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.

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.

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.

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.

Comparing directly measured versus mathematically calculated free serum 25-hydroxy vitamin D level in children.

INTRODUCTION: 25-Hydroxy vitamin D (25(OH)D) is essential for calcium homeostasis and bone metabolism. The majority of serum 25(OH)D is bound to vitamin D-binding protein (VDBP) (~ 85%) and to albumin (~ 15%), with only a miniscule amount circulating as free 25(OH)D. Free 25(OH)D can be calculated mathematically by Bikle method from the concentrations of total 25(OH)D, VDBP, and albumin or measured directly by ELISA. A direct head-to-head comparison between the two methods has not been done in children. MATERIALS AND METHODS: The objective of the study was to compare the mathematically calculated versus directly measured free 25(OH) vitamin D in children. Serum samples from 74 children (ages 1-19 years) were simultaneously analyzed for total 25(OH)D, serum albumin, VDBP, and free 25(OH)D. Pearson correlation analysis and Bland-Altman plot were used to evaluate agreement between the two methods. RESULTS: The mean age was 9.1 ± 5.1 years, with 61% boys, 76% Caucasians, and 24% African-Americans. The mean ± SD for total 25(OH)D was 38.7 ± 12.8 ng/mL, bioavailable 25(OH)D 3.1 ± 1.1 ng/mL, mathematically calculated free 25(OH)D 8.4 ± 3.2 pg/mL, and directly measured free 25(OH)D 8.9 ± 3.6 pg/mL. Pearson correlation reflected a significant correlation between mathematically calculated and directly measured free 25(OH)D (r = 0.66, p < 0.0005). Bland-Altman plot reflected a tight agreement within a 95% limit of agreement (mean = - 0.026 ± 2SD). CONCLUSIONS: The directly measured and mathematically calculated free 25(OH)D are in close agreement and are interchangeable. Depending on the local availability of instruments and methods, free 25(OH)D can be either directly measured or mathematically calculated.

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.

Use of calcimimetics in children with normal kidney function.

The calcium-sensing receptor (CaSR) plays an important role in the homeostasis of serum ionized calcium by regulating parathyroid hormone (PTH) secretion and tubular calcium handling. Calcimimetics, which act by allosteric modulation of the CaSR, mimic hypercalcemia resulting in suppression of PTH release and increase in calciuria. Mostly used in children to treat secondary hyperparathyroidism associated with advanced renal failure, we have shown that calcimimetics can also be successfully used in children with bone and mineral disorders in which elevated PTH plays a detrimental role in skeletal pathophysiology in the face of normal kidney function. The current review briefly discusses the role of the CaSR and calcimimetics in calcium homeostasis, and then addresses the potential applications of calcimimetics in children with normal kidney function with disorders in which suppression of PTH is beneficial.

Successful Brace Treatment of Pectus Carinatum in Osteogenesis Imperfecta Using the Dynamic Compression System.

Osteogenesis imperfecta (OI) is a genetic disorder of collagen resulting in a "fragile" skeleton with increased fracture risk and other complications, dependent on the specific variant. Pectus deformities of the chest wall, while not common, can be associated with OI. The use of a pectus carinatum brace in a patient with OI poses unknown risks for fractures and adverse treatment outcomes. We successfully applied external compression bracing using the dynamic compression system to one such patient. This case illustrates the ability to treat an OI patient with pectus carinatum using a nonsurgical brace, without complications, resulting in an excellent cosmetic result.

Risk Factors for Short- and Long-Term Outcomes in Children With STEC-HUS/D+ HUS: A Single-Center Experience.

Background. Hemolytic uremic syndrome (HUS) is one of the common causes for acute kidney injury in childhood. Objective. The goals of our study were to identify risk factors for short-term complications and long-term outcomes of chronic kidney disease (CKD) in Shiga toxin-producing Escherichia coli (STEC)-HUS and other diarrhea positive (D+) HUS. Methods. Retrospective chart review was obtained of 58 pediatric patients treated for STEC-HUS and other D+ HUS between February 2002 and January 2011. Results. Thirty-three patients (56.9%) required dialysis. Dialysis was more likely initiated if a patient was a female (P < .012), oliguric (urine output < 0.5 mL/kg/h, P < .0005), or hemoglobin (HGB) level >10 g/dL (P = .009) at admission. Neurological complications developed only among 5 dialyzed patients (P < .042), and were more common if the patient received hemodialysis (HD) compared with peritoneal dialysis (P < .0005). CKD was noted during the subsequent follow-up clinic visits in 5 patients (8.6%). Those who developed CKD received HD (P = .002), dialysis for >10 days (P = .0004), or HGB level >10 g/dL (P = .034) at admission. Conclusions. Children with STEC-HUS/D+ HUS who may need dialysis are identified by female gender, lower urine output, higher serum creatinine level, and higher HGB at admission. They are at higher risk developing central nervous system complications especially if they needed HD. Children requiring >10 days of dialysis are at risk for development of CKD.

