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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 E 2 (PGE 2 ), cyclooxygenase-2 and PGE 2 receptor EP2 in cultured podocytes and in uninephrectomized mice. We conceptualized urinary PGE 2 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 PGE 2 , 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 PGE 2 [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 PGE 2 (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 PGE 2 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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

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 PGE 2 and its receptors in response to tensile stress and FFSS. Preliminary observations show increased urinary PGE 2 in children born with a solitary kidney. FFSS-induced COX2-PGE 2 -EP 2 signaling provides an opportunity to identify targets and, for developing novel agents to complement currently available treatment., (Published by Elsevier Inc.)

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-β1 (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., (Copyright © 2015 the American Physiological Society.)

Hyperfiltration subjects podocytes to increased tensile stress and fluid flow shear stress (FFSS). We showed a 1.5- to 2.0-fold increase in FFSS in uninephrectomized animals and altered podocyte actin cytoskeleton and increased synthesis of prostaglandin E2 (PGE2) following in vitro application of FFSS. We hypothesized that increased FFSS mediates cellular changes through specific receptors of PGE2. Presently, we studied the effect of FFSS on cultured podocytes and decapsulated isolated glomeruli in vitro, and on solitary kidney in uninephrectomized sv129 mice. In cultured podocytes, FFSS resulted in increased gene and protein expression of cyclooxygenase (COX)-2 but not COX-1, prostanoid receptor EP2 but not EP4, and increased synthesis and secretion of PGE2, which were effectively blocked by indomethacin. Next, we developed a special flow chamber for applying FFSS to isolated glomeruli to determine its effect on an intact glomerular filtration barrier by measuring change in albumin permeability (Palb) in vitro. FFSS caused an increase in Palb that was blocked by indomethacin (P < 0.001). Finally, we show that unilateral nephrectomy in sv129 mice resulted in glomerular hypertrophy (P = 0.006), increased glomerular expression of COX-2 (P < 0.001) and EP2 (P = 0.039), and increased urinary albumin excretion (P = 0.001). Activation of the COX-2-PGE2-EP2 axis appears to be a specific response to FFSS in podocytes and provides a mechanistic basis for alteration in podocyte structure and the glomerular filtration barrier, leading to albuminuria in hyperfiltration-mediated kidney injury. The COX-2-PGE2-EP2 axis is a potential target for developing specific interventions to ameliorate the effects of hyperfiltration-mediated kidney injury in the progression of chronic kidney disease.

Endothelin (ET) 1 is a potent vasoactive peptide implicated in the pathogenesis of hypertension and renal disease. The aim of the current study was to test the hypotheses that ET-1 increases albumin permeability of glomeruli isolated from normal rats and that chronic ET-1 infusion will increase glomerular permeability and inflammation independent of blood pressure. Glomerular permeability to albumin was determined from the change in glomerular volume induced by exposing isolated glomeruli to oncotic gradients. Incubation of glomeruli taken from normal rats with ET-1 at a concentration that did not produce direct glomerular contraction (1 nmol/L) significantly increased glomerular permeability to albumin, reaching a maximum after 4 hours. Chronic ET-1 infusion for 2 weeks in Sprague-Dawley rats significantly increased glomerular permeability to albumin and nephrin excretion rate, effects that were attenuated in rats given an ET(A) receptor antagonist (ABT-627, 5 mg/kg per day). Urinary protein and albumin excretion and mean arterial pressure (telemetry) were not changed by ET-1 infusion. Acute incubation of glomeruli isolated from ET-1-infused rats with the selective ET(A) antagonist significantly reduced glomerular permeability to albumin, an effect not observed with acute treatment with a selective ET(B) antagonist. Chronic ET-1 infusion increased glomerular and plasma soluble intercellular adhesion molecule 1 and monocyte chemoattractant protein 1 and elevated the number of macrophages and lymphocytes in renal cortices (ED-1 and CD3-positive staining, respectively). These effects were all attenuated in rats given an ET(A) selective antagonist. These data support the hypothesis that ET-1 directly increases glomerular permeability to albumin and renal inflammation via ET(A) receptor activation independent of changes in arterial pressure.

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.

Glomerular dysfunction and proteinuria characterize focal segmental glomerulosclerosis (FSGS) associated with chronic kidney disease. FSGS is resistant to treatment and a circulating permeability factor (FSPF) frequently causes post-renal transplantation recurrence. In order to explore the role of 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs), we determined their effect on FSPF-induced increase in glomerular albumin permeability (P alb) using an in vitro assay. Exogenous 8,9-EET (1-1000 nM) dose-dependently prevented the FSPF-induced increase in P alb. The other three EET regioisomers, 8,9-EET metabolite, 8,9-dihydroxyeicosatrienoic acid and unrelated 11,14-eicosadienoic acid (100 nM each) were not effective suggesting specificity of the observed glomerular protection by 8,9-EET. Synthetic analogs of 8,9-EET containing one double bond antagonized the effect of 8,9-EET on the FSPF-induced increase in P alb. Analogs containing two double bonds did not antagonize the effect of 8,9-EET and significantly blocked the FSPF-induced increase in P alb. These novel findings suggest a unique protective role for 8,9-EET in the glomerulus. Stable analogs of 8,9-EET may be valuable in developing effective management/treatment of glomerular dysfunction.

