces, namely, tensile stress and fluid flow shear stress (FFSS), can directly and distinctly alter podocyte structure and function. The ultrafiltrate flow over the major processes and cell body generates FFSS to podocytes. Our previous work suggests that the cyclooxygenase-2 (COX-2)-PGE2-PGE2 receptor 2 (EP2) axis plays an important role in mechanoperception of FFSS in podocytes. To address mechanotransduction of the perceived stimulus through EP2, cultured podocytes were exposed to FFSS (2 dyn/cm2) for 2 h. Total RNA from cells at the end of FFSS treatment, 2-h post-FFSS, and 24-h post-FFSS was used for whole exon array analysis. Differentially regulated genes ( P < 0.01) were analyzed using bioinformatics tools Enrichr and Ingenuity Pathway Analysis to predict pathways/molecules. Candidate pathways were validated using Western blot analysis and then further confirmed to be resulting from a direct effect of PGE2 on podocytes. Results show that FFSS-induced mechanotransduction as well as exogenous PGE2 activate the Akt-GSK3ß-ß-catenin (Ser552) and MAPK/ERK but not the cAMP-PKA signal transduction cascades. These pathways are reportedly associated with FFSS-induced and EP2-mediated signaling in other epithelial cells as well. The current regimen for treating hyperfiltration-mediated injury largely depends on targeting the renin-angiotensin-aldosterone system. The present study identifies specific transduction mechanisms and provides novel information on the direct effect of FFSS on podocytes. These results suggest that targeting EP2-mediated signaling pathways holds therapeutic significance for delaying progression of chronic kidney disease secondary to hyperfiltration.

Multiple Targets for Novel Therapy of FSGS Associated with Circulating Permeability Factor.

A plasma component is responsible for altered glomerular permeability in patients with focal segmental glomerulosclerosis. Evidence includes recurrence after renal transplantation, remission after plasmapheresis, proteinuria in infants of affected mothers, transfer of proteinuria to experimental animals, and impaired glomerular permeability after exposure to patient plasma. Therapy may include decreasing synthesis of the injurious agent, removing or blocking its interaction with cells, or blocking signaling or enhancing cell defenses to restore the permeability barrier and prevent progression. Agents that may prevent the synthesis of the permeability factor include cytotoxic agents or aggressive chemotherapy. Extracorporeal therapies include plasmapheresis, immunoadsorption with protein A or anti-immunoglobulin, or lipopheresis. Oral or intravenous galactose also decreases Palb activity. Studies of glomeruli have shown that several strategies prevent the action of FSGS sera. These include blocking receptor-ligand interactions, modulating cell reactions using indomethacin or eicosanoids 20-HETE or 8,9-EET, and enhancing cytoskeleton and protein interactions using calcineurin inhibitors, glucocorticoids, or rituximab. We have identified cardiotrophin-like cytokine factor 1 (CLCF-1) as a candidate for the permeability factor. Therapies specific to CLCF-1 include potential use of cytokine receptor-like factor (CRLF-1) and inhibition of Janus kinase 2. Combined therapy using multiple modalities offers therapy to reverse proteinuria and prevent scarring.

Role of biomechanical forces in hyperfiltration-mediated glomerular injury in congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) including solitary kidney constitute the main cause of progressive chronic kidney disease (CKD) in children. Children born with CAKUT develop signs of CKD only during adolescence and do not respond to renin-angiotensin-aldosterone system blockers. Early cellular changes underlying CKD progression to end-stage renal disease by early adulthood are not well understood. The mechanism of maladaptive hyperfiltration that occurs from loss of functional nephrons, including solitary kidney, is not clear. We re-examine the phenomenon of hyperfiltration in the context of biomechanical forces with special reference to glomerular podocytes. Capillary stretch exerts tensile stress on podocytes through the glomerular basement membrane. The flow of ultrafiltrate over the cell surface directly causes fluid flow shear stress (FFSS) on podocytes. FFSS on the podocyte surface increases 1.5- to 2-fold in animal models of solitary kidney and its effect on podocytes is a subject of ongoing research. Podocytes (i) are mechanosensitive to tensile and shear forces, (ii) use prostaglandin E2, angiotensin-II or nitric oxide for mechanoperception and (iii) use specific signaling pathways for mechanotransduction. We discuss (i) the nature of and differences in cellular responses to biomechanical forces, (ii) methods to study biomechanical forces and (iii) effects of biomechanical forces on podocytes and glomeruli. Future studies on FFSS will likely identify novel targets for strategies for early intervention to complement and strengthen the current regimen for treating children with CAKUT.