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.

The Effects of Diuretics on Mineral and Bone Metabolism.

The effects of diuretics on water and electrolyte metabolism are well-established, but less known to the clinician are their effects on bone and mineral metabolism, and in particular on that of calcium homeostasis. In general, and clinically most relevant, diuretics acting at the thick ascending limb of the loop of Henle cause loss of calcium into the urine, thus making them a useful tool in treating hypercalcemia. However the hypercalciuria caused by loop diuretics may lead to the development of urolithiasis and nephrocalcinosis, as well as secondary hyperparathyroidism and bone disease. On the other hand, thiazide diuretics that act more distally, increase tubular calcium reabsorption, thus providing protection against hypercalciuria, and with that may raise serum calcium, suppress PTH secretion and improve bone metabolism. Additional hypocalciuric effect may be observed with the use of potassium-sparing diuretics. This review will address the effects of diuretics on mineral metabolism in the kidney and consequently on systemic mineral and bone metabolism.

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.

Hypercalcemia: a consultant's approach.

Due to their daily involvement in mineral metabolism, nephrologists are often asked to consult on children with hypercalcemia. This might become even more pertinent when the hypercalcemia is associated with acute kidney injury and/or hypercalciuria and renal calcifications. The best way to assess the severity of hypercalcemia is by measurement of plasma ionized calcium, and if not available by adjusting serum total calcium to albumin concentration. The differential diagnosis of the possible etiologies of the disturbance in the mineral homeostasis starts with the assessment of serum parathyroid hormone concentration, followed by that of vitamin D metabolites in search of both genetic and acquired etiologies. Several tools are available to acutely treat hypercalcemia with the current main components being fluids, loop diuretics, and antiresorptive agents. This review will address the pathophysiologic mechanisms, clinical manifestations, and treatment modalities involved in hypercalcemia.

Successful Reversal of Furosemide-Induced Secondary Hyperparathyroidism With Cinacalcet.

Secondary hyperparathyroidism (SHPT) is a rare complication of furosemide therapy that can occur in patients treated with the loop diuretic for a long period of time. We report a 6-month-old 28-weeks premature infant treated chronically with furosemide for his bronchopulmonary dysplasia, who developed hypocalcemia and severe SHPT, adversely affecting his bones. Discontinuation of the loop diuretic and the addition of supplemental calcium and calcitriol only partially reversed the SHPT, bringing serum parathyroid hormone level down from 553 to 238 pg/mL. After introduction of the calcimimetic Cinacalcet, we observed a sustained normalization of parathyroid hormone concentration at 27 to 63 pg/mL and, with that correction, of all biochemical abnormalities and healing of the bone disease. No adverse effects were noted. We conclude that in cases of SHPT due to furosemide in which traditional treatment fails, there may be room to consider the addition of a calcimimetic agent.

Life-Threatening Hypercalcemia During Prodrome of Pneumocystis jiroveci Pneumonia in an Immunocompetent Infant.

Severe hypercalcemia in infants is usually attributed to genetic etiologies and less commonly to acquired ones. An 8-week-old girl presented with failure to thrive, mild respiratory distress, and life-threatening hypercalcemia (23.5 mg/dL). Serum 1,25(OH)2-vitamin D (1,25(OH)2-D) level was elevated and parathyroid hormone undetectable. Evaluation for genetic mutations and malignant etiologies of hypercalcemia was negative. Treatment with intravenous hydration, loop diuretic, and calcitonin failed to correct the hypercalcemia, which was subsequently controlled with bisphosphonate therapy. Due to progressive respiratory deterioration, a bronchopulmonary lavage was done on day 17 of her hospitalization disclosing Pneumocystis jiroveci infection. The subsequent immunological investigation showed no abnormalities. She was treated with trimethoprim/sulfamethoxazole resulting in gradual clearing of her lungs and normalization of serum 1,25(OH)2-D level. A year later, she remains healthy with normal biochemical parameters of mineral metabolism. We conclude that in a child with hypercalcemia with suppressed parathyroid hormone and elevated 1,25(OH)2-D, once the genetic etiology for elevated 1,25(OH)2-D and malignancy are ruled out, one should investigate closely for a chronic granulomatous disease. Among the latter Pneumocystis jiroveci pneumonia infection should be considered even in an immunocompetent child.

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.