Increased infiltration of the kidney by mast cells is associated with proteinuria, and interstitial fibrosis in various renal diseases. Mast cells produce serine proteases including tryptase and chymase (MCC) that act via protease-activated receptors (PARs) to induce synthesis of fibrogenic cytokines by renal cells. In the present study, we investigated direct effect of MCC and role of PARs on glomerular albumin permeability (P(alb)). Isolated rat glomeruli were incubated with MCC (0.1, 1, 10, and 100 ng/ml) for 5-30 min in presence or absence of PAR-1 and PAR-2 blocking antibodies. P(alb) was determined from the change in glomerular volume in response to an albumin oncotic gradient. The effect of direct activation of PARs on P(alb) was verified by incubating glomeruli with synthetic hexapeptide known to activate PAR-1 and PAR-2. MCC increased P(alb) of isolated rat glomeruli in a dose- and time-dependent manner. Blocking PAR-2 prevented MCC-mediated increase in P(alb). RT-PCR analysis of glomerular RNA demonstrated the presence of constitutively expressed PAR-1, -2, and -3 and low levels of PAR-4. In addition, direct activation of PAR-2 by hexapeptide SLIGKV increased P(alb) comparable to MCC, whereas PAR-1 activation by TFLLRN had no effect on P(alb). Our results document that MCC induces increase in P(alb) and that this effect is mediated through PAR-2. MCC may also play a role in renal scarring. We propose that inhibiting MCC activity or blocking the activation of PAR-2 may provide new targets for therapy in renal diseases.

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.

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.

This study examined the role of transforming growth factor-beta (TGF-beta) in altering the glomerular permeability to albumin (P(alb)) during hypertension development in Dahl salt-sensitive (Dahl S) rats and whether TGF-beta acts by inhibiting the glomerular production of 20-HETE. The results indicate that the renal expression of TGF-beta doubles in Dahl S rats fed a high-salt diet for 7 days, and this is associated with a marked rise in P(alb) from 0.19+/-0.04 to 0.75+/-0.01 and changes in the ultrastructure of the glomerular filtration barrier. Chronic treatment of Dahl S rats with a TGF-beta neutralizing antibody prevented the increase in P(alb) and preserved the structure of glomerular capillaries. It had no effect on the rise in blood pressure produced by the high-salt diet. In other studies, preincubation of glomeruli isolated from Sprague Dawley rats with TGF-beta1 (10 ng/mL) for 15 minutes increased P(alb) from 0.01+/-0.01 to 0.60+/-0.02. This was associated with inhibition of the glomerular production of 20-HETE from 221+/-11 to 3.4+/-0.5 mug per 30 minutes per milligram of protein. Pretreatment of Sprague Dawley glomeruli with a stable analog of 20-HETE, 20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid, reduced baseline P(alb) and opposed the effects of TGF-beta to increase P(alb). These studies indicate that upregulation of the glomerular formation of TGF-beta may contribute to the development of proteinuria and glomerular injury early in hypertension development in Dahl S rats by increasing P(alb) through inhibition of the glomerular production of 20-HETE.

Focal segmental glomerulosclerosis (FSGS) is characterized by steroid resistant nephrotic syndrome and progression to end-stage renal disease. Proteinuria in certain patients with FSGS may be caused by a circulating factor (FSGS permeability factor [FSPF]). The current report documents the biochemical characteristics and the biological and molecular effects of 70% ammonium sulfate supernatant of plasma from patients with recurrence of FSGS after transplantation (FSGS 70% supernatant). FS permeability activity, defined as the capacity of plasma from patients with FSGS to increase albumin permeability (P(alb)) of isolated glomeruli, was assessed in vitro. Permeability activity was not affected by lyophilization. FSPF bound strongly to matrices containing Mono-Q anion exchanger or protein A. It eluted from matrix-bound Cibacron blue F3GA over a wide range of salt concentrations, indicating a potential binding with other proteins, such as albumin. FSPF caused a maximal increase in P(alb) within 2 mins of incubation in vitro. Cellular proteins isolated from glomeruli with increased P(alb) showed decreased tyrosine phosphorylation of focal adhesion kinase, paxillin, and other proteins. Tyrosine phosphatase ]inhibition prevented the increase in P(alb). Intravenous administration of as little as 3 mg protein in FSGS 70% supernatant increased P(alb), while 9 mg or more were required to produce proteinuria. We conclude that FSPF is a low-molecular-weight protein, carries an anionic charge, and binds to protein A. Effects of FSPF on the glomerular permeability barrier are rapid and dose dependent and involve signaling through altered phosphorylation of cellular proteins. Identification of these biochemical and biological characteristics may be used to design strategies for removing FSPF from circulation and for purification and identification of this factor.