Hyperfiltration-associated biomechanical forces in glomerular injury and response: Potential role for eicosanoids.

Hyperfiltration is a well-known risk factor in progressive loss of renal function in chronic kidney disease (CKD) secondary to various diseases. A reduced number of functional nephrons due to congenital or acquired cause(s) results in hyperfiltration in the remnant kidney. Hyperfiltration-associated increase in biomechanical forces, namely pressure-induced tensile stress and fluid flow-induced shear stress (FFSS) determine cellular injury and response. We believe the current treatment of CKD yields limited success because it largely attenuates pressure-induced tensile stress changes but not the effect of FFSS on podocytes. Studies on glomerular podocytes, tubular epithelial cells and bone osteocytes provide evidence for a significant role of COX-2 generated PGE2 and its receptors in response to tensile stress and FFSS. Preliminary observations show increased urinary PGE2 in children born with a solitary kidney. FFSS-induced COX2-PGE2-EP2 signaling provides an opportunity to identify targets and, for developing novel agents to complement currently available treatment.

Normalization of Testosterone Levels After Testosterone Replacement Therapy Is Not Associated With Reduced Myocardial Infarction in Smokers.

OBJECTIVE: To examine the effect of cigarette smoking (CS) status and total testosterone (TT) levels after testosterone replacement therapy (TRT) on all-cause mortality, myocardial infarction (MI), and stroke in male smokers and nonsmokers without history of MI and stroke. PARTICIPANTS AND METHODS: Data from 18,055 males with known CS status and low TT levels who received TRT at the Veterans Health Administration between December 1, 1999, and May 31, 2014, were grouped into (1) current smokers with normalized TT, (2) current smokers with nonnormalized TT, (3) nonsmokers with normalized TT, and (4) nonsmokers with nonnormalized TT. Combined effect of CS status and TT level normalization after TRT on all-cause mortality, MI, and stroke was compared using propensity score-weighted Cox proportional hazard models. RESULTS: Normalization of serum TT levels in nonsmokers was associated with a significant decrease in all-cause mortality (hazard ratio [HR]=0.526; 95% CI, 0.477-0.581; P<.001) and MI (HR=0.717; 95% CI, 0.522-0.986; P<.001). Among current smokers, normalization of serum TT levels was associated with a significant decrease in only all-cause mortality (HR=0.563; 95% CI, 0.488-0.649; P<.001) without benefit in MI (HR=1.096; 95% CI, 0.698-1.720; P=.69). Importantly, compared with nonsmokers with normalized TT, all-cause mortality (HR=1.242; 95% CI, 1.104-1.396; P<.001), MI (HR=1.706; 95% CI, 1.242-2.342; P=.001), and stroke (HR=1.590; 95% CI, 1.013-2.495; P=.04) were significantly higher in current smokers with normalized TT. CONCLUSION: We conclude that active CS may negate the protective effect of testosterone level normalization on all-cause mortality and MI after TRT.

Association Between Testosterone Replacement Therapy and the Incidence of DVT and Pulmonary Embolism: A Retrospective Cohort Study of the Veterans Administration Database.

BACKGROUND: Testosterone replacement therapy (TRT) prescriptions have increased several-fold in the last decade. There have been concerns regarding a possible increased incidence of DVT and pulmonary embolism (PE) with TRT. Few data support the association between TRT and DVT/PE. We evaluated the incidence of DVT and PE in men who were prescribed TRT for low serum total testosterone (sTT) levels. METHODS: This is a retrospective cohort study, conducted using data obtained from the Veterans Affairs Informatics and Computing Infrastructure. We compared the incidence of DVT/PE between those who received TRT and subsequently had normal on-treatment sTT levels (Gp1), those who received TRT but continued to have low on-treatment sTT (Gp2), and those who did not receive TRT (Gp3). Those with prior history of DVT/PE, cancer, hypercoagulable state, and chronic anticoagulation were excluded. RESULTS: The final cohort consisted of 71,407 subjects with low baseline sTT. Of these, 10,854 did not receive TRT (Gp3) and 60,553 received TRT. Of those who received TRT, 38,362 achieved normal sTT (Gp1) while 22,191 continued to have low sTT (Gp2). The incidence of DVT/PE was 0.5%, 0.4%, and 0.4% in Gp1, Gp2, and Gp3, respectively. Univariate, multivariate, and stabilized inverse probability of treatment weights analyses showed no statistically significant difference in DVT/PE-free survival between the various groups. CONCLUSIONS: This study did not detect a significant association between testosterone replacement therapy and risk of DVT/PE in adult men with low sTT who were at low to moderate baseline risk of DVT/PE.

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.

Fluorescence dilution technique for measurement of albumin reflection coefficient in isolated glomeruli.

This study describes a high-throughput fluorescence dilution technique to measure the albumin reflection coefficient (σAlb) of isolated glomeruli. Rats were injected with FITC-dextran 250 (75 mg/kg), and the glomeruli were isolated in a 6% BSA solution. Changes in the fluorescence of the glomerulus due to water influx in response to an imposed oncotic gradient was used to determine σAlb. Adjustment of the albumin concentration of the bath from 6 to 5, 4, 3, and 2% produced a 10, 25, 35, and 50% decrease in the fluorescence of the glomeruli. Pretreatment of glomeruli with protamine sulfate (2 mg/ml) or TGF-ß1 (10 ng/ml) decreased σAlb from 1 to 0.54 and 0.48, respectively. Water and solute movement were modeled using Kedem-Katchalsky equations, and the measured responses closely fit the predicted behavior, indicating that loss of albumin by solvent drag or diffusion is negligible compared with the movement of water. We also found that σAlb was reduced by 17% in fawn hooded hypertensive rats, 33% in hypertensive Dahl salt-sensitive (SS) rats, 26% in streptozotocin-treated diabetic Dahl SS rats, and 21% in 6-mo old type II diabetic nephropathy rats relative to control Sprague-Dawley rats. The changes in glomerular permeability to albumin were correlated with the degree of proteinuria in these strains. These findings indicate that the fluorescence dilution technique can be used to measure σAlb in populations of isolated glomeruli and provides a means to assess the development of glomerular injury in hypertensive and diabetic models.

Normalization of testosterone level is associated with reduced incidence of myocardial infarction and mortality in men.

AIMS: There is a significant uncertainty regarding the effect of testosterone replacement therapy (TRT) on cardiovascular (CV) outcomes including myocardial infarction (MI) and stroke. The aim of this study was to examine the relationship between normalization of total testosterone (TT) after TRT and CV events as well as all-cause mortality in patients without previous history of MI and stroke. METHODS AND RESULTS: We retrospectively examined 83 010 male veterans with documented low TT levels. The subjects were categorized into (Gp1: TRT with resulting normalization of TT levels), (Gp2: TRT without normalization of TT levels) and (Gp3: Did not receive TRT). By utilizing propensity score-weighted Cox proportional hazard models, the association of TRT with all-cause mortality, MI, stroke, and a composite endpoint was compared between these groups. The all-cause mortality [hazard ratio (HR): 0.44, confidence interval (CI) 0.42-0.46], risk of MI (HR: 0.76, CI 0.63-0.93), and stroke (HR: 0.64, CI 0.43-0.96) were significantly lower in Gp1 (n = 43 931, median age = 66 years, mean follow-up = 6.2 years) vs. Gp3 (n = 13 378, median age = 66 years, mean follow-up = 4.7 years) in propensity-matched cohort. Similarly, the all-cause mortality (HR: 0.53, CI 0.50-0.55), risk of MI (HR: 0.82, CI 0.71-0.95), and stroke (HR: 0.70, CI 0.51-0.96) were significantly lower in Gp1 vs. Gp2 (n = 25 701, median age = 66 years, mean follow-up = 4.6 years). There was no difference in MI or stroke risk between Gp2 and Gp3. CONCLUSION: In this large observational cohort with extended follow-up, normalization of TT levels after TRT was associated with a significant reduction in all-cause mortality, MI, and stroke.

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.

Efficacy of galactose and adalimumab in patients with resistant focal segmental glomerulosclerosis: report of the font clinical trial group.