A serum or plasma factor in certain patients with focal segmental glomerulosclerosis (FSGS) has been found to increase glomerular albumin permeability (P(alb)) and causes proteinuria in experimental animals. High P(alb) is associated with recurrence of FSGS after transplantation, but serial studies of P(alb) activity in patients with native kidney FSGS have not been performed, and the relationship between P(alb) and remission of proteinuria is not known. This study was designed to determine P(alb) activity before, during, and after 24 wk of treatment with cyclosporine or placebo given as part of a randomized controlled trial in steroid-resistant FSGS patients with nephrotic range proteinuria. Pretreatment P(alb) averaged 0.36 +/- 0.22 and was not significantly different between treatment groups and was not altered during or after the test medication. There was no association between P(alb) activity and remission or relapse in proteinuria. The average P(alb) activity in native kidney FSGS was lower than in previously reported patients with posttransplant recurrence of the disease, and its level did not vary during the course of the study. The antiproteinuric effect of cyclosporine appeared independent of changes in P(alb). This finding is consistent with a direct effect of cyclosporine on glomerular barrier function and/or that within this group of patients the variations in proteinuria are not reflected in changes in Palb because of its limits in terms of reproducibility and responsiveness.

Metallothioneins (MT) are low-molecular-weight, heat-stable, cysteine-rich proteins with four isoforms. MT-I and MT-II are ubiquitous and are induced by oxidative, physical, and chemical stress. MT-I is an efficient scavenger of superoxide (*O2) and hydroxyl ion (OH(-)). We have demonstrated that *O2 and hypohalous acid can cause an increase in glomerular albumin permeability (P(alb)) in vitro. The purpose of this study was to document the protective effect of MT gene product on the *O2-mediated increase in P(alb). Glomeruli from Sprague-Dawley rats in 4% BSA medium were incubated for 4 hr at 37 degrees C in duplicate tubes. Each set contained glomeruli alone or with 5 microM Cd(++), 0.3 mM Spermine-NONOate (NO donor), 0.3 mM Sulfo-NONOate (nitrous oxide donor), 0.6 mM SNP (nonspecific NO donor) and SNP + carboxy-PTIO (10 mg/ml). After incubation, one set of tubes was used to isolate total RNA for the measurement of the mRNA levels of MT-I by reverse transcriptase polymerase chain reaction (RT-PCR). Duplicate tubes were incubated for an additional 10 min with 10 nM of *O2, and P(alb) was measured using video microscopy. RT-PCR of total RNA from Cd(++) and Spermine-NONOate treated glomeruli revealed a 2-fold induction of MT-I expression at the mRNA level. *O2 caused a significant increase in P(alb) (0.8 +/- 0.06 vs. control 0.0 +/- 0.12, P < 0.05) and induction of MT-I in glomeruli by Cd(++) or by Spermine-NONOate blocked this effect (0.21 +/- 0.12 and 0.24 +/- 0.19, respectively, P < 0.05 vs. *O2). In contrast, Sulfo-NONOate and SNP did not induce mRNA for MT-I in glomeruli and did not provide protection against *O2-mediated increase in P(alb.) We conclude that MT-I gene products may play an important role in protecting the glomerular filtration barrier from the injury induced by reactive oxygen species in immune and/or nonimmune renal diseases.

Demonstrates the presence of angiotensin II receptors in cultured glomerular epithelial cells by immunoblotting. How glomerular epithelial cell cultures was prepared; Measurement of cyclic adenosine monophosphate (cAMP) glomerular epithelial cells; Importance of glomerular epithelial cells in the regulation of glomerular function.

Background: Heavy proteinuria and progressive renal injury recur after transplantation in up to 40 percent of patients with renal failure caused by idiopathic focal segmental glomerulosclerosis. A circulating factor may be responsible for this recurrence. Methods: To determine whether patients with focal segmental glomerulosclerosis have a circulating factor capable of causing glomerular injury, we tested serum samples from 100 patients with the disorder in an in vitro assay of glomerular permeability to albumin. Of the 56 patients who had undergone renal transplantation, 33 had recurrences. Sixty-four patients, many of whom had undergone transplantation, were being treated with dialysis. Thirty-one patients with other renal diseases and nine normal subjects were also studied. Results: The 33 patients with recurrent focal segmental glomerulosclerosis after transplantation had a higher mean (±SE) value for permeability to albumin (0.47±0.06) than the normal subjects (0.06±0.07) or the patients who did not have recurrences (0.14±0.06). After plasmapheresis in six patients with recurrences, the permeability was reduced (from 0.79±0.06 to 0.10±0.05, P = 0.008), and proteinuria was significantly decreased. Patients with corticosteroid-sensitive nephrotic syndrome or with membranous nephropathy after transplantation had low levels of serum activity. The circulating factor bound to protein A and hydrophobic-interaction columns and had an apparent molecular mass of about 50 kd. Conclusions: A circulating factor found in some patients with focal segmental glomerulosclerosis is associated with recurrent disease after renal transplantation and may be responsible for initiating the renal injury. (N Engl J Med 1996;334:878-83.) [ABSTRACT FROM AUTHOR]