BACKGROUND: Patients with resistant focal segmental glomerulosclerosis (FSGS) who are unresponsive to corticosteroids and other immunosuppressive agents are at very high risk of progression to end stage kidney disease. In the absence of curative treatment, current therapy centers on renoprotective interventions that reduce proteinuria and fibrosis. The FONT (Novel Therapies for Resistant FSGS) Phase II clinical trial (NCT00814255, Registration date December 22, 2008) was designed to assess the efficacy of adalimumab and galactose compared to standard medical therapy which was comprised of lisinopril, losartan, and atorvastatin. METHODS: Key eligibility criteria were biopsy confirmed primary FSGS or documentation of a causative genetic mutation, urine protein:creatinine ratio >1.0 g/g, and estimated glomerular filtration rate (eGFR) >40 ml/min/1.73 m(2). The experimental treatments - adalimumab, galactose, standard medical therapy-- were administered for 26 weeks. The primary endpoint was a 50 % reduction in proteinuria with stable eGFR. RESULTS: Thirty-two subjects were screened and 21 were assigned to one of the three study arms. While none of the adalimumab-treated subjects achieved the primary outcome, 2 subjects in the galactose and 2 in the standard medical therapy arm had a 50 % reduction in proteinuria without a decline in eGFR. The proteinuria response did not correlate with serial changes in the serum glomerular permeability activity measured by the Palb assay or soluble urokinase plasminogen activator receptor (suPAR). There were no serious adverse effects related to treatments in the study. CONCLUSIONS: Recruitment into this trial that addressed patients with resistant FSGS fell short of the enrollment goal. Our findings suggest that future studies of novel therapies for rare glomerular diseases such as FSGS may benefit from enrollment of patients earlier in the course of their disease. In addition, better identification of patients who are likely to respond to a new treatment based on biomarkers suggesting involvement of the disease pathway targeted by the experimental agent may reduce the required sample size and increase the likelihood of a favorable outcome.

Janus kinase 2/signal transducer and activator of transcription 3 inhibitors attenuate the effect of cardiotrophin-like cytokine factor 1 and human focal segmental glomerulosclerosis serum on glomerular filtration barrier.

Recurrence of idiopathic focal segmental glomerulosclerosis (FSGS) after renal transplantation is believed to be caused by a circulating factor(s). We detected cardiotrophin-like cytokine factor 1 (CLCF1), a member of the interleukin 6 family, in the plasma from patients with recurrent FSGS. We hypothesized that CLCF1 contributes to the effect of FSGS serum on the glomerular filtration barrier in vitro. Presently, we studied the effect of CLCF1 on isolated rat glomeruli using an in vitro assay of albumin permeability (P(alb)). CLCF1 (0.05-100 ng/mL) increased P(alb) and caused maximal effect at 5-10 ng/mL (P < 0.001). The increase in Palb was analogous to the effect of FSGS serum. Anti-CLCF1 monoclonal antibody blocked the CLCF1-induced increase in P(alb) and significantly attenuated the effect of FSGS serum (P < 0.001). The heterodimer composed of CLCF1 and cosecreted molecule cytokine receptor-like factor 1 (CRLF1) attenuated the increase in P(alb) caused by CLCF1 or FSGS serum. Western blot analysis showed that CLCF1 upregulated phosphorylation of signal transducer and activator of transcription 3 (STAT3) (Tyr705) in glomeruli. This effect was diminished by the heterodimer CLCF1-CRLF1. Janus kinase 2 (JAK2) inhibitor BMS-1119543 or STAT3 inhibitor Stattic significantly blocked the effect of CLCF1 or FSGS serum on P(alb) (P < 0.001). These novel findings suggest that when monomeric CLCF1 increases P(alb), the heterodimer CLCF1-CRLF1 may protect the glomerular filtration barrier. We speculate that albuminuria in FSGS is related to qualitative or quantitative changes in the CLCF1-CRLF1 complex, and that JAK2 or STAT3 inhibitors may be novel therapeutic agents to treat FSGS.

Ethanol at low concentrations protects glomerular podocytes through alcohol dehydrogenase and 20-HETE.

Clinical studies suggest cardiovascular and renal benefits of ingesting small amounts of ethanol. Effects of ethanol, role of alcohol dehydrogenase (ADH) or of 20-hydroxyeicosatetraenoic acid (20-HETE) in podocytes of the glomerular filtration barrier have not been reported. We found that mouse podocytes at baseline generate 20-HETE and express ADH but not CYP2e1. Ethanol at high concentrations altered the actin cytoskeleton, induced CYP2e1, increased superoxide production and inhibited ADH gene expression. Ethanol at low concentrations upregulated the expression of ADH and CYP4a12a. 20-HETE, an arachidonic acid metabolite generated by CYP4a12a, blocked the ethanol-induced cytoskeletal derangement and superoxide generation. Ethanol at high concentration or ADH inhibitor increased glomerular albumin permeability in vitro. 20-HETE and its metabolite produced by ADH activity, 20-carboxy-arachidonic acid, protected the glomerular permeability barrier against an ADH inhibitor, puromycin or FSGS permeability factor. We conclude that ADH activity is required for glomerular function, 20-HETE is a physiological substrate of ADH in podocytes and that podocytes are useful biosensors to understand glomeruloprotective effects of ethanol.